NZ613457B2 - Systems and methods for sample use maximization - Google Patents
Systems and methods for sample use maximization Download PDFInfo
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- NZ613457B2 NZ613457B2 NZ613457A NZ61345712A NZ613457B2 NZ 613457 B2 NZ613457 B2 NZ 613457B2 NZ 613457 A NZ613457 A NZ 613457A NZ 61345712 A NZ61345712 A NZ 61345712A NZ 613457 B2 NZ613457 B2 NZ 613457B2
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Abstract
Disclosed is a method of measuring an analyte concentration in a sample fluid. The method comprises providing the sample contained in a container dimensioned with a plurality of distinct widths to permit transmission of light along a plurality of varying path lengths that correspond to the plurality of distinct widths. The container is then illuminated along at least one of the plurality of path lengths. The container is illuminated to measure a first light intensity transmitted across said at least one of the plurality of path lengths, for the determination of the concentration of the analyte based on the measured first light intensity. of distinct widths. The container is then illuminated along at least one of the plurality of path lengths. The container is illuminated to measure a first light intensity transmitted across said at least one of the plurality of path lengths, for the determination of the concentration of the analyte based on the measured first light intensity.
Description
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SYSTEMS AND METHODS FOR SAMPLE USE MAXIMIZATION
CROSS-REFERENCE
This application claims priority to US. Provisional Patent Application Serial No. 61/435,250, filed
January 21, 201 l, which ation is entirely incorporated herein by nce.
BACKGROUND OF THE INVENTION
The discovery of a vast number of disease kers, new therapies and the establishment of
miniaturized medical systems have opened up new avenues for the prediction, diagnosis and monitoring of
ent of diseases in a point-of-care or other buted test settings. Point-of-care systems can rapidly
deliver test results to medical personnel, other medical professionals and patients. Early diagnosis of a e or
disease progression and monitoring of therapy are often al for treatment of deadly conditions such as
certain cancers and infectious diseases.
Diagnosis and treatment of diseases can take advantage of multiplexed biomarker measurements,
which provide additional knowledge of the condition of a patient. For example, when monitoring the effects of a
drug, three or more biomarkers can be measured in parallel. Typically, microtiter plates and other similar
apparatuses have been used to perform multiplexed separation-based assays. A microtiter plate (for example, a
384 well microtiter plate) can perform a large number of assays in parallel.
In a Point-of-Care (POC) device, the number of assays that can be performed in parallel is often
limited by the size of the deVice and the volume of the sample to be analyzed. In many POC devices, the number
assays performed is about 1 to 10. A POC device capable of performing multiplexed assays on a small sample
would be desirable.
A shortcoming ofmany multiplexed POC assay devices is the high cost of manufacturing the
components of the device. If the device is disposable, the cost of the components can make the manufacturing of
a POC deVice impractical. Further, for multiplexed POC devices that incorporate all of the ary reagents
onboard of the deVice, if any one of those reagents exhibit instability, an entire manufactured lot of devices may
have to be discarded even if all the other reagents are still usable.
When a customer is interested in customizing a POC device to a particular set of analytes,
manufacturers of multiplexed POC assay systems are often confronted with the need to mix and match the
assays and ts of the device. A multiplexed POC assay suitable to each customer can be very expensive,
difficult to calibrate, and difficult to maintain quality control.
POC methods have proven to be very valuable in monitoring disease and y (for e,
blood glucose systems in diabetes therapy, Prothrombin Time ement in anticoagulant therapy using
Warfarin). By ing multiple markers, it is ed that x diseases (such as cancer) for which
multi-drug therapies are required can be better monitored and controlled.
There exists the need to use multiple sources of information for monitoring the health status or
disease condition of individuals as well as treatments of various diseases. Especially ant is the
measurement of concentrations of several selected analytes (biomarkers, dies, gene expression levels,
metabolites, therapeutic drug concentrations and the like) over time. To make this process convenient and
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maximally ive, logies that enable measurement of any and all needed analytes (of whatever types)
using a small blood sample (blood drop obtained by finger-stick) or other le sample are particularly
valuable. Such technology will ideally be operable by non-technically trained users in distributed test settings,
e. g., homes, s, doctor’s offices, pharmacies, and retail shops. The present invention addresses these issues
and allows for one to be able to make such measurements routinely in patient’s home or other non-laboratory
setting.
There also exists the need to make the greatest use of available samples, particularly in the ce
where samples (e.g., blood samples) are limited by sample size. Blood s are used for the great ty
of medical/clinical tests. Blood cells have to be separated from plasma (or serum) prior to most types of
analysis since the presence of cells would compromise the assay chemistries. For example, glucose and
cholesterol are often ed by forming chemistries which would be interfered with by the presence of
formed ts, especially red cells, or hemoglobin (from lysed red cells).
Distributed test systems ideally require a small blood sample ed by fingerstick methods.
Such samples may be as small as 20 microliters (uL) (one drop) or less. Larger volume samples (say up to 200
uL) usually cannot be taken by fingerstick methods without repeated, inconvenient (“milking") of fingers.
Alternatively venous samples of several milliliters (mL) can be taken but this requires a medically trained
tomist.
It is usually very difficult to perform more than a single assay using small blood sample with 20
uL or less. This is especially so when the blood sample has to be filtered to remove cells and the recovery of
usable plasma from such small s is inefficient. Typically only about 5 uL or less of plasma can be
recovered. Samples as large as 200 uL can be efficiently separated by automated POC systems (Abaxis, Biosite
etc.) but this cannot be done routinely unless a technician is available to draw the .
SUMMARY OF THE ION
In view of the limitations of current methods, there is a pressing need for improved methods of
automatically separating plasma and/or other materials from blood cells. There is also a need for improved
accuracy of these measurements on analyte concentration. In measurements of biomarkers and other
components of blood for the purposes of monitoring therapy and diagnosis, it is important that the correct
volume of sample be used. In a laboratory setting, this is achieved by ing complex automated ments
and skilled professional staff members. In contrast in -of-care" settings such as homes, retail pharmacies
and shops, and the like, the methods and equipment used must enable non-technically trained people reliably to
obtain and process samples.
The present invention addresses the aforementioned needs and provides related advantages.
In some embodiments, the present invention relates to point-of-care and/or point-of-service
devices. In some embodiments, the present invention relates to systems, devices, user interfaces, and methods
for assaying samples using a point-of-care and/or point-of—service .
In one aspect, the devices and methods disclosed herein are designed to identify the sample type
(blood versus plasma and etc.) to measure the volume of sample early enough in the assay procedure to ensure
an appropriate sample is used is an intended assay. In another aspect, the present invention also allows for one
to be able to correct for significant volume errors that occur in performing an assay.
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In yet another aspect, this invention allows for simultaneous measurements on several es of
different types with high accuracy.
An aspect of the invention may be directed to an ted system for separating one or more
components in a biological fluid. The automated system may comprise a pipette tip or closed tube adapted to
engage with an tor wherein said pipette tip or tube comprises two opposing ends, at least one of which is
closed or sealable; and a fuge configured to e said sealed pipette tip or closed tube to effect said
separating of one or more components in a biological fluid. In an embodiment, the one or more ents are
selected from the group consisting of blood plasma, blood serum, blood cells, and particulates. In another
embodiment, when the pipette tip is engaged with the aspirator to effect a draw of the biological fluid. In
another embodiment, the pipette tip has an open end that forms an ht seal with the aspirator. In another
embodiment, the system further comprises an imaging device; and at least one other pipette tip ioned to
allow sing of a liquid into the pipette tip or tube of (a) or to allow the aspiration of a liquid from the
pipette tip or tube of (a). In another embodiment, the pipette tip or closed tube is oriented vertically when the
centrifuge is at rest. In another embodiment, the pipette tip or closed tube is oriented ntally when the
centrifuge is ng at a predetermined rotational velocity.
Another aspect of the ion may be a method for isolating components in a sample comprising
one or more of the following steps: loading a sample into a pipette tip or a tube comprising two opposing ends,
at least one of which is sealable or sealed; sealing the pipette tip on the at least one end of the pipette tip;
fuging the sealed pipette tip, thereby forming an interfacial region that separates the sample into a
atant and a pellet; imaging the centrifuged pipette tip to determine the location of the interfacial region;
and automatically aspirating the supernatant based on the location of the interfacial region. In an ment,
the method further comprises determining the location of the supernatant by said imaging step and automatically
aspirating the supernatant based on the location of the supernatant. In another embodiment, the determination
occurs with the aid of a processor, and said processor provides instructions to an aspirating device which
performs the automated aspiration step. In another embodiment, the imaging occurs by use of a camera that is
configured to capture the image of the side profile of the pipette tip or the tube. In another embodiment, the
supernatant includes one or more of the following: blood plasma or blood serum. In another embodiment, the
pellet includes one or more of the following: blood cells or particulates.
A computer-assisted method for characterizing an analyte suspected to be present in a sample may
be provided in accordance with an additional aspect of the invention. The computer-assisted method may
comprise obtaining a l image of the sample, wherein the digital image comprises at least a two-
dimensional array of pixels, and wherein each pixel comprises a plurality of intensity values, each of which
ponds to a distinct detection spectral region; correlating, with the aid of a mmable device, the
obtained intensity values with a predetermined set of values that define a c range of each ion
spectral region; and predicting the presence and/or ty of said analyte in the sample based on said
correlating of the obtained intensity values with a predetermine set of values. In an embodiment, the ity of
intensity values comprise intensity values for red, green, and blue detection spectral regions. In another
embodiment, the method further comprises selecting an illumination wavelength, and illuminating the sample
with the selected illumination wavelength prior to and/or concurrently with obtaining the digital image. In
another embodiment, the method further comprises, subsequent to obtaining the digital image, (a) selecting
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another illumination wavelength; (b) illuminating the sample with the other selected illumination wavelength;
(c) ing another digital image of the sample, wherein the digital image comprises at least a two-
dimensional array of pixels, and wherein each pixel comprises a plurality of intensity values, each of which
ponds to a distinct detection spectral region; and (d) predicting the presence and/or quantity of said
analyte in the sample based on the obtained intensity values from the digital image and said another digital
image.
Also, an aspect of the invention may be directed to a method of measuring an analyte
concentration in a sample fluid comprising providing the sample contained in a container dimensioned with a
plurality of distinct widths to permit ission of light along a plurality of varying path lengths that
correspond to the plurality of distinct widths; nating the container along at least one of the plurality of path
lengths; and imaging the container to measure a first light intensity transmitted across said at least one of the
plurality of path lengths, for the determination of the concentration of the analyte based on the measured first
light intensity.
In accordance with another aspect of the invention, a method of detecting the presence or
concentration of an analyte in a sample fluid ned in a container (e. g., cuvette) may comprise nating
the container along a first region having a first path length to yield a first measurement of light ity
itted across the first path length; moving the sample fluid to another region in the container having
another path length if the first measurement falls outside a predetermined dynamic range of transmitted light
intensity; illuminating the container along the another region to yield r measurement of light intensity
transmitted across the r path length; and optionally repeating second and third steps until a measurement
of light ity falls within the predetermined dynamic range, thereby detecting the presence or concentration
of the analyte. In an embodiment, the method further comprises deconvoluting a line scan of the image, y
detecting the presence or tration of an analyte. In another embodiment, the sample is moved from a first
region of the container having a first path length to a second region of the container having another path length
by aspirating the . In another embodiment, an end of the container is attached to a pipette which is
configured to aspirate the sample. In another embodiment, the sample is moved up or down the length of the
container. In another embodiment, the container is a pipette tip. In another embodiment, the container is
conically shaped. In another embodiment, the container has two open ends. In another embodiment, a first
open end has a greater diameter than a second open end. In r embodiment, the container has a plurality of
distinct widths to permit transmission of light along a plurality of varying path lengths. In r embodiment,
the container volume is less than 100 microliters. In another embodiment, a plurality of distinct path lengths are
imaged simultaneously.
A method may be provided as an onal aspect of the ion. The method may be provided
for characterizing an analyte suspected to be present in a sample of biological fluid, said method comprising:
providing said sample of biological fluid; allowing said analyte to react with one or more reagents that
specifically react with said analyte to generate an lly detectable signal; and measuring said optically
detectable signal with a ity of detection spectral regions, n the presence of said optically detectable
signal within a dynamic range of at least one detection spectral region is indicative of the concentration of said
analyte in said sample of biological fluid. In an embodiment, the measuring is performed by an imaging device
red to measure a plurality of detection spectral regions. In another embodiment, the imaging deVice is
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configured to measure the plurality of ion spectral s simultaneously. In another embodiment, the
imaging device is configured to measure the plurality of detection spectral regions sequentially.
An aspect of the invention provides a method for increasing the accuracy of an assay comprising
imaging a sample in a first tip to ine the volume of the first sample; imaging one or more reagents in a
second tip to determine the volume of the one or more reagents; mixing the sample and the one or more reagents
to form a reaction mixture; g the reaction mixture; correcting a calibration based on said determined
volumes of the sample and the one or more reagents; and calculating a concentration of an analyte using the
ted calibration. In an embodiment, the method r comprises imaging the reaction mixture to
determine the volume of the reaction mixture. In another embodiment, the imaging of the sample in the first tip
is conducted using a camera configured to e a side profile of the first tip. In another embodiment, imaging
of the one or more reagents in the second tip is ted using a camera configured to capture a side profile of
the second tip. In another embodiment, the height of the sample and the one or more reagents is calculated
based on the captured profiles. In another embodiment, determining the volume is based on the height of the
sample and the one or more reagents and the known cross-sectional areas of the sample and the one or more
reagents respectively. In r embodiment, the calibration is also based on the determined volume of the
reaction mixture.
Another aspect of the ion provides a setup, comprising: a vessel configured to accept and
confine a sample, wherein the vessel ses an interior surface, an exterior surface, an open end, and an
opposing closed end; and a tip configured to extend into the vessel through the open end, wherein the tip
comprises a first open end and second open end, wherein the second open end is inserted into the vessel,
wherein the vessel or the tip further comprises a protruding surface feature that prevents the second open end of
the tip from contacting the bottom of the interior e of the closed end of the vessel. In an embodiment, the
surface feature is integrally formed on the bottom interior surface of the vessel. In r embodiment, the
surface feature comprises a ity of bumps on the bottom interior surface of the . In r
embodiment, the protruding surface feature is at or near the closed end.
Another aspect of the invention provides a sample processing apparatus comprising a sample
preparation station, assay station, and/or ion station; a control unit having computer-executable commands
for performing a point-of-service service at a designated location with the aid of at least one of said sample
preparation n, assay station and detection station; and at least one centrifuge configured to perform
centrifilgation of a sample from a fingerstick. In an embodiment, the centrifuge is ned within the sample
preparation station and/or the assay station. In another embodiment, the computer-executable commands are
configured to perform the point-of-service e at a site selected from the group consisting of a retailer site,
the subject’s home, or a health assessment/treatment location.
Another aspect of the invention provides a method for dynamic feedback, said method comprising:
taking an initial measurement of a sample within a container using a detection mechanism; based on said initial
measurement, determining, using a processor, whether the sample concentration falls into a desired range, and
determining, using a processor, (a) a degree of dilution to be performed if the sample concentration is higher
than the desired range or (b) a degree of concentration to be performed if the sample concentration is lower than
the desired range; and adjusting the sample tration according to the determined degree of dilution or the
determined degree of concentration. In an embodiment, the method further ses taking a subsequent
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measurement of the sample within the container. In another embodiment, the method further comprises, based
on the subsequent measurement determining, using a processor, whether the sample concentration falls into a
desired range. In another embodiment, the subsequent measurement is made using the detection mechanism. In
another embodiment, the method further comprises determining a teristic of the sample based on the
uent measurement. In another embodiment, the characteristic is selected from one or more of the
ing: the ce or concentration of an analyte, the presence or concentration of a cell, and the
morphology of the cell. In another embodiment, the subsequent measurement is made using a separate detection
mechanism from the initial detection mechanism. In another embodiment, the initial measurement provides a
crude cell concentration measurement of the sample. In another embodiment, the subsequent measurement
provides a measurement of cell concentration of the sample of greater resolution than the initial measurement.
In another embodiment, the initial measurement is taken by g the . In another embodiment, the
ing of the sample concentration permits detection of analyte that would otherwise fall outside the desired
range.
r aspect of the invention provides a method for providing quality control, said method
comprising capturing an image of conditions under which a detection ism es a characteristic of a
sample; and determining, using a processor, based on the image whether there are undesirable conditions under
which the detection mechanism is operated. In an embodiment, the undesirable conditions es the presence
of one or more undesirable materials. In another embodiment, the undesirable als includes one or more of
the following: bubbles, particles, fibers, debris, and precipitates that interfere with the measurement of the
characteristic of the sample. In another embodiment, the ion ism is a different mechanism from a
mechanism used to capture the image. In another embodiment, the image is captured using a camera. In
another embodiment, the method further comprises ing an alert if an rable condition is detected. In
another embodiment, the method further comprises adjusting the sample if an undesirable condition is ed.
In another embodiment, the image includes an image of the sample. In another ment, the image includes
an image of one or more of the following: the sample container or the detection mechanism.
Another aspect of the invention is an automated system for ting one or more components in
a biological fluid comprising a centrifuge comprising one or more bucket configured to e a container to
effect said separating of one or more components in a fluid sample; and the container, wherein the container
es one or more shaped feature that is complementary to a shaped feature of the bucket. In an embodiment,
the shaped feature of the bucket includes one or more shelf upon which a protruding portion of the container is
ured to rest. In another embodiment, the bucket is configured to be capable of accepting a plurality of
containers having different configurations, and wherein the shaped feature of the bucket includes a plurality of
shelves, wherein a first container having a first configuration is configured to rest upon a first shelf, and a
second container having a second configuration is configured to rest upon a second shelf.
Another aspect of the invention provides an assay unit comprising a first end and a second end; an
outer surface; and an inner surface comprising one or more selected patterns each of which is lized
thereon or therein with a capture reagent capable of capturing an analyte suspected to be present in a biological
sample, wherein the first end and the second end are of different dimensions.
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Another aspect of the ion provides an assay unit comprising an identifier that is used to
determine (a) the one or more capture reagents immobilized on the inner surface; and (b) source of the
biological sample if the assay unit contains said sample.
Another aspect of the invention provides an assay unit comprising a plurality of selected patterns,
each pattern of said plurality comprises a distinct capturing agent.
Other goals and advantages of the invention will be further appreciated and understood when
considered in conjunction with the following ption and accompanying drawings. While the ing
description may contain specific details describing particular embodiments of the invention, this should not be
construed as limitations to the scope of the invention but rather as an exemplification of preferable
embodiments. For each aspect of the invention, many variations are le as suggested herein that are known
to those of ordinary skill in the art. A variety of changes and modifications can be made within the scope of the
invention without departing from the spirit thereof The various compounds/devices disclosed herein can be
used separately or conjunctively in any combination, for any methods disclosed herein alone or in any
combinations.
ORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this specification are herein
incorporated by reference to the same extent as if each individual publication, patent, or patent application was
ically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with ularity in the appended claims. A better
tanding of the features and advantages of the present invention will be ed by reference to the
following detailed description that sets forth illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawing(s) of which:
Figure 1 shows a side view of a centrifuge.
Figure 2 shows a face on view of a centrifuge.
Figure 3 shows a perspective view of the back of a fuge.
Figure 4 shows a top view of a sample tip.
Figure 5 shows a side view of a sample tip.
Figure 6 shows a sectional view of a sample tip.
Figure 7 shows a diagram of a sample tip positioned in a sample above a /packed cell
interface.
Figure 8 shows a graph of centrifugation time as a function of revolutions per minute.
Figure 9 shows a graph of centrifugation time as a function of the radius of the centrifuge rotor.
Figure 10 shows an empty capped sample tip.
Figure 11 shows a capped sample tip containing a sample of a bodily fluid, e.g., blood.
Figure 12 shows a capped sample tip ning a sample of about 23% hematocrit blood after
centrifugation.
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Figure 13 shows a capped sample tip containing a sample of about 31% hematocrit blood after
fugation.
Figure 14 shows a capped sample tip containing a sample of about 40% hematocrit blood after
centrifugation.
Figure 15 shows a capped sample tip ning a sample of about 52% hematocrit blood after
centrifugation.
Figure 16 shows a capped sample tip containing a sample of 68% hematocrit blood after
centrifugation.
Figure 17 shows a comparison of hematocrit measured using by digitally imaging system a
centrifuged sample (“hematocrit, % reported") and hematocrit ed by standard ematocrit apparatus
(“hematocrit, % target")
Figure 18 shows a diagram of a tip used for reactions and a tip used for blood/plasma (dimensions
shown in mm).
Figure 19 shows a cylindrical capillary ning a sample.
Figure 20 shows angles and dimensions for calculating volumes within a conical container, e. g. a
capillary.
Figure 21 shows angles and ions for calculating volumes within a conical container, e. g., a
capillary.
Figure 22 shows angles and dimensions for calculating volume of a spherical cap.
Figure 23 shows dimensions for calculating the volume of a sample contained within a cylindrical
tip, where the sample has a single meniscus.
Figure 24 shows dimensions for calculating the volume of a sample contained within a cylindrical
tip, where the sample has two i.
Figure 25 shows dimensions for calculating the volume of a sample contained within and/or
associated with a cylindrical tip, where the sample has two i and one of which is external to the
cylindrical tip.
Figure 26 shows dimensions for calculating the volume of a sample ned within a cylindrical
tip, where there is a bubble in the sample.
Figure 27 shows dimensions for calculating the volume of a sample contained within a cylindrical
tip, where there is a bubble in the sample that spans the width of the cylindrical tip.
Figure 28 shows dimensions for calculating the volume of a sample contained within and/or
associated with a cylindrical tip, where the sample includes a pendant droplet of sample outside the rical
tip.
Figure 29 shows dimensions for calculating the volume of a residual sample contained within a
cylindrical tip.
Figure 30 shows a blood sample within a tip prior to being mixed with a magnetic reagent.
Figure 31 shows a blood sample being mixed with a magnetic reagent.
Figure 32 shows a blood sample mixed with a magnetic reagent.
Figure 33 shows a blood sample mixed with a magnetic reagent contained within a tip.
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Figure 34 shows a blood sample mixed with a magnetic reagent moved to a selected position
within a tip.
Figure 35 shows a magnetic force d by a magnet (M) to a blood sample mixed with a
magnetic reagent.
Figure 36 shows a blood sample that has been separated into a red cell component and a plasma
component using magnetic force.
Figure 37 shows a well positioned beneath a tip ning a blood sample that has been separated
into a red cell component and a plasma component.
Figure 38 shows a depiction of blood plasma being transferred from a tip to a well.
Figure 39 shows a tip after dispensing of blood plasma to a well.
Figure 40 shows a high contrast image of a cylindrical tip ning a liquid with low absorbance.
Figure 41 shows an image of a conical tip containing a liquid with high absorbance.
Figure 42 shows a tip with a high absorbance liquid showing two menisci within the tip.
Figure 43 shows a tip with a sample liquid and large s that span the diameter of the tip.
Figure 44 shows a tip containing water showing a clear upper us in a transparent tip or
capillary.
Figure 45 shows a graph of computed Protein-C concentration as a function of sample volume.
Figure 46 shows an image of a sample transfer device with a capillary, housing, plunger, groove,
and raised feature. The raised feature may help locate the plunger in the housing.
Figure 47 shows a sample contained with the capillary of a sample transfer device.
Figure 48 shows a sample transfer device after a sample has been ejected by a plunger.
Figure 49 shows a sample transfer device after a sample has been incompletely ejected.
Figure 50 shows a conical tip containing a sample, with the position L3 ted by the arrow
shown.
Figure 51 shows a graph of the ratio of the distance between L2 and L1 and the distance between
L3 and L1 as a function of sample volume.
Figure 52 shows a schematic of a chemical reaction that es a colored product.
Figure 53 shows a tic of a chemical reaction that produces a colored product from
cholesterol.
Figure 54 shows a schematic of a chemical reaction that uses reducing equivalents to produce a
colored product.
Figure 55 shows an example of a compound that changes color upon being xed with a
metal ion.
Figure 56 shows a series of images of tips with two-fold decreasing concentration of n from
right to left, except for the left-most tip, which has no albumin.
Figure 57 shows a series of images of tips with two-fold decreasing concentration of cholesterol
from right to left, except for the left-most tip, which has no cholesterol.
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Figure 58 shows a series of hemispherical wells machined from a block of white opaque plastic,
which each well having two-fold decreasing concentration of analyte from right to left, except for the left-most
well, which has no analyte. In some embodiments, the analyte may be calcium.
Figure 59 shows a series of hemispherical wells machined from a block of white opaque plastic,
which each well having two-fold decreasing concentration of analyte from right to left, except for the left-most
well, which has no analyte. In some embodiments, the analyte may be magnesium.
Figure 60 shows a series of hemispherical wells machined from a block of white opaque plastic,
which each well having two-fold decreasing concentration of analyte from right to left, except for the left-most
well, which has no analyte. In some embodiments, the analyte may be urea.
Figure 61 shows a series of tips containing bromophenol blue solutions.
Figure 62 is an illustration of tips having a plurality of distinct optical path lengths.
Figure 63 shows a light path through a rectangular e.
Figure 64 shows a light path through a iter well.
Figure 65 shows a light path through a conically shaped cuvette.
Figure 66 shows a graph of light intensity as a function of on as measured on tips containing
samples with varying concentration of henol blue solutions for red, green, and blue color channels.
Figure 67 shows an image of the tips that were analyzed in Figure 66.
Figure 68 shows a graph of signal as a on of bromophenol blue concentration as measured by
red, green, and blue color ls. The optical density may be measured at 589 nm.
Figure 69 shows a log scale graph of signal response as a function of bromophenol blue
concentration as measured by blue (diamonds) and red es) color channels.
Figure 70 shows a graph of tration measured by color analysis of digital images as a
function actual concentration.
Figure 71 shows a graph of signal response as measured by red (squares), green (diamonds), and
blue (triangles) color ls as a function of albumin concentration.
Figure 72 shows three graphs of signal se as measured for green, red, and blue color
channels for polystyrene latex particles.
Figure 73 shows tips that each separately n reagents NADH, WST-l, PMS, and two tips
containing a mixture of the reagents.
] Figure 74 shows a digital image of tips containing two-fold sing concentration of lactate
dehydrogenase (LDH) from left to right.
Figure 75 shows a graph of optical density measured at 450 nm as a function of LDH.
Figure 76 shows solutions of potassium chloride added to potassium assay strips.
Figure 77 shows tips containing blood s mixed with blood typing reagents for , Anti-
B, Anti-D, and Control (from left to right).
Figure 78 shows measured signals for signal as a function of position for red (left column), green
(middle column), and blue (right column) for samples mixed with Anti-A, Anti B, Anti-D, and Control reagents.
Figure 79 shows normalized signal as a function of relative concentration measured for narrow and
wide path lengths using red, green, and blue color channels.
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Figure 80 shows a graph of log of measured concentration as a function of actual concentration,
illustrating the accuracy of the measurement algorithm.
Figure 81 shows a fluorescence image of assay ts in tubes.
Figure 82 shows an image of on products in tips.
Figure 83 shows an image of reaction products in tips.
Figure 84 shows an image of reaction products in tips.
Figure 85 shows an image of reaction products in tips.
Figure 86 shows an image of reaction products in tips.
Figure 87 shows an image of reaction products in tips.
Figure 88 shows a background color image obtained for calibration.
Figure 89 shows a fluorescence image of reaction products in tips.
Figure 90 shows red and blue color channel response and fluorescence response as a on of
DNA copy number.
Figure 91 shows graph of transformed 3-color signal as a function of fluorescence signal.
Figure 92 shows a graph of green channel signal response as a function of pixel position.
Figure 93 shows an image of tips containing solutions of bromophenol blue and water.
Figure 94 shows an image of additional tips that may contain solutions of bromophenol blue and
water.
] Figure 95 shows an schematic of a tip containing reaction mixtures to perform le assays.
Figure 96 shows an image of tips containing solutions of bromophenol blue and water.
Figure 97 shows a graph of signal response for sample, water, and control in multiple standards.
The samples may be aqueous calibrators containing known concentrations of analyte.
Figure 98 shows tips containing assays for both Ca2+ (upper region of the tip) and Mg2+ (lower
region of the tip).
Figure 99 shows four tips with s types of serum samples: zed (reddish in color),
lipemic (gray), icteric (yellow in , and normal (from left to .
Figure 100 shows a schematic of a camera and optical components.
Figure 101 shows a cross-sectional View of a camera and optical components ing a white
light source, an aperture, and a .
Figure 102 shows a tic of an optical setup for measuring light signal using (A) a sensor that
is positioned to detect light at a perpendicular angle to an tion beam, and (B) a sensor that is positioned in
line with an tion beam.
Figure 103 shows images taken using (A) an excitation beam perpendicular to a sensor and (B) an
excitation beam that is in line with a .
Figure 104 shows an array of printed dyes that can be used to calibrate the optical setup.
Figure 105 shows a graph of signal as a function of sample volume. Series 1-5 may correspond to
different analyte concentrations, such as 0, 20, 40, 60, and 80 respectively.
Figure 106 shows a graph of signal as a function of sample volume. Series 1-5 may correspond to
different analyte concentrations, such as 0, 20, 40, 60, and 80 respectively.
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Figure 107 shows a graph of signal as a function of sample volume. Series 1-5 may correspond to
different e concentrations, such as 0, 20, 40, 60, and 80 respectively.
Figure 108 shows a graph of measured analyte concentration as a function of actual analyte
concentration.
Figure 109 shows a graph of ed analyte concentration as a function of actual analyte
concentration.
Figure 110 schematically illustrates an exemplary method for an ELISA assay.
Figure 111 shows an example of a rotor at rest with buckets vertical.
Figure 112 shows an example of a rotor at a speed with buckets at a small angle to horizontal.
Figure 113 shows an example of a bucket configuration.
Figure 114 shows an example of a centrifugation vessel mated with the bucket.
Figure 115 shows an example of another centrifugation vessel that can be mated with the bucket.
Figure 116 shows an example of a centrifugation vessel.
Figure 117 shows an example of an extraction tip.
Figure 118 provides an example ofhow the centrifugation vessel and extraction tip may mate.
] Figure 119 is an image that was taken of the original reaction e prior to centrifugation.
Figure 120 is another image that was taken of the original reaction mixture prior to centrifugation
Figure 121 is an additional image that was taken of the original reaction mixture prior to
centrifugation
Figure 122 shows results as distance of the interface from the plasma meniscus.
Figure 123 es an example of a fluorescence micrograph showing labeled leukocytes.
Figure 124 provides an example of intracellular patterns using eld images.
Figure 125 provides an example of multi-parameter acquisition of data from labeled cell samples.
Figure 126 provides an example of field images of human whole blood.
Figure 127 provides an example of quantitative multi-parametric data acquisition and analysis.
Figure 128 shows ion in light distribution.
Figure 129 shows data from five assays.
Figure 130 shows a parameter plotted against tration of the e, as well as graphs
relating to accuracy, precision, and ted concentration.
Figure 131 shows images collected by a l camera.
Figure 132 illustrates examples of images taken of reaction product.
Figure 133 provides examples of images that were analyzed before spinning in the centrifuge, and
after spinning in the centrifuge.
Figure 134 illustrates examples of images taken of reaction product.
Figure 135 illustrates spectra of several serum samples.
Figure 136 illustrates an example detection process of the invention using an array.
Figure 137 rates an example detection process of the invention using beads.
Figure 138 illustrates an example ion process of the ion using tagged aptamers.
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Figure 139 illustrates detection of aptamer binding to a complementary probe.
Figure 140 illustrates e of binding n aptamer and a non-complementary probe.
] Figure 141 illustrates binding city of aptamers on an array.
Figure 142 shows a more detailed view of analyte detection on an array.
Figure 143 shows an example array.
Figure 144 shows a plot of chemiluminescence against concentration for a vitamin D assay.
Figure 145 shows a plot of chemiluminescence against concentration for an estradiol assay.
Figure 146 shows a spectrophotometric measurement ofWBC concentration.
Figure 147 shows plots of turbidity as a function of time.
Figure 148 is a plot of inflection points for three experiments at 800 copies/uL and 80 copies/uL.
Figure 149 is a plot of an e in which magnetic beads are used for the analysis of ns
and small molecules via ELISA assays.
Figure 150 is a plot of an example in which magnetic beads are used for the analysis of proteins
and small molecules via ELISA assays.
DETAILED PTION OF THE INVENTION
While preferable embodiments of the invention have been shown and described herein, it will
be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous
variations, changes, and substitutions will now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the embodiments of the invention described
herein can be employed in practicing the ion.
] The invention es mobile applications for system and methods for sample use maximization.
Various aspects of the invention described herein may be applied to any of the ular applications set forth
below or for any other types of diagnostic or therapeutic applications. The ion may be applied as a
standalone system or method, or as part of an integrated pre-clinical, clinical, laboratory or medical application.
It shall be understood that different aspects of the invention can be appreciated individually, tively, or in
combination with each other.
The devices and systems herein can provide an effective means for real-time detection of analytes
present in a bodily fluid from a subject. The detection methods may be used in a wide y of circumstances
including identification and quantification of analytes that are associated with specific biological processes,
physiological conditions, disorders or stages of disorders. As such, the systems have a broad spectrum of utility
in, for example, drug screening, disease diagnosis, phylogenetic classification, parental and forensic
identification, disease onset and recurrence, individual response to treatment versus population bases, and/or
monitoring of therapy. The subject devices and systems are also particularly useful for advancing preclinical and
clinical stage of development of therapeutics, improving patient compliance, monitoring ADRs associated with
a prescribed drug, developing dualized medicine, outsourcing blood testing from the l laboratory to
the home or on a prescription basis, and/or ring therapeutic agents following regulatory approval. The
subject devices and system can be utilized by payors outsourcing blood tests from a l laboratory. The
devices and systems can provide a flexible system for personalized medicine. Using the same system, a device
can be changed or interchanged along with a protocol or instructions to a programmable processor of the
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s to perform a wide y of assays as described. The systems and devices described herein, while
being much smaller and/or portable, embody novel features and offer many functions of a laboratory instrument.
In an aspect, a system of the invention ses a device sing assay units and reagent
units, which include reagents, e. g., both liquid and/or solid phase reagents. In some embodiments, at least one
of the whole device, an assay unit, a reagent unit, or a combination thereof is disposable. In a system of the
invention, the detection of an analyte with the subject device is typically automated. Such automation can be
effected by a built-in protocol or a protocol provided to the system by the manufacturer.
The s and systems as described herein can offer many features that are not available in
existing POC s or integrated is systems. For example, many POC cartridges rely on a closed fluidic
system or loop to handle small volumes of liquid in an efficient manner. The fluidic devices such as cartridges
described herein can have open fluid movement between units within a given cartridge. For example, a t
can be stored in a unit, a sample stored in a sample collection unit, a diluent stored in a t unit, and the
capture surface can be in an assay unit, where in one state of cartridge, none of the units are in fluid
communication with any of the other units. The units can be movable relative to each other in order to bring
some units into fluid communication using a fluid transfer device of the system. For example, a fluid transfer
device can comprise a head that engages an assay unit and brings the assay unit in fluidic communication with a
reagent unit. In some cases, the head is a pipette head that moves the assay unit (e.g., tip) in fluid
ication with a reagent unit.
] Accordingly, in an embodiment, the present invention provides a method of detecting and /or
measuring the concentration of an analyte in a bodily fluid or tissue sample, the method typically comprises the
steps of providing a sample (e.g., blood, urine, saliva, tissue) to a device or system of the invention, allowing the
sample to react within at least one assay unit of the device, and detecting the detectable signal generated from
the analyte in the blood sample.
One aspect of the invention es for analyzing samples using a point-of-care device that is
configured to maximize sample utilization. For example, more than about 15, 25, 50, 75, or 100 assays can be
performed on a sample having a volume of less than about 1, 20, 50, 100, or 500 uL. The sample can be a blood
sample taken from a finger prick. The sample can be collected in a sealable capillary or tip. The sample can be
prepared for one or more assays by subjecting the sample to a separation (e. g., centrifugation) and/or on
process. The one or more assays can be prepared by combining the sample, which may have been separated and
diluted, with one or more reagents in a reaction chamber. The reaction chamber can be a pipette tip, vial, a
sample transfer device, and/or a cuvette. The one or more assays can be configured such that an optical signal
can be measured which is tive of the concentration of one or more analytes in the sample. The reaction
chamber can contain a plurality of assays, which may be spatially separated. A plurality of optical s can
be generated within a single reaction chamber from one assay, or from a plurality of spatially separated assays.
The one or more l signals can be measured by a digital imaging camera that can measure a plurality of
detection spectral regions or detection bands, e. g., red, green and blue. The optical signal can be measured on
the assay reaction product in the reaction chamber, which can be a pipette tip or other sample containers. The
systems, devices, and methods can be fully automated or semi-automated by programmable logic.
Another aspect of the invention provides for s, devices, and methods for preparing s
for analysis. Samples can be prepared for analysis by one or more separation devices. For example, a sample
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can be prepared for analysis by centrifugation within a centrifuge. Other separations based on charge, size,
hydrophobicity/hydrophilicity, and/or volatility can also be implemented.
One aspect of the invention provides for sample and reaction product analysis using image-based
analysis. The system can include a camera that can measure an optical signal using one or more detection
spectrum regions. For example, a camera can measure an optical signal using red, green, and blue detection
spectrum regions. The measured signal can include three measured values that can be interpreted using one or
more thms described herein. The use of more than one detection spectrum region can increase the
dynamic range of an assay and can increase the accuracy of a measurement as compared to measurements using
a single detection spectrum region.
The invention also es for s, devices, and methods for performing optical
measurements on samples and assay reaction products that are contained within reaction chambers, each with a
plurality of distinct path lengths. The reaction rs can have a plurality of distinct path lengths such that a
greater or lower amount of light absorbance is observed. The plurality of distinct path lengths (such as, for
example, through the sample and/or reaction r) allows for an increase in the dynamic range of a selected
assay protocol. The image of the reaction chamber can be ed as described herein to obtain information on
the sample or the assay reaction products. The ation of utilizing the plurality of available path lengths
within a single reaction r and the use of three channel detection spectrum regions y enhances the
dynamic range of a given assay.
A system for performing sample preparation and analysis can include instrumentation, disposable
components, and reagents. The system can accept samples and automatically performs a plurality of assays
without user intervention. Where desired, the instrumentation can include a graphical user interface, a
mechanism for ucing cartridges, which may be disposable, a motorized stage, which may have mobility in
three dimensions, one or more -head liquid handling devices, one or more multi-head liquid handling
devices, one or more devices for performing sample preparation, optical s, which can include a PMT
and/or an imaging device, temperature controllers, and communication devices. The disposable ent can
include a able cartridge that contains sample tips, tip seals, and reagents. In some embodiments, the
disposable cartridge may also n neutralizing assemblies configured to absorb and lize liquid assay
products.
] The instrumentation, disposable ents, and reagents can be housed within a closeable
environment, such as a case or a cabinet. In some embodiments, the case has a cross-sectional area less than
about 4 m2, 2 m2, 1 m2, 0.5 m2, 0.1 m2, 0.05 m2, or lower. The invention provides for a distributed test system,
such as a point-of—care device, which can include one or more of the following aspects:
1. Efficient (centrifugal) separation of blood and recovery of the separated plasma
2. Dilution of the plasma sample to one or more levels (for example 1:10, 1:100, 1:1000) so that
each assay can be performed at an optimal dilution
3. Optimized distribution of sample to several different assays which may involve several different
methodologies
4. Optimal assay protocols
5. Use of open-ended ar section cuvettes for sample analysis, mixing with reagents,
incubation and presentation to optical systems
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6. Analysis of assays using imaging technology (scanning and/or raphy, and/or copy)
In one embodiment, the device of the invention is self-contained and comprises all ts,
liquid- and solid-phase reagents, required to perform a plurality of assays in parallel. Where desired, the device
is configured to perform at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 500, 1000 or more assays. One
or more control assays can also be incorporated into the device to be performed in parallel if desired.
Calibrators can also be provided for assay system ation. Some examples of dried controls and calibrators
useful for assay system calibration can include aqueous solutions of analytes, serum, or plasma samples with
known levels of analytes, known quantities of such calibrators and controls can also be dried by lyophilization,
vacuum drying, and other manufacturing processes (and dissolved during the assay).
By incorporating these components within a point-of-care system, a patient or user can have a
plurality of analytes, for example more than about 10, 20, 30, 50, 75, 100, 150, or 200 analytes, quantified
within less than about 0.5, l, 2, 3, 4, 5, 10, 20, 30, 60, 120, 180, 240, 480 or 600 minutes.
The subject s and systems can be utilized for conducting quantitative immunoassays, which
can be ted in a short period of time. Other assay type can be performed with a device of the invention
including, but not d to, measurements of c acid sequences and measurements of metabolite, such as
cholesterol or electrolytes such as magnesium and chloride ions. In some embodiments, the assay is completed
in no more than one hour, preferably less than 120, 60, 30, 15, 10, 5, 4, 3, 2, or 1 minute. In other embodiments,
the assay is performed in less than 5 s. The duration of assay ion can be adjusted ingly to the
type of assay that is to be d out with a device of the invention. For example, if needed for higher
sensitivity, an assay can be incubated for more than one hour or up to more than one day. In some examples,
assays that require a long duration may be more cal in other POC applications, such as home use, than in a
clinical POC setting.
In other embodiments of the invention the reagent units of a subject device are configured to be a
set of d-match ents. A reagent unit typically stores liquid or solid reagents necessary for
conducting an assay that detect a given analyte. The assay units can mes (or optionally not always)
comprise at least one capture surface capable of reacting with an analyte from the sample of bodily fluid. The
assay unit may be a tubular tip with a capture surface within the tip. Examples of tips of the invention are
described herein. Each individual assay and reagent unit can be configured for assay function independently. To
assemble a device, the units can be assembled in a just-in-time fashion for use in an integrated device, which can
take the format of cartridge.
A housing for a device of the invention can be made of a polymeric material, a metallic material or
a composite material, such as, e.g., aluminum, yrene or other moldable or machinable plastic, and can
have defined locations to place assay units and reagent units. The housing may include a metal or any other
material. The housing may partially or entirely enclose the assay units and/or reagent units. The housing may
support the weight of the assay units and/or reagent units. In an embodiment, the housing has means for blotting
tips or assay units to remove excess liquid. The means for blotting can be a porous membrane, such as cellulose
acetate, or a piece bibulous material such as filter paper.
In some embodiments, at least one of the ents of the device may be constructed of
polymeric materials. Non-limiting examples of polymeric materials include polystyrene, polycarbonate,
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polypropylene, polydimethysiloxanes (PDMS), polyurethane, polyvinylchloride (PVC), polysulfone,
thylmethacrylate , acrylonitrile-butadiene-styrene (ABS), and glass.
The device or the subcomponents of the device may be ctured by variety of methods
including, without tion, stamping, injection molding, embossing, casting, blow molding, machining,
welding, ultrasonic welding, and thermal bonding. In an embodiment, a device in ctured by injection
molding, thermal bonding, and ultrasonic welding. The subcomponents of the device can be affixed to each
other by thermal bonding, ultrasonic welding, friction fitting (press fitting), adhesives or, in the case of certain
substrates, for example, glass, or semi-rigid and non-rigid polymeric substrates, a natural adhesion between the
two components.
] A system as described can run a y of assays, regardless of the analyte being detected from a
bodily fluid sample. A protocol dependent on the identity of the device may be transferred from an al
device where it can be stored to a reader assembly to enable the reader assembly to carry out the specific
protocol on the device. In some embodiments, the device has an identifier (ID) that is detected or read by an
identifier detector bed herein. The identifier can enable two-way communication between the device and
a sensor or receiving system. The identifier or can communicate with a communication assembly via a
controller which transmits the identifier to an external device. Where desired, the external device sends a
ol stored on the external device to the communication assembly based on the identifier. The ol to be
run on the system may comprise instructions to the controller of the system to perform the protocol, including
but not limited to a particular assay to be run and a detection method to be performed. Once the assay is
med by the system, a signal indicative of an analyte in the bodily fluid sample is generated and detected
by a detection assembly of the . The detected signal may then be communicated to the communications
assembly, where it can be itted to the external device for processing, including without limitation,
calculation of the analyte concentration in the sample.
Systems, devices and methods for performing sample analysis using point-of-care devices and tips
that can function as reaction chambers are described in US. Patent Publication No. 088336 and US.
Provisional Application No. 60/997,460, each of which is incorporated herein by reference in its entirety for all
purposes.
Sample ng and Reaction Chambers
Samples, reagents, and assembled assays described herein can be handled and contained by a
variety of reaction chamber types. A sample handing device and a reaction r can be a well, a tube, or an
open ended tip, which may also be a cuvette. As used herein, a tip can also referred to as a sample tip, a cuvette
tip, a reaction chamber, a cuvette, a capillary, a sample handing device, or a sample transfer device. Samples
may be ted from a source into a tip or a tube. The tips may be sealed. Such seals may be permanent or
reversible. Diluted samples can be combined with one or more ts and mixed (as described in previous
applications) within “assay elements" such as tips (open-ended cuvettes) or open or covered wells. Once the
assay is ready for reading, the assay element can be presented to the optical system for image analysis or other
types of reading. Alternatively, assay reaction es can be transferred from one type of element to another.
For example, assays incubated in tips can be blotted onto an absorbent or us medium or assays incubated
in wells can be aspirated into tips. Many assays can be processed in parallel. Assay readout can be serial or
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simultaneous ing on the assay protocol and/or incubation time. For assays involving measurement of a
rate of change, the assay element can be presented to the optical system more than once at different times.
Fluid and Material Transfer Devices
A fluid transfer device can be part of a system. The fluid transfer device can se a plurality
of heads. Any number of heads as is necessary to detect a plurality of analytes in a sample is envisioned for a
fluid transfer device of the invention. In an example, a fluid transfer device has about eight heads mounted in a
line and separated by a distance. In an embodiment, the heads have a d nozzle that engages by press fitting
with a variety of tips, such as assay unit or sample collection units as described . The tips can have a
feature that enables them to be removed automatically by the instrument and disposed into in a housing of a
device as described after use. In an embodiment, the assay tips are clear and transparent and can be similar to a
cuvette within which an assay is run that can be ed by an optical detector such as a photomultiplier tube or
camera sensor.
In an example, a programmable processor (e. g., central sing unit, CPU) of a system can
comprise or be configured to accept (such as, e. g., from a memory location) instructions or commands and can
operate a fluid transfer device according to the instructions to transfer liquid samples by either awing (for
drawing liquid in) or extending (for expelling liquid) a piston into a closed air space. The processor can be
configured to facilitate aspiration and/or dispensing. Both the volume of air moved and the speed of movement
can be ely lled, for example, by the programmable processor.
Mixing of samples (or reagents) with diluents (or other reagents) can be achieved by ting
components to be mixed into a common tube and then repeatedly aspirating a significant fraction of the
combined liquid volume up and down into a tip. Dissolution of reagents dried into a tube can be done is similar
fashion. tion of liquid s and reagents with a capture surface on which is bound a capture reagent
(for example an antibody) can be achieved by drawing the appropriate liquid into the tip and holding it there for
a predetermined time. Removal of samples and ts can be achieved by expelling the liquid into a reservoir
or an absorbent pad in a device as described. Another reagent can then be drawn into the tip according to
instructions or protocol from the programmable processor.
A system can comprise a holder or engager for moving the assay units or tips. An engager may
comprise a vacuum assembly or an assembly designed to fit snugly into a boss of an assay unit tip. For example,
a means for moving the tips can be moved in a manner similar to the fluid transfer device heads. The device can
also be moved on a stage according to the on of an engager or holder.
In an embodiment, an ment for moving the tips is the same as an instrument for moving a
volume of sample, such as a fluid transfer device as described herein. For example, a sample collection tip can
be fit onto a pipette head according to the boss on the collection tip. The collection tip can then be used to
distribute the liquid throughout the device and system. After the liquid has been distributed, the collection tip
can be disposed, and the pipette head can be fit onto an assay unit according to the boss on the assay unit. The
assay unit tip can then be moved from reagent unit to reagent unit, and reagents can be distributed to the assay
unit according to the aspiration- or pipette-type action provided by the pipette head. The pipette head can also
m mixing within a collection tip, assay unit, or reagent unit by aspiration- or syringe-type .
In another embodiment, tips containing liquids including assay reaction mixtures can be
disconnected from the pipetting device and “parked" at ic ons within the instrument or within a
[Annotation] eaa
disposable unit. If needed, tips can be capped using a seal (as used in the centrifuge) to prevent liquids from
draining out. In some embodiments, the seal can be a vinyl seal.
Exemplary Sample Tips
A variety of ner shapes can be utilized as sample tips, reaction chambers, and cuvettes. For
example, a cuvette can be circular, cylindrical, square, rectangular, l, conical, pyramidal, or any other
shape capable of holding a sample of fluid. Rectangular cuvettes where a light beam es at right angles to
the cuvette surfaces as shown in plan and section views in Figure 63 can be employed. In such rectangular
cuvettes, the liquid sample that is illuminated is also gular and is defined by the cuvette. Cuvettes with
circular cross-sections can also be used. For example, some types of microtiter plates where the illuminated
sample volume is in part defined by the sample meniscus as shown below in plan and section view in Figure 64.
Variable pathlength cuvettes can be used to optimize and extend the assay response and minimize
the volume of sample required to measure the assay. Cuvettes can be longer in relation to their cross-section in
at least one region. In some cases, the pathlength of a cuvette can be selected based on cuvette geometry and/or
al. Different cuvettes can be selected for ent .
] In the t invention, one preferred version of the assay cuvette has a circular cross-section in
the direction of the light beam as shown in Figure 65. The use of a cuvette with a circular cross-section has
several advantages, including, but not limited to the following:
1. The optical pathlength can be precisely d. Dimensional precision of ion-molded
parts have been found to be better than 1-2 % CV. In conventional microtiter plates the unconstrained liquid
meniscus can introduce imprecision in pathlength.
2. The open-ended ter and circular section of the tips confers excellent fluid handling
characteristics, making aspiration of liquids very precise.
3. The optical image of the tips provides for the ability to identify the tip location and boundaries
of the liquid column and to locate very precisely the center of the tip where the signal is maximal.
4. More than one liquid sample can be incubated and analyzed in the same tip. This is because
in the narrow part of the tip, very little material transfer occurs (in the axial direction) between nt “slugs"
of liquid.
An exemplary tip may have the following general features:
Tip length: 0.5 i 4 cm
Tip OD: 0.2 i 1.0 cm
Tip ID: 0.1 i 0.5 cm
Tip capacity for liquids: 5 i 50 uL
Tip dimensional precision: generally better than 2% or +/- 0.001 cm
Tip configuration: The tip will generally have a feature that engages with a pipette (cylindrical) so
as to form a fluid tight seal. There is a region generally cylindrical or conical which is used for imaging.
lly the optical part of the tip will have at least two ent sections with different pathlengths. The
lower end of the tip will typically be narrow so as to aid in retention of vertical liquid columns under gravity
Tip material: Clear or uniformly ar plastic (polystyrene, polypropylene etc.) (transmission of
light in the visible > 80%)
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For imaging purposes, the tip can generally be clear or translucent, but the tips do not have to be
clear to work well as assay cuvettes when three-color analysis is used. Tip cuvettes which appear “cloudy" may
on similarly to clear tips. The cloudy tips are made in injection molds with non-polished or textured
surfaces or by adding some light scattering material to the plastic used to fabricate the tips. The light scattering
intensity of such cloudy tips may be chosen to be not so great as to e the colored liquid to be measured.
In general, the impact of light scattering on transmitted light can be selected to be less than 10, (20, and 30 %)
relative to the impact of the colored material. The light ring effect can be selected such that the light
scattering of the cloudy tips is uniform.
The tips and reaction chambers described herein can be comprised of a rical (or conical)
shaft about 2 cm in length and having an inner diameter of about 1-5 mm corresponding to a capacity of about
7 50 uL.
In one example, at the upper end of the cylinder is a truncated cylindrical “boss" fluidically
connected to the er and adapted so as to be able to engage with the tapered feature of a pipetter. The
lower end of the tip may be narrowed to provide a feature that enables the tip to hold its liquid contents when
oriented vertically and not attached to the pipetter. The tip may be a pointed tip. The external shape of the
lower end of the tip is typically also somewhat pointed with the diameter being reduced from the main part of
the cylindrical shaft toward the end so as to be capable of being fluidically sealed with a e (vinyl) cap into
which the tip end is press fit. Tips are usually made of molded plastic (polystyrene, polypropylene and the
like). The tips can be clear or translucent such that information about the sample can be acquired by imaging.
Figure 4, Figure 5, and Figure 6 show an example of a tip. The tip is configured with (1) an upper
feature that can engage to form an air tight seal with a pipette head, (2) a basically cylindrical (actually conical
with a very slight draft angle) shaft and a narrow, d lower tip. This tip can form a liquid-tight seal with a
cap. The pointed shape aids in getting good conformance with the cap under moderate force. The material used
is injection-molded polystyrene. The overall ions are: 32 mm long, about 7.6 mm largest outer diameter,
useful capacity about 20 uL. The dimensions of the tip can be scaled to a larger . For example, for a 50
uL , the IDs can be increased by about 1.6-fold.
Sealing can be achieved using a cap made of vinyl or other als which is easily press-fit to the
narrow end of the sample containment means using force generated by motion of the instrument stage in the z-
direction. A bubble of air can become trapped within the tip when the tip is capped. A centrifugation step can
be used to drive the bubble to the top of the column of blood so as to eliminate the effects of the bubble. The
dimensions of the tip and/or the dimensions of the tip holder in a centrifuge can be matched such that a tip can
be secured for centrifugation.
] Sample ation
The invention provides for systems, methods, and devices for the processing and analysis of
samples can be collected from a variety of s. For example, the sample can be collected from patients,
animals, or the environment. The sample can be a bodily fluid. Any bodily fluids suspected to contain an
analyte of interest can be used in conjunction with the system or devices of the invention. Commonly employed
bodily fluids include but are not limited to blood, serum, saliva, urine, gastric and digestive fluid, tears, stool,
semen, vaginal fluid, titial fluids derived from tumorous tissue, and cerebrospinal fluid.
[Annotation] eaa
In some embodiments, the bodily fluid is a blood sample from a human patient. The blood source
can be collected from a finger prick and have a volume ofless than about 0.5, l, 5, 10, 20, 50, 100, 200, 300,
400, 500, or 1000 uL.
A bodily fluid may be drawn from a patient and ed to a device in a y of ways,
including but not limited to, lancing, injection, or pipetting.
] As used herein, the terms “subject" and “patient" are used interchangeably herein, and refer to a
vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines,
simians, humans, farm animals, sport animals, and pets.
In one embodiment, a lancet punctures the skin and withdraws a sample using, for example,
gravity, capillary action, aspiration, or vacuum force. The lancet may be part of the device, or part of a system
or a stand-alone ent. Where needed, the lancet may be activated by a variety of mechanical, electrical,
electromechanical, or any other known activation mechanism or any combination of such methods. In r
embodiment where no active mechanism is required, a patient can simply provide a bodily fluid to the , as
for example, could occur with a saliva sample. The collected fluid can be placed in the sample collection unit
within the . In yet another embodiment, the device comprises at least one microneedle which res
the skin.
The volume of bodily fluid to be used with a device can be less than about 500 microliters,
typically between about 1 to 100 microliters. Where desired, a sample of l to 50 microliters, l to 40 microliters,
l to 30 microliters, l to 10 microliters or even 1 to 3 microliters can be used for detecting an analyte using the
device. In an embodiment, the volume of bodily fluid used for detecting an e utilizing the subject s
or systems is one drop of fluid. For example, one drop of blood from a pricked finger can provide the sample of
bodily fluid to be analyzed with a device, system or method described .
A sample of bodily fluid can be collected from a subject directly into a tip of the described herein,
or can be later transferred to a tip.
Sample Dilution
In some ces, the configuration of the processor to direct fluid transfer effects a degree of
dilution of the bodily fluid sample in the array of assay units to bring signals indicative of the plurality of
analytes being detected within a detectable range, such that said plurality of analytes are detectable with said
system. In an example, the bodily fluid sample comprises at least two analytes that are present at concentrations
that differ by at least 1, 2, 5, 10, 15, 50, 100, 500, 1000, 10,000, 105, 106, 107, 108, 109, or 1010 fold. In an
example the bodily fluid sample is a single drop of blood. In an embodiment, the concentrations of at least two
analytes present in a sample differs by up to 10 orders of magnitude (for example, a first analyte is present at 01
pg/mL and a second analyte is present at 500 ug/mL). In another example, some protein analytes are found at
concentrations of greater than 100 mg/mL, which can extend the range of interest to about twelve orders of
ude. In the case of measurement of nucleic acid analytes such as DNA and RNA using exponential
amplification methods such as rase reaction, the number of copies of analyte can be increased by a
n fold prior to ement.
Where desired, a degree of dilution of the bodily fluid sample can bring the signals indicative of
the at least two analytes within the detectable range.
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As described, the systems and s herein can enable many features of the flexibility of
laboratory setting in a POC environment. For example, samples can be collected and manipulated automatically
in a table top size or smaller device or system. A common issue in FCC devices is achieving different dilution
ranges when conducting a plurality of assays, wherein the assays may have icantly different sensitivity or
specificity. For example, there may be two analytes in a sample, but one analyte has a high tration in the
sample and the other analyte has a very low concentration. As provided, the s and devices herein can
dilute the sample to significantly different levels in order to detect both analytes. Alternatively, a sample may
be split into two or more samples, which may enable individual analytes to be detected at various levels of
dilution.
For example, if the analyte is in a high concentration, a sample can be serially d to the
appropriate detection range and provided to a capture surface for detection. In the same system or device, a
sample with an analyte in a low concentration may not need to be diluted. In this manner, the assay range of the
FCC devices and systems provided herein can be expanded from many of the current POC devices.
In POC assay systems using disposable cartridges containing the t there is often a practical
limit to the extent of dilution. For example, if a small blood sample is obtained by fingerstick (for example,
about 20 microliters) is to be diluted and the maximum volume of diluent that can be placed in a tube is 250
microliters, the practical limit of dilution of the whole sample is about 10-fold. In an e herein, a system
can aspirate a smaller volume of the sample (for example about 2 iters) making the maximum dilution
factor about lOO-fold. For many assays, such dilution factors are acceptable but for an assay like that of CRP (as
described in the examples herein) there is a need to dilute the sample much more. Separation-based ELISA
assays can have an intrinsic limitation in the capacity of the capture e to bind the analyte (for example
about a few hundred ng/ml for a typical protein analyte). Some analytes are present in blood at hundreds of
micrograms/ml. Even when diluted by ld, the analyte concentration may be outside the range of
calibration. In an exemplary embodiment of a system, device, and fluid transfer device herein, multiple dilutions
can be ed by performing multiple fluid transfers of the diluent into an individual assay unit or sample
tion unit. For example, if the concentration of an analyte is very high in a sample as described above, the
sample can be diluted multiple times until the concentration of the analyte is within an able detection
range. The systems and methods herein can provide te measurements or estimations of the dilutions in
order to calculate the original concentration of the analyte.
Sample Separation
In some embodiments of the invention, a sample can be ed for analysis by an initial
separation step. For example, if the assay is to analyze DNA, a DNA separation step can be employed to
ate or reduce contaminants or unwanted source material. The separation step can utilize chromatography,
centrifugation, liquid-liquid extraction, solid-liquid extraction, affinity binding, or any other mechanisms known
to one skilled in the art.
In some embodiments, a blood sample to be analyzed is first sed by separating the plasma
ent from the blood sample. This step can be performed using a variety of techniques, such as filtration,
centrifugation, and affinity binding. Centrifugation can be an efficient method for separation of blood sample
components, and can be ed in the present invention.
Plasma separation
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Blood can be introduced into a close ended or sealable tip in a variety of ways, for example
samples can be provided in a tube and a sealable tip can receive the sample from the tube via capillary action or
via pneumatic force. One preferred means of introduction is the use of capillary action. Alternatively, container
used to hold the sample for centrifugal separation can be configured with only one opening as in a conventional
centrifuge.
The tip, once filled with blood, can be moved automatically to a location in a disposable cartridge
where there is a sealing element. The sealing element can be a small “cup" made of a able t)
material (vinyl, silicone or the like) conformed to fit on the lower end of the tip and to seal it. The tip is pressed
into the seal by the instrument thus g a liquid-tight junction. The sealed tip is then moved to a small
centrifuge (typically located in and forming part of the instrument) and press-fit into a positioning e in the
centrifuge rotor such that the lower (sealed) end of the tip butts up to a rigid shelf that will support the tip during
the centrifugation step.
The centrifuge rotor can be ar having about 10 cm in er. The mass of the blood-
containing tip is either (1) small relative to the rotor or (2), where desired, balanced by a counter weight located
on the opposite part of the rotor such that any vibration during the centrifugation step is minimized. One
exemplary orientation of the centrifuge rotor is vertical (axis of rotation horizontal). The rotor is mounted in a
drive shaft with is driven by an electric motor.
Centrifugation can be achieved by spinning the rotor at about 15,000 rpm for 5 minutes. During
this process, the particular elements in the blood (red cells and white cells) sediment to the sealed end of the tip
and form a closely packed column with cell free plasma ted at the part of the tip distal from the seal.
The tip containing the separated sample can then be placed vertically in a on accessible to a
fluid handling device comprised of a narrow pipette tip (“sample acquisition tip") mounted on a pipetting device
in turn mounted on an x-y-z stage.
] Plasma can now be efficiently recovered from the centrifuged sample. This is achieved by moving
the sample acquisition tip vertically along the axis of the centrifuge tip so that it comes into fluid t with
the plasma and can draw the plasma upwards using, e.g., pneumatic means.
Optionally, this operation can be monitored using a camera or other imaging device which can be
used both to measure the sample hematocrit and to provide information as to the location of the plasma/red cell
boundary to the stage/pipetter controller. With the aid of imaging the separated blood, a narrow pipette tip fitted
to a pipette is slowly moved vertically down, such that the tip is directed axially down the sample containment
means until it contacts the plasma. The tip is then moved further until it is close (within less than about 3, 2, l,
0.5, or 0.1 mm) of the packed cell interface. At the same time, plasma is aspirated into the narrow pipette tip
under er control. The plasma can be aspirated simultaneously while moving the narrow pipette tip into
the plasma column so that the plasma does not become ced into the upper part of the sample nment
means. The aspiration can be controlled to avoid air being aspirated during the plasma removal step.
] In general, a pipette tip with a very narrow end, such as those used to apply samples to an
electrophoresis system, can be used to te the plasma from the centrifuged sample tip. The narrow tip is
typically conical or tapered and has dimensions 1 , 3 x 0.1 , 0.5 cm (length x er) and made of any of a
variety of materials (polypropylene, polystyrene etc.). The material can be clear or translucent in the visible.
One end of the tip engages with a pipetting device. The other is very narrow (0.05 , 0.5 mm OD) such that it
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can move into the sample tip without touching the inner e of the sample tip. Even if there is contact
n the plasma aspiration tip and the sample tip, plasma aspiration is not hindered.
A schematic of the plasma aspiration process at the stage where the plasma aspiration tip is d
just above the plasma-packed cell interface during the aspiration step is shown in Figure 7.
In this way we have found that almost all of the plasma can be removed leaving as little as e.g. l
uL in the centrifuged sample tip. This corresponds to about 11 uL of plasma (90% recovery) from 20 uL of
blood with a 40% hematocrit. Additionally the quality of the plasma sample (with t to hemolysis, lipemia
and icteria) can be determined from an image of the centrifuged sample.
The aspirated plasma can be moved to other locations for dilution and mixing with assay reagents
so that assays for analytes ry but not limited to metabolites, electrolytes, protein biomarkers, drugs and
nucleic acids may be performed.
Separation ofwhz'te blood cells
A further use of the invention is to isolate and concentrate the white cells from blood. In one
aspect of the invention, the blood sample is first subject to a process which lyses the red cells (and optionally
fixes the white cells) by adding a reagent (For example, BD PharmlyseTM 555899 or BD FACSTM Lysing
Solution 349202)_to the blood and mixing. Following a brief incubation, the lysed sample is subject to
centrifugation as described above such that the white cells are trated at the sealed end of the blood tip.
The lysed red cell solution can then be removed by aspiration. Recovery of the white cells is achieved by either
(1) addition of a small amount of a buffer solution and repeated up and down aspiration to re-suspend the cells
followed by displacement into a receptacle or (2) removal of the seal and downward displacement of the packed
cells into a receptacle using air pressure.
] An ate scheme allows recovery of white cells without lysis of the red cells. After
centrifugation of blood (as is well known) the white cells form a layer on top of the packed red cells known as
the Buffy Coat. Following removal of most of the plasma (as above) the white cells can be efficiently recovered
by (l) optionally adding a small volume (e. g. about 5 uL) of isotonic buffer, or (2) using the residual plasma and
re-suspending the white cells by repeated aspiration and/or mechanical stirring using the sample acquisition tip.
Once suspended, the resulting mixture of white cells er with a small tion of red cells also re-
suspended can be acquired by aspiration for analysis of the white cells. In this way most of the white cells
(typically all) can be recovered with only a small (contaminating) quantity of red cells (typically less than 5% of
the original).
fuges
Figure 1, Figure 2, and Figure 3 show scale perspectives of a centrifuge (Figure l - side view,
Figure 2 - front face view, Figure 3 - rear view) that can be integrated into the . The centrifuge may
contain an electric motor capable of turning the rotor at 15,000 rpm. One type of centrifuge rotor is shaped
somewhat like a fan blade is mounted on the motor spindle in a vertical plane. Affixed to the rotor is an element
which holds the sample g means (tip) and provides a ledge or shelf on which the end of the tip distal to
the motor axis rests and which provides t during the centrifugation so that the sample cannot escape. The
tip may be r supported at its proximal end by a mechanical stop in the rotor. This can be ed so that
the force generated during centrifugation does not cause the tip to cut through the soft vinyl cap. The tip can be
inserted and removed by standard pick and place mechanisms but preferably by a pipette. The rotor is a single
[Annotation] eaa
piece of acrylic (or other material) shaped to ze Vibration and noise during operation of the centrifuge.
The rotor is (optionally) shaped so that when it is ed in particular angles to the vertical, other movable
components in the instrument can move past the fuge. The sample holding means are centrifugally
balanced by counter masses on the opposite side of the rotor such that the center of rotational inertia is axial
relative to the motor. The centrifuge motor may provide positional data to a computer which can then control
the rest position of the rotor ally vertical before and after centrifugation).
As may be seen from the two graphs in Figure 8 and Figure 9, to minimize centrifugation time
(without generating too much ical stress during centrifugation) according to hed standards (DIN
58933-1; for the US. the CLSI standard H07-A3 “Procedure for Determining Packed Cell Volume by the
Microhematocrit Method"; Approved rd - Third Edition) convenient dimensions for the rotor are in the
range of about 5 , 10 cm spinning at abouth - 20 thousand rpm giVing a time to pack the red cells of about 5
min.
An exemplary equation for calculating centrifugation force is shown below:
i '
] 3?
Where:
gis earth's gravitational acceleration,
] ‘Y‘is the rotational radius,
imirIS the rotational speed, measured in revolutions per unit of time.
Where:
Teams the rotational radius measured in centimeters (cm),
Aliphilis rotational speed measured in revolutions per minute (RPM).
, ,5.
REF 3 1 .1. 15:
>< 19—5 gm. ; gem
In some ments, a centrifuge may be a horizontally oriented centrifuge with a swinging
bucket design. In some preferable embodiments, the axis of rotation of the centrifuge is vertical. In alternate
embodiments, the axis of rotation can be horizontal or at any angle. The centrifuge may be capable of
aneously spinning two or more vessels and may be designed to be fully ated into an automated
system employing computer-controlled pipettes. In some embodiments, the vessels may be close-bottomed.
The swinging bucket design may permit the centrifugation vessels to be ely oriented in a vertical position
when stopped, and spin out to a fixed angle when spinning. In some embodiments, the swinging buckets may
permit the centrifugation vessels to spin out to a horizontal orientation. Alternatively they may spin out to any
angle between a vertical and horizontal position (e.g., about 15, 30, 45, 60, or 75 degrees from vertical. The
centrifuge with swinging bucket design may meet the positional accuracy and repeatability requirements of a
robotic system a number of positioning s are employed.
A computer-based control system may use position information from an optical encoder in order to
spin the rotor at controlled slow speeds. Because an appropriate motor could be designed for high-speed
mance, accurate static positions need not be held using position feedback alone. In some embodiments, a
cam in ation with a solenoid-actuated lever may be employed to achieve very accurate and stable
[Annotation] eaa
stopping at a fixed number of positions. Using a separate control system and feedback from Hall-Effect sensors
built into the motor, the velocity of the rotor can be very tely controlled at high speeds.
Because a number of sensitive instruments must function simultaneously within the assay
instrument system, the design of the centrifuge preferably minimizes or reduces vibration. The rotor may be
namically designed with a smooth exterior , fully enclosing the buckets when they are in their horizontal
position. Also, vibration dampening can be employed in multiple locations in the design of the case.
Rotor
A centrifuge rotor can be a component of the system which may hold and spin the centrifugation
vessel(s). The axis of on can be vertical, and thus the rotor itself can be positioned ntally. However,
in alternate embodiments, different axes of rotation and rotor positions can be employed. There are two
components known as buckets positioned symmetrically on either side of the rotor which hold the centrifugation
vessels. Alternative configurations are possible in which s are oriented with radial symmetry, for
example three s oriented at 120 degrees. Any number of buckets may be provided, including but not
limited to l, 2, 3, 4, 5, 6, 7, 8, or more buckets. The buckets can be evenly spaced from one another. For
example, if n buckets are provided where n is a whole number, then the buckets may be spaced about 360/n
degrees apart from one another. In other embodiments, the buckets need not be spaced evenly around one
another or with radial symmetry.
When the rotor is stationary, these buckets, influenced by gravity, may passively fall such as to
position the vessels vertically and to make them accessible to the pipette. Figure 111 shows an example of a
rotor at rest with buckets vertical. In some embodiments, the buckets may passively fall to a predetermined
angle that may or may not be al. When the rotor spins, the s are forced into a nearly horizontal
position or to a predetermined angle by fugal forces. Figure 112 shows an example of a rotor at a speed
with buckets at a small angle to horizontal. There can be physical hard stops for both the vertical and ntal
positions acting to enforce their accuracy and positional repeatability.
The rotor may be aerodynamically designed with a disk shape, and as few physical features as
possible in order to minimize vibration caused by air turbulence. To achieve this, the outer geometry of the
bucket may exactly match that of the rotor such that when the rotor is ng and the bucket can be forced
horizontal the bucket and rotor can be tly aligned.
To facilitate plasma extraction, the rotor may be angled down toward the ground relative to the
horizon. e the angle of the bucket can be matched to that of the rotor, this may enforce a fixed spinning
angle for the bucket. The resulting pellet from such a configuration could be angled ve to the vessel when
placed upright. A narrow extraction tip may be used to aspirate plasma from the top of the centrifugation vessel.
By placing the extraction tip near the bottom of the slope d by the angle , the final volume of plasma
can be more efficiently extracted without disturbing the sensitive buffy coat.
A variety of tubes designs can be odated in the buckets of the device. In some
embodiments, the s tube designs may be closed ended. Some are shaped like conventional centrifuge
tubes with conical bottoms. Other tube designs may be cylindrical. Tubes with a low ratio of height to cross-
sectional area may be favored for cell processing. Tubes with a large ratio (>10: 1) may be suitable for accurate
measurement of hematocrit and other imaging requirements. However, any height to cross-sectional area ratio
may be employed. The buckets can be made of any of several plastics (polystyrene, polypropylene), or any
[Annotation] eaa
other material discussed ere herein. s have capacities ranging from a few microliters to about a
milliliter. The tubes may be inserted into and removed from the centrifuge using a “pick and place" mechanism.
Control System
Due to the spinning and positioning requirements of the centrifuge deVice, a dual control system
approach may be used. To index the rotor to specific rotational orientations, a position based control system may
be implemented. In some embodiments, the control system may employ a PID (Proportional Integral
Derivative) l system. Other feedback control systems known in the art can be employed. Positional
feedback for the position controller may be provided by a high-resolution optical encoder. For operating the
centrifuge at low to high speeds, a ty controller may be implemented, while employing a PID l
system tuned for velocity control. Rotational rate feedback for the velocity controller may be provided by a set
of simple Hall-Effect sensors placed on the motor shaft. Each sensor may generate a square wave at one cycle
per motor shaft rotation.
ng Mechanism
To consistently and firmly position the rotor in a particular position, a physical stopping
ism may be employed. In one embodiment, the stopping mechanism may use a cam, coupled to the
rotor, along with a solenoid-actuated lever. The cam may be shaped like a circular disk with a number of “C"
shaped notches machined around the perimeter. To position the centrifuge rotor, its onal velocity may first
be lowered to, at most, 30RPM. In other embodiments, the rotational velocity may be lowered to any other
, including but not d to about 5 rpm, 10 rpm, 15 rpm, 20 rpm, 25 rpm, 35 rpm, 40 rpm, or 50 rpm.
Once the speed is iently slow, the lever may be actuated. At the end of the lever is a cam follower which
may glide along the perimeter of the cam with minimal friction. Once the cam follower reaches the center of a
particular notch in the cam, the force of the solenoid-actuated lever can overcome that of the motor and the rotor
may be brought to a halt. At that point the motor may be electronically braked, and, in combination with the
stopping mechanism a rotational position can be very accurately and firmly held indefinitely.
Centrifuge buckets
The centrifuge swing-out buckets may be ured to accommodate different type of centrifuge
tubes. In preferable embodiments, the various tube types may have a collar or flange at their upper (open) end.
This collar or flange feature may rests on the upper end of the bucket and support the tube during centrifugation.
As shown in Figures 113, 114, and 115, conical and cylindrical tubes of various lengths and volumes can be
accommodated. Figures 113, 114, and 115 provide examples of buckets and other bucket designs may be
ed. For example, Figure 113, shows an example of a bucket configuration. The bucket may have side
portions that mate with the fuge and allow the bucket to swing freely. The bucket may have a closed
bottom and an opening at the top. Figure 114 shows an example of a fugation vessel mated with the
bucket. As previously mentioned, the bucket may be shaped to accept various configurations of centrifugation
vessels. The centrifugation vessel may have one or more protruding member that may rest upon the .
The centrifugation vessel may be shaped with one or more feature that may mate with the centrifugation bucket.
The feature may be a shaped feature of the vessel or one or more protrusion. Figure 115 shows an example of
another centrifugation vessel that can be mated with the bucket. As previously described, the bucket can have
one or more shaped e that may allow different configurations of centrifugation vessels to mate with the
bucket.
[Annotation] eaa
Centrifuge tubes and sample extraction means:
] The centrifuge tube and extraction tip may be provided individually and can be mated together for
tion of al following centrifugation. The centrifugation tube and extraction tip may be ed to
deal with complex processes in an automated system. Figure 116 shows an example of a centrifugation vessel.
Figure 117 shows an example of an extraction tip. Figure 118 provides an example ofhow the centrifugation
vessel and extraction tip may mate. Any dimensions are provided by way of example only, and other
dimensions of the same or differing proportions may be ed.
The system can enable one or more of the ing:
1. Rapid processing of small blood s (typically 5 — 50 uL)
9‘95”!“ Accurate and precise measurement of hematocrit
Efficient l of plasma
Efficient re-suspension of formed elements (red and white blood cells)
Concentration of white cells (following labeling with fluorescent antibodies and fixation plus
lysis of red cells)
6. Optical confirmation of red cell lysis and recovery of white cells
fugation Vessel and Extraction Tip Overview
A custom vessel and tip may be used for the operation of the centrifuge in order to satisfy the
variety of constraints placed on the system. The centrifugation vessel may be a closed bottom tube designed to
be spun in the centrifuge. In some embodiments, the centrifugation vessel may be the vessel illustrated in
Figure 116 or may have one or more features illustrated in Figure 116. It may have a number of unique features
enabling the wide range of required functionality including hematocrit measurement, RBC lysing, pellet resuspension
and efficient plasma extraction. The extraction tip may be designed to be inserted into the
centrifugation vessel for precise fluid extraction, and pellet re-suspension. In some embodiments, the extraction
tip may be the tip illustrated in Figure 117 or may have one or more es rated in Figure 117.
Exemplary specifications for each tip are discussed herein.
Centrifugation Vessel
The centrifugation vessel may be designed to handle two separate usage scenarios, each associated
with a different anti-coagulant and whole blood volume.
A first usage scenario may e that 40uL of whole blood with Heparin be pelleted, the
m volume of plasma be recovered, and the hematocrit measured using computer vision. In the case of
60% hematocrit or below the volume of plasma required or preferable may be about 40uL*40%=16uL.
In some ments, it will not be possible to r 100% of the plasma because the buffy coat
must not be bed, thus a minimum distance must be maintained between the bottom of the tip and the top of
the pellet. This minimum distance can be determined mentally but the volume (V) sacrificed as a function
of the required safety distance (d) can be estimated using: V(d) = d*n1.25mm2. For example, for a required
safety distance of 0.25 mm, the sacrificed volume could be 1.23uL for the 60% hematocrit case. This volume
can be decreased by decreasing the internal radius of the hematocrit portion of the centrifugation vessel.
However, because in some embodiments, that narrow portion must fully accommodate the outer radius of the
extraction tip which can be no smaller than 1.5 mm, the existing dimensions of the centrifugation vessel may be
close to the minimum.
ation] eaa
Along with plasma extraction, in some embodiments it may also be ed that the hematocrit be
measured using computer vision. In order to facilitate this process the total height for a given volume of
hematocrit may be maximized by minimizing the internal diameter of the narrow portion of the vessel. By
maximizing the height, the relationship between changes in hematocrit volume and physical change in column
height may be optimized, thus increasing the number of pixels that can be used for the measurement. The height
of the narrow portion of the vessel may also be long enough to accommodate the worst-case scenario of 80%
hematocrit while still leaving a small portion of plasma at the top of the column to allow for efficient extraction.
Thus, 40uL*80% = 32uL may be the required volume capacity for accurate measurement of the hematocrit. The
volume of the narrow n of the tip as designed may be about 35.3uL which may allow for some volume of
plasma to remain, even in the worst case.
A second usage scenario is much more involved, and may e one, more, or all of the
following:
0 whole bloodpelleted
0 plasma extracted
0 pellet re-suspended in lysing bufler and stain
0 remaining white blood cells (WBCs) pelleted
0 supernatant removed
0 WECs re-suspended
0 WEC suspensionfully extracted
In order to fully re-suspend a packed , ments have shown one can physically disturb
the pellet with a tip capable of completely reaching the bottom of the vessel containing the pellet. A preferable
geometry of the bottom of the vessel using for re-suspension seems to be a hemispherical shape r to
standard commercial PCR tubes. In other embodiments, other vessel bottom shapes may be used. The
centrifugation , along with the extraction tip, may be designed to facilitate the re-suspension s by
adhering to these geometrical requirements while also allowing the extraction tip to physically contact the
bottom.
During manual re-suspension experiments it was noticed that physical contact between the bottom
of the vessel, and the bottom of the tip may create a seal that its fluid movement. A te spacing may
be used in order to both fully disturb the pellet, while allowing fluid flow. In order to facilitate this process in a
robotic , a physical feature may be added to the bottom of the fugation vessel. In some
embodiments, this feature may comprise four small hemispherical nubs placed around the perimeter of the
bottom portion of the . When the extraction tip is fully inserted into the vessel and allowed to make
physical contact, the end of the tip may rest on the nubs, and fluid is allowed to freely flow between the nubs.
This may result in a small amount of volume (~.25uL) lost in the gaps.
During the lysing process, in some implementations, the maximum expected fluid volume is 60uL,
which, along with 25uL displaced by the extraction tip may demand a total volume capacity of 85uL. A design
with a current maximum volume of 100uL may exceed this requirement. Other aspects of the second usage
io require similar or already discussed tip characteristics.
The upper geometry of the centrifugation vessel may be designed to mate with a e nozzle.
Any pipette nozzle described elsewhere herein or known in the art may be used. The external geometry of the
[Annotation] eaa
upper portion of the vessel may exactly match that of a reaction tip which both the current nozzle and dge
may be designed around. In some ments, a small ridge may circumscribe the internal surface of the
upper portion. This ridge may be a visual marker of the maximum fluid height, meant to facilitate automatic
error ion using computer vision system.
In some embodiments, the distance from the bottom of the fully mated nozzle to the top of the
maximum fluid line is 2.5mm. This distance is 1.5mm less than the 4mm recommended distance adhered to by
the extraction tip. This decreased ce may be driven by the need to ze the length of the tion
tip while adhering to minimum volume requirements. The justification for this decreased distance stems from
the particular use of the vessel. e, in some implementations, fluid may be ged with the vessel
from the top only, the m fluid it will ever have while mated with the nozzle is the maximum amount of
whole blood expected at any given time (40uL). The height of this fluid may be well below the bottom of the
nozzle. Another concern is that at other times the volume of fluid in the vessel may be much greater than this
and wet the walls of up to the height of the nozzle. In some embodiments, it will be up to those using the vessel
to ensure that the meniscus of any fluids contained within the vessel do not exceed the max fluid height, even if
the total volume is less than the maximum specified. In other embodiments, other features may be provided to
keep the fluid contained within the vessel.
Any dimensions, sizes, volumes, or distances provided herein are provided by way of example
only. Any other dimension, size, volume or distance may be utilized which may or may not be proportional to
the amounts mentioned herein.
The centrifugation vessel can be subjected to a number of forces during the process of exchanging
fluids and rapidly inserting and removing tips. If the vessel is not constrained, it is possible that these forces
will be strong enough to lift or otherwise ge the vessel from the centrifuge . In order to prevent
movement, the vessel should be secured in some way. To accomplish this, a small ring circumscribing the
bottom exterior of the vessel was added. This ring can easily be mated with a compliant mechanical feature on
the bucket. As long as the retaining force of the nub is greater than the forces experienced during fluid
manipulations, but less than the friction force when mated with the nozzle then the problem is solved.
Extraction Tip
The Extraction Tip may be designed to interface with the centrifugation vessel, efficiently
extracting , and re-suspending pelleted cells. Where desired, its total length (e.g., 34.5 mm) may exactly
match that of another blood tip including but not limited to those described in US. Serial No. 12/244,723
(incorporated herein by reference) but may be long enough to physically touch the bottom of the centrifugation
vessel. The ability to touch the bottom of the vessel may be required in some embodiments, both for the re-
suspension s, and for complete recovery of the white cell suspension.
The required volume of the extraction tip may be determined by the maximum volume it is
expected to aspirate from the centrifugation vessel at any given time. In some embodiments, this volume may
be approximately 60uL, which may be less than the maximum capacity of the tip which is 85uL. In some
embodiments, a tip of r volume than required volume may be provided. As with the centrifugation vessel,
an internal feature circumscribing the interior of the upper portion of the tip may be used to mark the height of
this m volume. The distance between the maximum volume line and the top of the mated nozzle may
[Annotation] eaa
be 4.5mm, which may be considered a safe distance to prevent nozzle ination. Any sufficient distance to
prevent nozzle contamination may be used.
The fuge may be used to sediment precipitated LDL-cholesterol. Imaging may be used to
verify that the supernatant is clear, indicating complete removal of the precipitate.
In one example, plasma may be diluted (e. g., 1:10) into a mixture of dextran sulfate (25mg/dL) and
magnesium sulfate (100mM), and may be then incubated for 1 minute to precipitate LDL-cholesterol. The
reaction product may be aspirated into the tube of the centrifuge, capped then and spun at 3000 rpm for three
minutes. Figures 119, 120, and 121 are images that were taken of the original reaction mixture prior to
centrifugation (showing the white itate), following centrifugation ng a clear atant) and of the
LDL-cholesterol pellet (after removal of the cap), tively.
Other examples of centrifuges that can be employed in the present invention are described in US.
Patent Nos. 5,693,233, 5,578,269, 6,599,476 and US. Patent ation Nos. 2004/0230400, 2009/0305392,
and 2010/0047790, which are incorporated by reference in their entirety for all purposes.
Example protocols
Many variations of protocol may be used for centrifugation and processing. For example, a typical
protocol for use of the fuge to process and concentrate white cells for cytometry may include one or more
of the following steps. The steps below may be ed in varying orders or other steps may be substituted for
any of the steps below:
1. e 10 uL blood anti-coagulated with EDTA (pipette injects the blood into the bottom of
the centrifuge bucket)
2. Sediment the red and white cells by centrifugation (< 5 min x 10,000 g).
3. Measure hematocrit by imaging
4. Remove plasma slowly by aspiration into the pipette (4 uL corresponding to the worst case
scenario [60 % hematocrit]) t disturbing the cell pellet.
. Re-suspend the pellet after adding 20 uL of an appropriate cocktail of up to five fluorescently
d antibodies1 dissolved in buffered saline + BSA (1 mg/mL) (total reaction volume
about 26 uLZ).
6. Incubate for 15 minutes at 37C.
7. Prepare lysing/fixative reagent by mixing red cell lysing solution (ammonium
chloride/potassium bicarbonate) with white cell fixative reagent (formaldehyde).
8. Add 30 uL lysing/fixative reagent (total on volume about 60 uL).
9. Incubate 15 minutes at 37C
. Sediment the white cells by fugation (5 min, 10,000 g).
11. Remove the supernatant sate (about 57 uL).
12. Re-suspend the white cells by adding 8 uL buffer (isotonic buffered saline).
13. Measure the volume accurately.
14. Deliver sample (c 10 uL) t0 cytometry.
The steps may include receiving a sample. The sample may be a bodily fluid, such as blood, or
any other sample described elsewhere herein. The sample may be a small volume, such as any of the volume
measurements described elsewhere herein. In some instances, the sample may have an anti-coagulant.
1 Concentration will be adjusted appropriately to deal with the different volume ratio relative to
standard laboratory method (specifically about 5 x lower)
2 If
ary, this volume can be bigger to have optimal staining but not more than 50 uL.
[Annotation] eaa
A separation step may occur. For example, a density-based separation may occur. Such
separation may occur via centrifugation, magnetic separation, lysis, or any other separation que known in
the art. In some embodiments, the sample may be blood, and the red and white blood cells may be separated.
] A measurement may be made. In some instances, the measurement may be made via imaging, or
any other detection mechanism described elsewhere . For example, the crit of a ted blood
sample may be made by imaging. Imaging may occur via a digital camera or any other image capture device
described herein.
One or more component of a sample may be removed. For example, if the sample is separated into
solid and liquid components, the liquid component may be moved. The plasma of a blood sample may be
removed. In some instances, the liquid component, such as plasma, may be removed via a e. The liquid
component may be removed without disturbing the solid component. The imaging may aid in the removal of
the liquid component, or any other selected component of the sample. For example, the imaging may be used to
determine where the plasma is located and may aid in the ent of the pipette to remove the plasma.
In some embodiments, a reagent or other al may be added to the sample. For example, the
solid portion of the sample may be resuspended. A material may be added with a label. One or more incubation
step may occur. In some instances, a lysing and/or fixative t may be added. Additional separation and/or
resuspending steps may occur. As needed, dilution and/or concentration steps may occur.
The volume of the sample may be measured. In some instances, the volume of the sample may be
measured in a precise and/or accurate fashion. The volume of the sample may be measured in a system with a
low coefficient of ion, such as coefficient of variation values described elsewhere herein. In some
instances, the volume of the sample may be measured using imaging. An image of the sample may be captured
and the volume of the sample may be calculated from the image.
The sample may be delivered to a desired process. For example, the sample may be delivered for
cytometry.
In another e, a typical protocol that may or may not make use of the centrifuge for nucleic
acid purification may include one or more of the following steps. The system may enable DNA/RNA extraction
to deliver nucleic acid template to exponential amplification reactions for detection. The process may be
designed to extract nucleic acids from a variety of s including, but not limited to whole blood, serum,
viral transfer medium, human and animal tissue samples, food samples, and bacterial cultures. The process may
be completely automated and may extract DNA/RNA in a consistent and quantitative manner. The steps below
may be provided in varying orders or other steps may be substituted for any of the steps below:
1. Sample Lysis. Cells in the sample may be lysed using a chaotropic-salt buffer. The chaotropic-
salt buffer may include one or more of the ing: chaotropic salt such as, but not limited to, 3-6 M
guanidine hydrochloride or guanidinium anate; sodium dodecyl sulfate (SDS) at a typical concentration of
0.1-5% v/v; nediaminetetraacetic acid (EDTA) at a typical concentration of l-SmM; lysozyme at a typical
concentration of 1 mg/mL; proteinase-K at a typical concentration of 1 mg/mL; and pH may be set at 7-7.5
using a buffer such as HEPES. In some embodiments, the sample may be incubated in the buffer at typical
ature of 20-95 °C for 0-30 minutes. Isopropanol (50%-100% v/v) may be added to the mixture after lysis.
2. e Loading. Lysed sample may be exposed to a functionalized surface (often in the form
of a packed bed of beads) such as, but not limited to, a resin-support packed in a chromatography style column,
[Annotation] eaa
magnetic beads mixed with the sample in a batch style manner, sample pumped through a suspended resin in a
fluidized-bed mode, and sample pumped through a closed l in a tangential flow manner over the surface.
The surface may be functionalized so as to bind nucleic acids (e.g. DNA, RNA, DNA/RNA hybrid) in the
ce of the lysis buffer. Surface types may include silica, and ion-exchange functional groups such as
diethylaminoethanol (DEAE). The lysed mixture may be exposed to the surface and nucleic acids bind.
] 3. Wash. The solid surface is washed with a salt solution such as 0-2 M sodium chloride and
ethanol (20-80% v/v) at pH 7.0 - 7.5. The g may be done in the same manner as loading.
4. Elation. Nucleic acids may be eluted from the surface by exposing the e to water or
buffer at pH 7-9. Elution may be performed in the same manner as loading.
Many variations of these protocols or other protocols may be employed by the system. Such
protocols may be used in combination or in the place of any protocols or s bed herein.
In some embodiments, it is important to be able to recover the cells packed and concentrated by
centrifugation for cytometry. In some ments, this may be achieved by use of the pipetting device.
Liquids (typically isotonic buffered saline, a lysing agent, a mixture of a lysing agent and a fixative or a cocktail
of labeled antibodies in buffer) may be dispensed into the centrifuge bucket and repeatedly ted and re-
dispensed. The tip of the pipette may be forced into the packed cells to facilitate the process. Image analysis
aids the process by objectively verifying that all the cells have been re-suspended.
] Use offhe pipette and centrifuge to process samples prior to is:
In accordance with an embodiment of the invention, the system may have pipetting, pick-and-place
and centrifugal capabilities. Such capabilities may enable almost any type of sample pretreatment and complex
assay procedures to be performed efficiently with very small volumes of sample.
Specifically, the system may enables separation of formed ts (red and white cells) from
plasma. The system may also enable re-suspension of formed elements. In some embodiments, the system may
enable concentration of white cells from fixed and hemolysed blood. The system may also enable lysis of cells
to release nucleic acids. In some embodiments, purification and concentration of nucleic acids by filtration
h tips packed with (typically beaded) solid phase reagents (e.g. silica) may be enabled by the system. The
system may also permit elution of purified nucleic acids following solid phase extraction. Removal and
collection of precipitates (for example LDL-cholesterol precipitated using polyethylene glycol) may also be
enabled by the system.
In some embodiments, the system may enable ty purification. Small molecules such as
vitamin-D and serotonin may be adsorbed onto beaded (particulate) hydrophobic substrates, then eluted using
organic solvents. Antigens may be provided onto antibody-coated substrates and eluted with acid. The same
methods can be used to concentrate analytes found at low concentrations such as thromboxane-BZ and 6-keto-
glandin F l a. Antigens may be ed onto dy or aptamer-coated substrates and then eluted.
In some ents, the system may enable chemical modification of analytes prior to assay. To
assay nin (5-Hydroxytryptamine) for example, it may be ed to convert the analyte to a derivative
(such as an acetylated form) using a reagent (such as acetic anhydride). This may be done to produce a form of
the analyte that can be recognized by an antibody.
] Liquids can be moved using the pipette (vacuum aspiration and pumping). The pipette may be
limited to relatively low positive and negative pressures (approximately 0.1 , 2.0 atmospheres). A centrifuge
[Annotation] eaa
can be used to generate much higher pressures when needed to force liquids through beaded solid phase media.
For example, using a rotor with a radius of 5 cm at a speed of 10,000 rpm, forces of about 5,000 X g (about 7
atmospheres) may be generated, sufficient to force liquids through resistive media such as packed beds. Any of
the centrifuge designs and configurations discussed elsewhere herein or known in the art may be used.
Measurement of hematocrit with very small volumes of blood may occur. For example,
inexpensive digital cameras are capable of making good images of small s even when the contrast is poor.
Making use of this capability, the system of the present invention may enable ted measurement of
hematocrit with a very small volume of blood.
For e, 1 uL of blood may be drawn into a microcap glass capillary. The capillary may then
be sealed with a curable adhesive and then subject to centrifugation at 10,000 x g for 5 minutes. The packed cell
volume may be easily measured and the plasma meniscus (indicated by an arrow) may also be visible so
hematocrit can be accurately measured. This may enable the system to not waste a relatively large volume of
blood to make this measurement. In some embodiments, the camera may be used “as is" without operation with
a microscope to make a larger image. In other embodiments, a microscope or other optical techniques may be
used to magnify the image. In one implementation, the hematocrit was determined using the digital camera
without additional optical interference, and the hematocrit measured was cal to that determined by a
conventional microhematocrit laboratory method requiring many microliters of sample. In some embodiments,
the length of the sample column and of that of the column of packed red cells can be measured very precisely
(+/- < 0.05 mm). Given that the blood sample column may be about 10 - 20 mm, the standard deviation of
hematocrit may be much better than 1 % matching that obtained by standard laboratory methods.
The system may enable measurement of erythrocyte sedimentation rate (ESR). The y of
digital cameras to measure very small distances and rates of change of distances may be ted to measure
ESR. In one example, three blood samples (15 uL) were aspirated into “reaction tips". Images were captured
over one hour at two-minute intervals. Image analysis was used to measure the movement of the interface
between red cells and plasma. Figure 122 shows results as distance of the interface from the plasma meniscus.
The precision of the measurement may be estimated by fitting the data to a polynomial function
and calculating the standard deviation of the difference between the data and the fitted curve (for all samples).
In the e, this was ined to be 0.038 mm or < 2 % CV when related to the distance moved over one
hour. Accordingly, ESR can be measured ely by this method. Another method for determination of ESR
is to measure the maximum slope of the distance versus time relationship.
] Assay Preparation
In some embodiments, tips can be designed to accommodate a plurality of reactions or assays.
Simultaneous measurement of l different assay mixtures and one or more controls or one or more
calibrators can be made within one tip of the present invention. In doing this we exploit the y to sample
l liquid sources by tial aspiration of liquids into the same tip. Effective tation and
separation of the liquids is greatly improved by aspirating in sequence a small volume of air and a small volume
of a wash solution which cleans the surface of the tips prior to aspiration of the next liquid of interest.
As described above, tips can have conical shapes. In some embodiments, an assay can e
oxygen as a reactant. In such reactions, sing availability of oxygen within a reaction can be achieved by
moving the sample and/or assay mixture to a wide part of tip to increase surface area to volume ratio.
[Annotation] eaa
In Figure 93 and Figure 94, solutions of bromphenol blue were aspirated into tips. The uppermost
segments (aspirated first) 6 uL are from a ld dilution series (highest concentration (0.0781 mg/mL) to the
right of the image, with the exception of the left-most tip which is a water blank). Then air (2 uL), wash
on (2 uL) respectively were ted followed by a 6 uL volume of a fixed concentration control solution
(0.625 mg/mL).
Using this approach several alternative assay configurations can be ed, for example:
1. Simultaneous measurement of reagent and/or sample blank and assay
2. Simultaneous measurement of sample, blank and control
3. Within-tip calibration of assay
The table below illustrates some “multiplex types" in which preferred combinations of ,
controls and calibrators are led within a tip.
——--
—----“mm
—--—----
—-—---—----
_----I------
Case 2 is shown in Figure 95.
] Serial measurements of blank solutions, sample, controls and calibrators can also be made with
single tips. In this scenario, the tip is filled with the first solution, read then emptied. The tip can then be re-
filled and read with other samples etc. in sequence. The impact of “carry-over" of colored product from one
sample to the next is minimized by either or both:
] 1. Reading the liquid column in the middle portion well away from that part that first comes into
contact with the preceding sample
2. Washing the tip between samples.
In order to measure the extent of ‘carry-over" from one liquid segment to the next, the following
procedure was be performed. A small amount (e. g. 6 uL) of a high concentration of bromphenol blue (e. g.
0.625 mg/mL) was aspirated into tips, followed by 2 uL of air and 2 uL of wash solution. Finally 6 uL of serial
two-fold dilutions of enol blue is aspirated with the ing results (highest concentration (0.0781
mg/mL) to the right; left most tip is a water blank).
As can be seen from the images shown in Figure 96 and the 3-color analysis shown in Figure 97,
able amounts of the high concentration solution is transferred into the wash solution.
Average carry-over (from high concentration control to the water wash) is calculated at 1.4 %.
Since, in effect, the leading zone (proximal to the earlier slug) of a later slug of liquid acts as second wash step
and the color reading is taken at a location remote from this leading zone (typically at a central zone of the slug),
the effective carryover from one slug to the next is typically much less than 1 % and ore generally
insignificant. When the dilution series is measured using only dilution series samples to fill the tips, results are
identical with those obtained above. The above represents a s test" designed to evaluate the extent of
carry-over.
ation] eaa
Figure 98 shows a tip containing reaction products for two commercially ble assays for
ionized calcium, Ca2+ (upper segment) and ium Mg2+ (lower segment) that were aspirated into tips for
measurement. Ca2+ concentrations used in this experiment are 0, 2.5, 5, 10, 20, and 40 mg/dL; Mg2+
concentrations are 0, 1.25, 2.5, 5, 10, 20 mg/dL. Assay reaction mixtures (6 uL [Ca2+] and 4 uL [Mg2+]) are
well separated using 2 uL of air, 3 uL of wash and a r 4 uL of air. Results for each assay read in this way
are essentially identical to those measured having only one assay reaction e per tip.
As noted above, the present invention allows for simultaneous evaluation of a plurality of .
Images can be made ofmany assay cuvettes in the same field of view. Specifically, simultaneous evaluation of
assays and controls in the same assay cuvette can be med. Simultaneous evaluation of several assays in
the same assay e can be also performed.
Reaction Environment
A system can comprise a heating block for heating the assay or assay unit and/or for control of the
assay temperature. Heat can be used in the incubation step of an assay reaction to promote the reaction and
shorten the on necessary for the incubation step. A system can comprise a heating block configured to
receive an assay unit of the invention. The heating block can be configured to receive a plurality of assay units
from a device of the invention. For e, if 8 assays are desired to be run on a device, the heating block can
be configured to receive 8 assay units. In some embodiments, assay units can be moved into thermal contact
with a heating block using the means for moving the assay units. The heating can be performed by a heating
means known in the art.
Protocol Optimization
Assay protocols for analyzing samples can be optimized in a variety of manners. When multiple
assays are to be run on a sample, all ols can be optimized to the most stringent reaction conditions, or
each assay protocol can be optimized based on the desired performance of a particular assay.
In some embodiments, a single protocol that can be designed to meet the test requirements under
all possible use cases. For example, on a multiplex cartridge, a single protocol may be specified based on the
case when all tests on the cartridge are to be performed (i.e., the limiting case). This protocol can be designed to
meet the l test requirements, such as the precision and dynamic range for each test on the cartridge.
However, this ch can be suboptimal for alternate use cases, for example, when only a subset of tests on
the dge is to be performed. In these cases, by using more sample, some assays can achieve improved
performance in terms of sensitivity and precision. There can be a trade-off between how much sample is
allocated to an assay and assay sensitivity. For example, an assay which has a sensitivity of 1 unit/mL when the
sample is diluted 1:100 may be able to detect 0.1 unit/mL if the on factor is increased to 1:10. One
downside of using a lower dilution factor in a multiplexed assay system with restricted sample volume can be
that the fraction of the sample required for this assay is increased by 10-fold even when using the minimal
volume to perform the assay. se, assay precision may be improved by using a higher sample
tration. For example, an assay which results in a signal of (say) 0.1 absorbance +/- 0.02 (20 % signal
imprecision) at its limit of detection can be improved by use of 10 times the sample concentration such that the
signal produced is 10 times greater giving a signal of 0.1 +/1 0.02 OD at an analyte concentration ten times
lower and at signal of 1.0, +/- 0.02 the imprecision is now only 2 %. The reason this is the case is that typically
assay signal (at the lower range of analyte concentrations) is directly proportional to the analyte concentration
[Annotation] eaa
(and therefore to the sample concentration) s the signal imprecision can be typically related to the square
root of the signal and so increases as the square root of e concentration (and sample concentration). Thus,
the coefficient of ion (CV) of the signal can be inversely proportional to the square root of the signal; such
that a 10-fold increase in signal corresponds to approximately three-fold decrease in signal CV. Since
concentration CV is typically directly related to signal CV, the concentration CV will decrease with increased
sample concentration (decreased dilution).
Protocols can be zed to specific use cases rather than the typical one-size fits all approach
described above. For example, the ol may be optimized to enhance the precision of each test being
performed in the multiplex device. Moreover, some tests may be prioritized relative to other tests for
optimization. ol optimization can be pre-computed for use cases that are known a priori. Protocol
optimization can also be performed in real-time for new use cases not known a priori. System validation can be
performed to span the suite of use cases.
] One example of protocol optimization is described below comparing two uses cases. For both use
cases, 8 uL of ted sample is available to run the required tests. In this example, the multiplex cartridge
has 20 tests on board, where 5 of the tests require 1:5 on and 15 of the tests require 1:25 dilution.
In the first use case, all tests are required to be run on the sample. The protocol in this use case
(Use-case B) is as follows:
] 1) Prepare 1:5 dilution (8 uL sample + 32 uL diluent)
2) Prepare 1:25 on (15uL 1:5 sample + 60 uL diluent)
3) For each test (n = 20), mix 5 uL of riately diluted sample with 10 uL of the reagent This
protocol results in concentration imprecision of 10% CV for all 20 tests, meeting the minimal requirements.
The sample usage is 1 uL for each 1:5 dilution assay and 0.2 uL for each 1:25 on assay (for a total of 5*1 +
*0.2 = 8 uL, using all the available sample).
In the second use case (Use-case “B") with the same cartridge type, only 10 tests are required to be
run for the sample, not all 20. Moreover, all these 10 tests would be performed at the 1:25 dilution level in use-
case A. The protocol is zed for this use case to maximize precision for all the tests by using a lower
dilution (1:5). The optimized protocol for this specific use case is as follows:
1) Prepare 1:5 dilution (8 uL sample + 32 uL diluent)
2) For each test (n = 10), mix 4 uL of diluted sample with 11 uL of reagent
Sample usage is 0.8 uL undiluted sample per assay for a total of 8 uL. Since the sample
concentration in the assay is increased by 5-fold relative to that for use-case A, the assay sensitivity is improved
by a factor of 5 and the assay imprecision is reduced by about 2.4 (5A0.5) fold to about 4.5 %.
By re-optimizing the protocol, in use case B employs s as much original sample for each
test, thereby improving l performance. Note that the above discussion does not account for any
imprecision due to errors in metering of volumes but only addresses errors due to imprecision in measurement
of optical signal. Use-case B would have a lower imprecision due to imprecision in volumes since it uses fewer
pipetting steps. For example if the volume imprecision introduces 5 % imprecision in the reported analyte
concentration in both use cases there would be a total analyte imprecision of 11.2 % (10A2 + 5A2)A0.5 in use-
case A compared with 6.5 % (4.5A2+5A2)A0.5 in use-case B (assuming, as is generally true, that s causing
imprecision in assays aggregate as the square root of the sum of squares of each source of imprecision).
[Annotation] eaa
The effects illustrated above can more easily be seen in the case of luminescence-based assays
where the assay signal is expressed as a number of photons emitted per unit time. As is the case for counting of
radioactive emissions in for e radioimmunoassay, the signal imprecision is equal to the square root of the
signal and thus the signal CV is 100/ (square root of signal). For example, a signal of 10,000 counts will have a
CV of 1 %. In many assays which produce photons (for example chemiluminescence immunoassays, the signal
is almost exactly proportional to analyte concentration, at least at the lower concentration range). Thus the
measured analyte imprecision scales with 1/ (square root of ) for concentrations significantly above the
limit of detection. In assays which utilize dilution of the sample, the measured analyte imprecision will
therefore scale as 1/ (sample dilution). For example, an assay using a 1:100 dilution of sample will have signal
and concentration CVs about 3.2 fold (10A0.5) higher than an assay using a on 1: 10 (and will also have a
sensitivity about es higher).
Reaction Chemistries
A variety of assays may be performed on a fluidic device according to the present invention to
detect an e of interest in a sample. Where a label is utilized in the assay, one may choose from a wide
diversity of labels is available in the art that can be employed for conducting the subject assays. In some
embodiments labels are detectable by spectroscopic, hemical, biochemical, electrochemical,
chemical, or other chemical means. For example, useful nucleic acid labels include the, fluorescent
dyes, electron-dense ts, and enzymes. A wide variety of labels le for labeling biological components
are known and are ed extensively in both the scientific and patent literature, and are generally applicable
to the present invention for the labeling of biological components. Suitable labels e, enzymes, fluorescent
moieties, chemiluminescent moieties, bioluminescent labels, or colored labels. Reagents defining assay
specificity optionally include, for example, monoclonal antibodies, polyclonal antibodies, aptamers, proteins,
nucleic acid probes or other polymers such as affinity matrices, carbohydrates or lipids. Detection can proceed
by any of a y of known methods, including spectrophotometric or optical tracking of fluorescent, or
luminescent markers, or other methods which track a molecule based upon size, charge or ty. A detectable
moiety can be of any material having a detectable physical or chemical property. Such detectable labels have
been well-developed in the field of gel electrophoresis, column chromatography, solid substrates, spectroscopic
techniques, and the like, and in general, labels useful in such methods can be applied to the present invention.
Thus, a label includes without limitation any composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, nucleic acid probe-based, electrical, optical thermal, or other chemical means.
] In some embodiments the label (such as a colored compound, fluor or ) is coupled directly
or indirectly to a molecule to be detected, according to methods well known in the art. In other embodiments,
the label is attached to a receptor for the e (such as an dy, nucleic acid probe, aptamer etc.). As
ted above, a wide variety of labels are used, with the choice of label depending on the sensitivity required,
ease of conjugation of the compound, stability requirements, available mentation, and disposal provisions.
Non-radioactive labels are often attached by indirect means. Generally, a receptor specific to the e is
linked to a signal-generating moiety. mes the analyte receptor is linked to an adaptor molecule (such as
biotin or ) and the assay t set includes a binding moiety (such as a biotinylated reagent or avidin)
that binds to the adaptor and to the analyte. The analyte binds to a specific receptor on the reaction site. A
labeled reagent can form a sandwich complex in which the analyte is in the center. The reagent can also
[Annotation] eaa
e with the analyte for receptors on the reaction site or bind to vacant receptors on the reaction site not
occupied by analyte. The label is either inherently detectable or bound to a signal system, such as a detectable
enzyme, a fluorescent compound, a chemiluminescent compound, or a chemiluminogenic entity such as an
enzyme with a luminogenic ate. A number of ligands and anti-ligands can be used. Where a ligand has a
natural anti-ligand, for example, biotin, thyroxine, digoxigenin, and ol, it can be used in conjunction with
labeled, anti-ligands. Alternatively, any haptenic or antigenic compound can be used in ation with an
antibody.
s of interest as labels will primarily be ases, particularly phosphatases, esterases and
glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its
derivatives, ine and its derivatives, dansyl , and umbelliferone. Chemiluminescent compounds
include dioxetanes, acridinium esters, luciferin, and 2,3-dihydrophthalazinediones, such as luminol.
Methods of detecting labels are well known to those of skilled in the art. Thus, for example, where
the label is fluorescent, it may be ed by exciting the fluorochrome with light of an appropriate wavelength
and detecting the resulting fluorescence by, for example, microscopy, visual tion, via photographic film,
by the use of onic detectors such as digital cameras, charge coupled devices (CCDs) or photomultipliers
and phototubes, or other detection devices. Similarly, enzymatic labels are detected by providing appropriate
substrates for the enzyme and detecting the resulting reaction t spectroscopically or by digital imaging
(the subject of the present invention). Finally, simple colorimetric labels are often detected simply by observing
the color associated with the label. For example, colloidal gold sols often appear pink, while s beads
doped with dyes are strongly colored.
In some embodiments the detectable signal may be ed by luminescence sources.
scence is the term commonly used to refer to the emission of light from a substance for any reason other
than a rise in its temperature. In general, atoms or molecules emit photons of electromagnetic energy (e. g., light)
when they transition from an excited state to a lower energy state ly the ground state). If the exciting agent
is a photon, the luminescence process is referred to as photoluminescence or fluorescence. If the exciting cause
is an electron, the luminescence process can be referred to as electroluminescence. More specifically,
electroluminescence results from the direct injection and l of electrons to form an electron-hole pair, and
subsequent recombination of the electron-hole pair to emit a photon. Luminescence which results from a
chemical reaction is usually referred to as chemiluminescence. Luminescence produced by a living organism is
y referred to as bioluminescence. If photoluminescence is the result of a spin allowed transition (e. g., a
single-singlet transition, triplet-triplet transition), the photoluminescence process is usually referred to as
fluorescence. Typically, fluorescence emissions do not persist after the ng cause is removed as a result of
short-lived excited states which may rapidly relax through such spin d transitions. If uminescence
is the result of a spin forbidden transition (e. g., a triplet-singlet transition), the uminescence process is
usually referred to as phosphorescence. Typically, phosphorescence emissions persist long after the exciting
cause is removed as a result of long-lived excited states which may relax only through such spin-forbidden
transitions. A luminescent label may have any one of the above-described properties.
Suitable chemiluminescent sources include a compound which becomes electronically excited by a
chemical reaction and may then emit light which serves as the detectible signal or donates energy to a
fluorescent acceptor. A diverse number of families of nds have been found to provide
[Annotation] eaa
chemiluminescence under a variety of conditions. One family of compounds is 2,3-dihydro-l,4-
phthalazinedione. A frequently used compound is luminol, which is a 5-amino compound. Other s of the
family include the 5-amino-6, 7, 8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can
be made to luminesce with alkaline hydrogen de or calcium hypochlorite and base. Another family of
compounds is the 2,4,5-triphenylimidazoles, with lophine as the common name for the parent t.
Chemiluminescent analogs include para-dimethylamino and -methoxy substituents. Chemiluminescence may
also be obtained with oxalates, usually oxalyl active esters, for example, p-nitrophenyl and a de such as
en peroxide, under basic ions. Other useful chemiluminescent compounds that are also known
e -N-alkyl acridinum esters and dioxetanes. Alternatively, luciferins may be used in conjunction with
luciferase or lucigenins to e bioluminescence. Especially preferred chemiluminescent sources are
“luminogenic" enzyme substrates such as dioxetane-phosphate esters. These are not luminescent but produce
luminescent products when acted on by phosphatases such as alkaline phosphatase. The use of luminogenic
substrates for enzymes is particularly red because the enzyme acts as an amplifier capable of converting
thousands of substrate molecules per second to product. Luminescence methods are also preferred because the
signal (light) can be detected both very sensitively and over a huge dynamic range using PMTs.
The term analytes as used herein includes without limitation drugs, prodrugs, pharmaceutical
agents, drug metabolites, biomarkers such as expressed proteins and cell s, antibodies, serum proteins,
cholesterol and other metabolites, electrolytes, metal ions, polysaccharides, c acids, biological analytes,
biomarkers, genes, proteins, hormones, or any combination thereof Analytes can be combinations of
polypeptides, roteins, ccharides, , and nucleic acids.
The system can be used to detect and/or quantify a variety of analytes. For example, analytes that
can be detected and/or quantified include Albumin, Alkaline Phosphatase, ALT, AST, Bilirubin (Direct),
Bilirubin (Total), Blood Urea Nitrogen (BUN), Calcium, Chloride, Cholesterol, Carbon Dioxide (COZ),
Creatinine, Gamma-glutamyl-transpeptidase (GGT), Globulin, Glucose, HDL-cholesterol, Hemoglobin,
Homocysteine, Iron, Lactate Dehydrogenase, Magnesium, Phosphorous, Potassium, , Total Protein,
cerides, and Uric Acid. The detection and/or quantification of these analytes can be med using
optical, electrical, or any other type of ements.
] Of particular interest are biomarkers which are associated with a particular disease or with a
specific e stage. Such analytes e but are not limited to those associated with autoimmune diseases,
obesity, hypertension, diabetes, al and/or muscular degenerative diseases, cardiac diseases, endocrine
disorders, metabolic disorders, inflammation, vascular diseases, sepsis, angiogenesis, cancers,
Alzheimer’s disease, athletic complications, and any combinations thereof.
Of also interest are biomarkers that are present in varying abundance in one or more of the body
tissues including heart, liver, prostate, lung, , bone marrow, blood, skin, bladder, brain, muscles, nerves,
and selected tissues that are affected by various disease, such as different types of cancer (malignant or non-
metastatic), autoimmune diseases, inflammatory or degenerative diseases.
Also of interest are analytes that are indicative of a microorganism, virus, or Chlamydiaceae.
Exemplary microorganisms include but are not limited to bacteria, viruses, fungi and protozoa. Analytes that
can be ed by the subject method also include blood-bom pathogens selected from a non-limiting group
that consists of Staphylococcus epidermidis, Escherichia coli, methicillin-resistant Staphylococcus aureus
[Annotation] eaa
(MSRA), Staphylococcus aureus, Staphylococcus hominis, Enterococcusfaecalis, Pseudomonas nosa,
Staphylococcus s, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influenzae,
Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.
Analytes that can be ed by the subject method also encompass a variety of sexually
transmitted diseases selected from the following: gonorrhea (Neisseria gonorrhoeae), is (Treponena
pallidum), clamydia (Clamyda tracomitis), nongonococcal urethritis (Ureaplasm urealyticum), yeast infection
(Candida albicans), chancroid (Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis), genital herpes
(HSV type I & II), HIV I, HIV II and hepatitis A, B, C, G, as well as tis caused by TTV.
Additional analytes that can be ed by the subject methods encompass a diversity of
respiratory ens including but not limited to Pseudomonas aeruginosa, methicilliniresistant
Staphlococccus aureus (MSRA), Klebsiella pneumoniae, Haemophilis influenzae, Staphlococcus aureus,
Stenotrophomonas maltophilia, Haemophilis parainfluenzae, Escherichia coli, Enterococcusfaecalis, Serratia
marcescens, Haemophilis parahaemolyticus, Enterococcus cloacae, Candida albicans, Moraxiella catarrhalis,
Streptococcus pneumoniae, Citrobacterfreundii, Enterococcusfaecium, Klebsella oxytoca, Pseudomonas
fluorscens, Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii, lla pneumoniae
Legionella pneumophila, Mycoplasma pneumoniae, and Mycobacterium ulosis.
Listed below are additional exemplary markers according to the present invention: Theophylline,
CRP, CKMB, PSA, bin, CA125, Progesterone, TxBZ, -PGF-l-alpha, and Theophylline, Estradiol
, Lutenizing e, Triglycerides, Tryptase, Low density lipoprotein Cholesterol, High density lipoprotein
Cholesterol, Cholesterol, IGFR.
] Exemplary liver markers include without limitation LDH, (LD5), Alanine-aminotransferase
(ALT), Arginase 1 (liver type), Alpha-fetoprotein (AFP), Alkaline phosphatase, Lactate ogenase, and
bin.
Exemplary kidney markers include without tion TNFa or, Cystatin C, Lipocalin-type
urinary prostaglandin D, synthatase (LPGDS), Hepatocyte growth factor receptor, Polycystin 2, Polycystin l,
Fibrocystin, Uromodulin, Alanine, aminopeptidase, N-acetyl-B-D-glucosaminidase, Albumin, and Retinolbinding
protein (RBP).
Exemplary heart markers include t limitation Troponin I (TnI), Troponin T (TnT), Creatine
dinase (CK), CKMB, bin, Fatty acid binding protein (FABP), C-reactive protein (CRP), Fibrinogen D-
dimer, S-100 protein, Brain natriuretic peptide (BNP), NT-proBNP, PAPP-A, Myeloperoxidase (MPO),
Glycogen phosphorylase isoenzyme BB (GPBB), Thrombin Activatable Fibrinolysis Inhibitor (TAFI),
Fibrinogen, Ischemia modified n (IMA), Cardiotrophin-l, and MLC-I (Myosin Light Chain-I).
Exemplary pancrease markers include without limitation Amylase, Pancreatitis-Associated protein
(PAP-l), and Regeneratein proteins (REG).
Exemplary muscle tissue markers include without limitation Myostatin.
Exemplary blood markers e without limitation Erythopoeitin (EPO).
] Exemplary bone s include without limitation, linked N-telopeptides of bone type I
en (NTx), Carboxyterminal cross-linking telopeptide of bone collagen, Lysyl-pyridinoline
(deoxypyridinoline), Pyridinoline, Tartrate-resistant acid phosphatase, Procollagen type I C propeptide,
Procollagen type IN propeptide, Osteocalcin (bone gla-protein), Alkaline phosphatase, Cathepsin K, COMP
[Annotation] eaa
llage eric Matrix Protein), Osteocrin, Osteoprotegerin (OPG), RANKL, sRANK TRAP 5 (TRACP
), last Specific Factor 1 (OSF-1, Pleiotrophin), Soluble cell adhesion les, sTfR, sCD4, sCD8,
sCD44, and last Specific Factor 2 (OSF-2, Periostin).
In some embodiments markers according to the present ion are disease specific. Exemplary
cancer markers include Without tion PSA (total prostate specific antigen), Creatinine, Prostatic acid
phosphatase, PSA complexes, Prostrate-specific gene-1, CA 12-5, Carcinoembryonic Antigen (CEA), Alpha
feto n (AFP) hCG (Human chorionic gonadotropin), Inhibin, CAA Ovarian C1824, CA 27.29, CA 15-3,
CAA Breast C1924, Her-2, atic, CA 19-9, CAA pancreatic, Neuron-specific enolase, Angiostatin DcR3
(Soluble decoy receptor 3), Endostatin, Ep-CAM (MK-1), Free Immunoglobulin Light Chain Kappa, Free
Immunoglobulin Light Chain Lambda, Herstatin, Chromogranin A, Adrenomedullin, Integrin, Epidermal
growth factor receptor, Epidermal grth factor or-Tyrosine kinase, Pro-adrenomedullin N-terminal 20
peptide, Vascular endothelial growth factor, Vascular endothelial grth factor receptor, Stem cell factor
receptor, c-kit/KDR, KDR, and Midkine.
Exemplary infectious disease conditions include Without tion: Viremia, Bacteremia, Sepsis,
and markers: PMN Elastase, PMN elastase/ (11-PI complex, Surfactant Protein D (SP-D), HBVc antigen, HBVs
antigen, Anti-HBVc, Anti-HIV, T-supressor cell antigen, T-cell antigen ratio, T-helper cell antigen, Anti-HCV,
Pyrogens, p24 antigen, l-dipeptide.
Exemplary diabetes markers include Without limitation C-Peptide, Hemoglobin A1c, ed
n, Advanced glycosylation end products (AGEs), 1,5-anhydroglucitol, Gastric Inhibitory Polypeptide,
Glucose, Hemoglobin A1c, ANGPTL3 and 4.
Exemplary inflammation s include Without limitation Rheumatoid factor (RF), Antinuclear
Antibody (ANA), tive protein (CRP), Clara Cell Protein (Uteroglobin).
Exemplary allergy markers include Without limitation Total IgE and Specific IgE.
Exemplary autism markers include Without limitation Ceruloplasmin, Metalothioneine, Zinc,
Copper, B6, B12, Glutathione, Alkaline phosphatase, and Activation of apo-alkaline phosphatase.
Exemplary coagulation disorders markers e Without limitation b-Thromboglobulin, Platelet
factor 4, Von Willebrand .
In some embodiments a marker may be y specific. Markers indicative of the action of COX
inhibitors include Without limitation TxB2 (Cox-1), 6-keto-PGFalpha (Cox 2), 11-Dehydro-TxB-1a (Cox-1).
Other markers of the present invention e Without limitation Leptin, Leptin receptor, and
Procalcitonin, Brain S100 protein, Substance P, 8-Iso-PGF-2a.
Exemplary geriatric markers include Without limitation, Neuron-specific enolase, GFAP, and
S 100B.
Exemplary s of nutritional status include Without limitation Prealbumin, Albumin, l-
g protein (RBP), Transferrin, Acylation-Stimulating n (ASP), Adiponectin, Agouti-Related Protein
(AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP (Adipocyte Fatty Acid Binding
Protein, FABP4), Acylation-Stimulating Protein (ASP), EFABP (Epidermal Fatty Acid Binding Protein,
FABP5), Glicentin, Glucagon, Glucagon-Like Peptide-1, Glucagon-Like Peptide-2, n, Insulin, Leptin,
Leptin Receptor, PYY, RELMs, Resistin, amd sTfR (soluble Transferrin Receptor).
ation] eaa
ary markers of Lipid metabolism e without limitation Apo-lipoproteins (several),
Apo-Al, Apo-B, Apo-C-CII, Apo-D, Apo-E.
Exemplary coagulation status markers include t limitation Factor I: Fibrinogen, Factor 11:
ombin, Factor III: Tissue factor, Factor IV: Calcium, Factor V: Proaccelerin, Factor VI, Factor VII:
Proconvertin, Factor VIII:, Anti-hemolytic factor, Factor IX: Christmas factor, Factor X: Stuart-Prower factor,
Factor XI: Plasma thromboplastin antecedent, Factor XII: Hageman factor, Factor XIII: Fibrin-stabilizing factor,
Prekallikrein, High-molecular-weight kininogen, Protein C, Protein S, r, Tissue plasminogen activator,
Plasminogen, iplasmin, Plasminogen tor inhibitor 1 (PAIl).
Exemplary monoclonal antibodies include those for EGFR, ErbB2, and IGFlR.
Exemplary tyrosine kinase inhibitors include without limitation Abl, Kit, PDGFR, Src, ErbB2,
ErbB 4, EGFR, EphB, VEGFR1-4, PDGFRb, FLt3, FGFR, PKC, Met, Tie2, RAF, and TrkA.
Exemplary Serine/Threonine Kinase Inhibitors include without limitation AKT, Aurora A/B/B,
CDK, CDK (pan), CDK1-2, VEGFRZ, PDGFRb, CDK4/6, MEK1-2, mTOR, and ta.
GPCR targets include without limitation Histamine Receptors, Serotonin Receptors, Angiotensin
Receptors, Adrenoreceptors, Muscarinic Acetylcholine Receptors, GnRH Receptors, Dopamine Receptors,
Prostaglandin Receptors, and ADP Receptors.
Cholesterol
ement of metabolites can be performed by production of a d product using es
(such as cholesterol oxidase) (to make H202) and radish peroxidase plus a chromogen (such as N-Ethyl-
N-(2-hydroxy-3 -sulfopropyl)-3,5-dimethoxyaniline, sodium salt [“DAOS" plus amino anti-pyrene] to form a
colored product such as a Trinder dye). One e of such chemistry is shown in Figure 52 and Figure 53.
NADH 0r NADPH
Production or consumption ofNADH or NADPH are frequently used in clinical assays. This is
because these coenzymes are common substrates for enzymes. For example, measurement of s of
clinical interest such as lactate dehydogenase (LDH) can be measured by the rate of production ofNADH.
Since NADH absorbs light maximally at 340 nm and (l) polystyrene and other plastics transmit light poorly in
the near UV, (2) White light sources produce little light in the near UV and (3) camera and scanner sensors have
low sensitivity to near UV light, it is not practical to measure NADH by three color image analysis. To deal
with this issue NADH can be ted to a colored product using tetrazolium salts such as Water Soluble
Tetrazolium (e.g.WST-l (Dojindo Molecular Technologies) plus an “electron mediator" such as l-
Methoxyphenazine methosulfate (PMS).
In some embodiments, assays that produce or consume NADH or NADPH can be paired with
other reactions that allow for colorimetric measurement. For example, NADH or NADPH can be used to reduce
compounds such as 2-(4-Iodophenyl)-3 -(4-nitrophenyl)(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt
(WST-l) to a colored formezan dye as shown below with the use of phenazine methosulfate as an electron
mediator, as shown in Figure 54.
] As shown in Figure 73, when NADH, WST-l and PMS are combined at millimolar trations,
a yellow product (shown in tips indicated as Mixture) is formed.
Using this chemistry, an assay for LDH was setup. Lactate (mM), NAD (mM) and LDH were
ed and incubated at 37C for 10 s before addition of WST-l and PMS. A good dose-response to
[Annotation] eaa
LDH was obtained as shown in Figure 74 for two-fold serial dilutions of LDH (1000 IU/L) (left to right)
ponding to the OD 450 nm values shown in the graph in Figure 75.
Alkaline Phosphatase
In other embodiments, assays utilizing s such as alkaline phosphatase can be measured
using a genic substrate such as p-nitrophenyl phosphate. The enzymatic reaction can make p-
nitrophenol which is yellow in alkaline conditions.
Metal ions
Measurements can also be performed on assays that form colored complex, such as between a
metal ion a chelating dye which changes color on binding. For example, o-Cresolphthalein Complexone (shown
in Figure 55) forms a complex with calcium, which has a different color than the reagent. The general scheme
of such assays is: Chelating dye (color 1) + MN+ <-> Chelating dye: MN+:(Color 2)
Optical signals can also be measured for metal ion assays using metal-dependant enzymes. For
example, sodium ions can be determined enzymatically via sodium dependent B-galactosidase activity with onitro-phenyl
oside (ONPG) as the substrate. The absorbance at 405 nm of the product o-nitrophenol is
proportional to the sodium tration.
ELISAs
Assays can be performed for analytes by color-forming ELISAs. Many ELISA methods are
known which generate color using enzymes such as horseradish dase, ne phosphatase and [3-
galactosidase with chromogenic substrates such as o-phenylene diamine, p-nitrophenyl phosphate, and onitrophenyl
galactoside respectively. Such assays can be readily performed and read by the subject invention.
Luminogenic immunoassays
Luminogenic immunoassays can also be med. Assays can utilize chemiluminogenic entities
such as an enzyme with a luminogenic substrate. For example, chemiluminescent compounds include
dioxetanes, acridinium esters, luciferin, and 2,3-dihydrophthalazinediones, such as luminol.
Furthermore, suitable chemiluminescent sources include a compound which becomes
electronically excited by a chemical reaction and may then emit light which serves as the detectible signal or
donates energy to a fluorescent acceptor. A diverse number of families of compounds have been found to
provide uminescence under a variety of conditions. One family of nds is 2,3-dihydro-l,4-
phthalazinedione. A frequently used compound is luminol, which is a 5-amino compound. Other members of the
family include the o-6, 7, 8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can
be made to luminesce with alkaline hydrogen peroxide or calcium hypochlorite and base. Another family of
compounds is the 2,4,5-triphenylimidazoles, with e as the common name for the parent product.
Chemiluminescent analogs e imethylamino and -methoxy substituents. uminescence may
also be obtained with oxalates, usually oxalyl active esters, for e, p-nitrophenyl and a peroxide such as
hydrogen peroxide, under basic conditions. Other useful chemiluminescent compounds that are also known
include N-alkyl acridinum esters and dioxetanes. Alternatively, luciferins may be used in conjunction with
rase or lucigenins to provide bioluminescence.
Nucleic Acid Amplification
] Assays that can be performed also include nucleic acid amplification. Among these assays,
isothermal ication and Loop-Mediated Isothermal Amplification Assays (LAMP) are examples. Nucleic
[Annotation] eaa
acid amplification can be used to produce visibly turbid, fluorescent or colored assay reaction products for
es such as nucleic acid targets (genes etc.). Nucleic acid amplification technology can be used for
rmal cation of specific DNA and RNA targets. Additional information on isothermal nucleic acid
cation is described in Goto et al., “Colorimetric detection of ediated isothermal amplification
reaction by using y naphthol blue", BioTechniques, Vol. 46, No. 3, March 2009, 167-172.
Nucleic acid amplification can be used to measure DNA and, coupled with the use of reverse
transcriptase, RNA. Once the reaction has occurred, the amplified product can be ed optically using
intercalating dyes or chromogenic reagents that react with released pyrophosphate generated as a side product of
the amplification.
The reaction can be visualized by changes (increases) in color, fluorescence or turbidity. Very
small copy numbers of DNA can be ed in less than one hour. This technology can advantageously be read
out in the present invention using three-color image analysis. As shown below, images of isothermal nucleic
acid amplification assay reaction products can be ed by (1) back lit-illumination (transmission optics)
ing absorbance of light, (2) images captured by a digital camera of light transmitted through a reaction
product or (3) fluorescent light images generated by illumination of reaction products with a UV source (or any
other riate light source) captured by a digital .
The nucleic acid amplification assay is generally performed in a “one-pot" format where sample
and reagents are combined in a sealed tube and incubated at elevated temperature. In some formats, the reaction
can be monitored in real time by changes in l properties. In other assay formats the reaction is stopped
and reaction products visualized after adding a chromogenic or fluorogenic reagent. The present invention
allows for the reading of nucleic acid amplification assay products directly in the reaction vessel or after
aspiration into the tips described herein.
Turbidity
The invention also provides for optical turbidimetric assays. For example, assays can be
set up by measurement of the ination of small latex particles (50 - 300 nm). In these assays the particles
can be coated with an antigen and/or antibody and agglutination occurs when a binding counterpart in the
sample such as antibody or antigen is added. Assays can be set up as direct (e. g. antibody on the particle
reacting with a multi-epitope protein or ker) or the competitive mode (e.g. drug hapten on particle reacts
with rug antibody in competition with free drug in the ). The dispersion of latex becomes more
turbid and the turbidity can be measured as decreased transmission of light using 3-color optics.
Similarly, assays based on the agglutination of large latex particles (diameter about 1 um) or red
blood cells can be measured. Assay configuration is similar to turbidimetric assays as disclosed above, but the
measurement can be by image analysis er or camera measurement) using re to interpret the number
and size of the agglutinates.
Reagents for ming reaction chemistries can be included in the cartridges described here, such
as in pipette tips. The reagents can be stored as liquids or in dried, lyophilized, or glassy forms.
] Localized Reagents
In some embodiments, the location and configuration of a reaction site is an important element in
an assay device. Most, if not all, disposable immunoassay devices have been configured with their capture
surface as an integral part of the device.
[Annotation] eaa
In one embodiment, a molded plastic assay unit is either commercially available or can be made by
injection molding with precise shapes and sizes. For example, the characteristic dimension can be a diameter of
0.05 i 3 mm or can be a length of 3 to 30 mm. The units can be coated with capture reagents using method
similar to those used to coat microtiter plates but with the advantage that they can be processed in bulk by
placing them in a large vessel, adding coating reagents and processing using sieves, holders, and the like to
recover the pieces and wash them as needed.
The assay unit (e.g. encompassing the tip disclosed herein, tips, vessels, or any other containers)
can offer a rigid t on which a reactant can be immobilized. The assay unit is also chosen to provide
appropriate characteristics with respect to interactions with light. For example, the assay unit can be made of a
material, such as onalized glass, Si, Ge, GaAs, GaP, SiOZ, SiN4, modified silicon, or any one of a wide
y of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,
polycarbonate, polypropylene, polymethylmethacrylate (PMMA), acrylonitrile-butadiene-styrene (ABS), or
ations thereof. In an embodiment, an assay unit comprises polystyrene. In some embodiments, the assay
unit may be formed from a homogeneous material, heterogeneous material, clad al, coated material,
impregnated material, and/or embedded material. Other riate materials may be used in accordance with
the present invention. A transparent reaction site may be advantageous. In on, in the case where there is an
optically transmissive window permitting light to reach an optical detector, the surface may be advantageously
opaque and/or preferentially light scattering. In some embodiments, the assay unit may be formed from a
transparent material. Alternatively, a portion of the assay unit may be formed from a transparent material.
The assay unit may have a reagent coated thereon and/or impregnated therein. In some
embodiments, the reagent may be a capture reagent capable of immobilizing a reactant on a e surface.
The nt may be a cell and/or analyte, or any other reactant described elsewhere herein. In some
embodiments, the reagent may be a molecule that may be a cell capture agent. A cell capture agent may anchor
to the e of desired cells during fluid transport. In some embodiments, the capture reagents may be an
antibody, peptide, organic le (e.g., which may have a lipid chain, lipophilic molecule), polymer matrix,
n, n composite, rotein, that may interact with the cell membrane. Capture reagents may be
molecules, cross-linked les, nanoparticles, nanostructures, and/or scaffolds. In some embodiments,
microstructures may be provided that may become an analysis mechanism in a . Capture reagents (which
may include capture ures formed by the assay unit material) may allow cells to be tethered, bound, and/or
trapped.
The capture reagents may immobilize a reactant, such as a cell, during processing. Capture
techniques may be chemical, physical, electrical, magnetic, mechanical, size-related, density-related, or any
combination thereof. In some embodiments, the capture reagents may be used to trate reactants, such as
cells, at a desired location. For example, an assay unit may be coated with the capture reagents, which may
cause cells to be ed at the assay unit surface, thus concentrating the cells on the captured surface. The
capture reagents may keep the captured reactant immobilized on the cell surface. This may aid in keeping the
reactants (e. g., cells, es) stationary during g.
Immobilizing the reactants may be useful for applications where there may be long acquisition
times for reactions and/or detection. For example, a number of imaging applications may require extended
exposure times (~l min) or imaging of small objects (<lum) which may have significant Brownian motion.
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In some embodiments, the capture reagents may be formed from materials that may provide little
or no background for imaging. In some instances, the material of the assay unit may provide little or no
background for imaging. The capture reagents may be selected so that they do not interfere with, or only have a
small interference with, imaging and/or ion.
A reactant immobilized at the capture surface can be anything useful for detecting an analyte of
interest in a sample of bodily fluid. For instance, such reactants e, without limitation, nucleic acid probes,
antibodies, cell membrane ors, monoclonal antibodies, ra, and aptamers reactive with a specific
analyte. Various cially available reactants such as a host of polyclonal and monoclonal antibodies
specifically developed for specific analytes can be used.
One skilled in the art will appreciate that there are many ways of immobilizing various reactants
onto a support where reaction can take place. The immobilization may be covalent or noncovalent, via a linker
moiety, or tethering them to an immobilized moiety. Non-limiting ary binding moieties for attaching
either nucleic acids or proteinaceous molecules such as antibodies to a solid support e streptavidin or
avidin/biotin linkages, carbamate linkages, ester linkages, amide, thiolester, (N)-functionalized thiourea,
functionalized maleimide, amino, disulfide, amide, hydrazone linkages, and among others. In addition, a silyl
moiety can be attached to a nucleic acid directly to a substrate such as glass using methods known in the art.
Surface immobilization can also be achieved via a Poly-L Lysine tether, which provides a charge-charge
coupling to the surface.
The assay units can be dried following the last step of incorporating a capture e. For
example, drying can be performed by passive exposure to a dry atmosphere or via the use of a vacuum ld
and/or application of clean dry air through a ld or by lyophilization.
A capture surface may be applied to an assay unit using any technique. For example, the capture
surface may be painted on, printed on, electrosprayed on, ed in the material, impregnating the material,
or any other technique. The capture reagents may be coated to the assay unit material, incorporated in the
material, co-penetrate the al, or may be formed from the al. For example, a t, such as a
capture reagent may be embedded in a polymer matrix that can be used as a sensor. In some embodiments, one
or more small particles, such as a nanoparticle, a microparticle, and/or a bead, may be coated and/or
impregnated with reagents. In some embodiments, the capture reagents may be part of the assay unit material
itself, or may be something that is added to the al.
In many embodiments, an assay unit is designed to enable the unit to be manufactured in a high
volume, rapid manufacturing processes. For example, tips can be mounted in large-scale arrays for batch g
of the capture surface into or onto the tip. In another e, tips can be placed into a moving belt or rotating
table for serial processing. In yet another e, a large array of tips can be connected to vacuum and/or
pressure manifolds for simple processing.
A capture reagent may be applied to an assay unit during any point in the process. For example,
the capture reagent may be applied to the assay unit during manufacturing. The capture reagent may be applied
to the assay unit prior to shipping the assay unit to a ation. Alternatively, the capture reagent may be
applied to the assay unit after the assay unit has been shipped. In some ces, the capture reagent may be
applied to the assay unit at a point of use, such as a point of service location.
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In some embodiments, the capture t may cover an entire surface or region of the assay unit.
The capture t may be provided on an inner surface of the assay unit. In some embodiments, the capture
reagent may cover portions or ns of an assay unit surface. The capture reagent may be provided on a
surface in a pattern. A unit may have portions of the surface that have a capture reagent d thereon, and
portions of the surface that do not have a capture reagent applied thereon. For example, there may be coated
and non-coated regions. A capture t may be applied in a surface in accordance with a geometric choice of
how the capture t is to be applied. For example, the capture reagent may be applied in dots, rows,
columns, arrays, regions, circles, rings, or any other shape or pattern. The capture reagents may be applied at
desired ons on the surface.
A plurality of e reagents may optionally be applied to an assay unit. In some embodiments,
the plurality of capture reagents may be applied so that the different capture reagents do not overlap (e. g., the
different capture reagents are not applied to the same region or area). Alternatively, they may overlap (e.g., the
different capture reagents may be applied to the same region or area). Space without any capture reagents may
or may not be provided between regions with different e reagents. The different capture reagents may be
used to immobilize different nts. For example, different capture reagents may be used to immobilize
different cells and/or analytes on the capture surface. By using a plurality of capture reagents patterned in
selected regions, a plurality of reactants may be detected from the same assay unit. In some embodiments, two
or more, three or more, four or more, five or more, seven or more, ten or more, fifteen or more, twenty or more,
thirty or more, forty or more, fifty or more, seventy or more, 100 or more, 150 or more, 200 or more, or 300 or
more different capture reagents may be applied to a surface of an assay unit. The ent capture reagents may
be applied in any pattern or shape. For example, different capture reagents may be applied as an array or series
of rings on an inner surface of an assay unit. For example, different capture reagents may be applied on an inner
surface of a tip, vessel, container, e, or any other container described elsewhere herein.
The location of the different capture reagents on the assay unit may be known prior to detection of
the captured reactants. In some embodiments, the assay unit may have an identifier that may indicate the type of
assay unit and/or the pattern of capture agents therein. Alternatively the location of the ent e
reagents of the assay unit may not be known prior to detection of the captured reactants. The location of the
ent capture reagents may be determined based on detected patterns of captured reactants.
The capture reagents may be d using any technique, such as those described elsewhere
herein. In some instances, masking or raphic ques may be used to apply different capture reagents.
Any description herein of a capture t and/or coating applied to an assay unit may apply to
any other units or containers bed elsewhere herein, including but not limited to tips, vessels, cuvettes, or
reagent units.
Reagent lies
In many embodiments of the invention the reagent units are modular. The reagent unit can be
designed to enable the unit to be manufactured in a high volume, rapid manufacturing processes. For example,
many t units can be filled and sealed in a large-scale process simultaneously. The reagent units can be
filled according to the type of assay or assays to be run by the device. For e, if one user desires different
assays than another user, the reagent units can be manufactured accordingly to the preference of each user,
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without the need to manufacture an entire device. In another example, reagent units can be placed into a moving
belt or rotating table for serial processing.
In another embodiment, the reagent units are accommodated directly into cavities in the housing of
a device. In this embodiment, a seal can be made onto areas of housing surrounding the units.
Reagents according to the present invention include without limitation wash buffers, enzyme
substrates, dilution buffers, conjugates, enzyme-labeled conjugates, DNA amplifiers, sample diluents, wash
ons, sample eatment reagents ing additives such as detergents, polymers, chelating agents,
albumin-binding reagents, enzyme inhibitors, enzymes, anticoagulants, red-cell agglutinating agents, antibodies,
or other materials necessary to run an assay on a device. An enzyme-labeled conjugate can be either a
polyclonal antibody or monoclonal dy d with an enzyme that can yield a detectable signal upon
reaction with an appropriate substrate. Non-limiting examples of such s are alkaline phosphatase and
horseradish peroxidase. In some embodiments, the reagents comprise immunoassay reagents. In general,
reagents, ally those that are relatively unstable when mixed with liquid, are confined separately in a
defined region (for example, a reagent unit) within the device.
In some embodiments, a reagent unit contains approximately about 5 microliters to about 1
milliliter of liquid. In some embodiments, the unit may contain about 20-200 microliters of liquid. In a further
embodiment, the reagent unit contains 100 microliters of fluid. In an embodiment, a reagent unit contains about
40 microliters of fluid. The volume of liquid in a reagent unit may vary depending on the type of assay being
run or the sample of bodily fluid ed. In an embodiment, the volumes of the reagents do not have to
predetermined, but must be more than a known minimum. In some embodiments, the reagents are initially
stored dry and dissolved upon initiation of the assay being run on the device.
In an embodiment, the reagent units can be filled using a siphon, a funnel, a pipette, a syringe, a
needle, or a ation thereof. The reagent units may be filled with liquid using a fill l and a vacuum
draw l. The reagent units can be filled individually or as part of a bulk manufacturing process.
In an embodiment, an individual reagent unit comprises a ent t as a means of isolating
reagents from each other. The reagent units may also be used to contain a wash solution or a substrate. In
addition, the reagent units may be used to contain a luminogenic substrate. In another ment, a plurality of
reagents are contained within a reagent unit.
] In some instances, the setup of the device enables the capability of pre-calibration of assay units
and the reagent units prior to assembly of disposables of the subject device.
Aptamer binding assays
] The subject invention enables a variety of assay methods based on the use of binding elements that
specifically bind to one or more analytes in a sample. In general, a binding element is one member of a binding
pair capable of specifically and selectively binding to the other member of the binding pair in the presence of a
plurality of different molecules. es of binding elements include, but are not limited to, antibodies,
antigens, metal-binding ligands, nucleic acid probes and primers, receptors and nts as described herein,
and aptamers. In some embodiments, a binding t used to detect an analyte is an aptamer. The term
“aptamer" is used to refer to a peptide, nucleic acid, or a combination thereof that is selected for the ability to
specifically bind one or more target analytes. e rs are affinity agents that generally comprise one or
more variable loop s displayed on the surface of a scaffold n. A nucleic acid aptamer is a specific
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binding oligonucleotide, which is an oligonucleotide that is capable of selectively forming a complex with an
intended target analyte. The complexation is target-specific in the sense that other materials, such as other
analytes that may accompany the target analyte, do not complex to the aptamer with as great an affinity. It is
recognized that complexation and affinity are a matter of ; however, in this context, “target-specific"
means that the aptamer binds to target with a much higher degree of affinity than it binds to contaminating
materials. The g of specificity in this context is thus similar to the meaning of specificity as applied to
antibodies, for example. The aptamer may be prepared by any known method, including synthetic,
recombinant, and purification s. Further, the term er" also includes “secondary aptamers"
containing a consensus sequence derived from comparing two or more known aptamers to a given target.
In general, nucleic acid aptamers are about 9 to about 35 nucleotides in length. In some
embodiments, a nucleic acid aptamer is at least 4, 5, 6, 7, 8, 9, 10, ll, 12, l3, 14, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 80, 90, 100, or more es in length. Although the oligonucleotides of the aptamers generally
are single-stranded or double-stranded, it is contemplated that aptamers may sometimes assume -stranded
or quadruple-stranded structures. In some embodiments, a nucleic acid aptamer is circular, such as in
0176940. The specific g oligonucleotides of the aptamers should contain the sequence-conferring
specificity, but may be extended with flanking regions and otherwise tized or modified. The aptamers
found to bind to a target analyte may be isolated, sequenced, and then re-synthesized as conventional DNA or
RNA moieties, or may be modified oligomers. These modifications include, but are not limited to incorporation
of: (1) d or analogous forms of sugars (e. g. ribose and deoxyribose); (2) ative linking groups; or (3)
ous forms of purine and pyrimidine bases.
Nucleic acid aptamers can comprise DNA, RNA, onalized or modified nucleic acid bases,
nucleic acid analogues, modified or alternative backbone tries, or combinations thereof The
oligonucleotides of the aptamers may contain the conventional bases e, e, cytosine, and thymine or
uridine. Included within the term aptamers are synthetic aptamers that incorporate ous forms of purines
and pyrimidines. gous" forms of purines and pyrimidines are those generally known in the art, many of
which are used as chemotherapeutic agents. Non-limiting examples of analogous forms of purines and
pyrimidines (i.e. base analogues) include aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-
carboxymethylaminomethylthiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine,
l-methyladenine, ylpseudouracil, l-methylguanine, l-methylinosine, 2,2-dimethylguanine, 2-
adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-
methylaminomethyl-uracil, 5-methoxyaminomethylthiouracil, beta-D-mannosquueosine, 5-methoxyuracil,
2-methyl-thio-N6-isopentenyladenine, uraciloxyacetic acid methylester, pseudouracil, queosine, 2-
thiocytosine, 5-methylthiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5 -oxyacetic acid, 5-
pentynyl-uracil, and 2,6-diaminopurine. The use of uracil as a substitute base for thymine in deoxyribonucleic
acid (hereinafter referred to as “dU”) is considered to be an “analogous" form of pyrimidine in this invention.
Aptamer oligonucleotides may contain analogous forms of ribose or deoxyribose sugars that are
known in the art, including but not limited to 2' substituted sugars such as 2'-O-methyl-, llyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or
lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, locked nucleic acids (LNA), peptide nucleic acid
(PNA), acyclic analogs and abasic side analogs such as methyl riboside.
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Aptamers may also include intermediates in their synthesis. For example, any of the hydroxyl
groups ordinarily present may be replaced by phosphonate , phosphate groups, protected by a standard
protecting group, or ted to prepare additional linkages to additional nucleotides or substrates. The 5'
terminal OH is conventionally free but may be phosphorylated; OH substituents at the 3' terminus may also be
phosphorylated. The hydroxyls may also be derivatized to standard ting groups. One or more
phosphodiester linkages may be replaced by ative linking groups. These alternative linking groups
e, but are not limited to embodiments wherein P(O)O is replaced by P(O)S (“thioate”), P(S)S
(“dithioate”), P(O)NR 2 (“amidate”), P(O)R, P(O)OR', CO or CH 2 (“formacetal”), wherein each R or R' is
independently H or substituted or tituted alkyl (1-20C.) ally ning an ether (70*) linkage,
aryl, alkenyl, lkyl, cycloalkenyl or aralkyl.
One particular embodiment of aptamers that are useful in the present invention is based on RNA
aptamers as disclosed in US. Pat. Nos. 5,270,163 and 5,475,096, which are incorporated herein by reference.
The aforementioned patents disclose the SELEX method, which involves selection from a mixture of candidate
oligonucleotides and stepwise iterations of binding, partitioning and amplification, using the same general
selection , to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a
mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method
includes steps of contacting the mixture with a target, such as a target analyte, under conditions ble for
binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target
molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the
nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of
binding, partitioning, iating and amplifying through as many cycles as desired to yield highly specific,
high affinity nucleic acid s to the target molecule. In some embodiments, negative screening is employed
in which a plurality of aptamers are exposed to analytes or other materials likely to be found together with target
es in a sample to be analyzed, and only aptamers that do not bind are retained.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing
modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or
improved delivery characteristics. Examples of such modifications e chemical tutions at the ribose
and/or phosphate and/or base positions. In some ments, two or more aptamers are joined to form a
single, multivalent aptamer le. Multivalent aptamer molecules can comprise multiple copies of an
aptamer, each copy targeting the same analyte, two or more ent aptamers targeting different analytes, or
ations of these.
Aptamers can be used as diagnostic and stic reagents, as reagents for the discovery of novel
therapeutics, as reagents for monitoring drug se in individuals, and as reagents for the ery of novel
therapeutic targets. rs can be used to detect, modify the function of, or interfere with or inhibit the
function of one or more target analytes. The term “analytes" as used herein includes without limitation drugs,
prodrugs, pharmaceutical agents, drug metabolites, biomarkers such as expressed proteins and cell markers,
antibodies, serum proteins, cholesterol and other lites, electrolytes, metal ions, polysaccharides, nucleic
acids, biological analytes, biomarkers, genes, proteins, hormones, or any combination thereof Analytes can be
combinations of polypeptides, glycoproteins, polysaccharides, lipids, and nucleic acids. Aptamers can t
the function of gene products by any one of, but not limited to only, the following mechanisms: (i) modulating
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the affinity of a protein-protein interaction; (ii) ting the expression of a protein on a transcriptional level;
(iii) ting the expression of a protein on a post-transcriptional level; (iv) modulating the activity of a
protein; and (v) modulating the location of a protein. The precise mechanism of action of peptide aptamers can
be determined by biochemical and c means to ain their specific function in the context of their
interaction with other genes, and gene products.
rs can be used to detect an analyte in any of the detection schemes described herein. In one
embodiment, apatamers are covalently or non-covalently coupled to a substrate. miting examples of
substrates to which aptamers may be coupled include microarrays, microbeads, pipette tips, sample transfer
devices, es, capillary or other tubes, reaction chambers, or any other suitable format ible with the
subject detection system. Biochip microarray production can employ various semiconductor fabrication
techniques, such as solid phase chemistry, combinatorial chemistry, molecular biology, and robotics. One
s typically used is a photolithographic manufacturing process for producing microarrays with millions of
probes on a single chip. Alternatively, if the probes are pre-synthesized, they can be attached to an array surface
using techniques such as channel pumping, et" spotting, template-stamping, or photocrosslinking.
An exemplary ithographic process begins by coating a quartz wafer with a light-sensitive chemical
compound to prevent coupling between the quartz wafer and the first nucleotide of the DNA probe being
d. A lithographic mask is used to either inhibit or permit the transmission of light onto specific ons
of the wafer surface. The surface is then contacted with a solution which may contain adenine, thymine,
cytosine, or guanine, and coupling occurs only in those regions on the glass that have been deprotected through
illumination. The coupled nucleotide bears a light-sensitive protecting group, allowing the cycle can be
repeated. In this manner, the microarray is created as the probes are synthesized via repeated cycles of
deprotection and coupling. The process may be repeated until the probes reach their full length. Commercially
available arrays are typically manufactured at a y of over 1.3 million unique features per array.
ing on the demands of the experiment and the number of probes required per array, each wafer, can be
cut into tens or hundreds of individual arrays.
Other methods may be used to produce the biochip. The biochip may be a Langmuir-Bodgett film,
onalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold, , membrane, nylon,
PVP, or any other material known in the art that is capable of having functional groups such as amino, carboxyl,
Alder reactants, thiol or hydroxyl incorporated on its surface. These groups may then be covalently
attached to crosslinking agents, so that the subsequent attachment of the nucleic acid ligands and their
interaction with target molecules will occur in solution without hindrance from the biochip. Typical
crosslinking groups include ethylene glycol oligomer, diamines, and amino acids. Alternatively, aptamers may
be coupled to an array using enzymatic procedures, such as described in U820100240544.
In some embodiments, aptamers are coupled to the surface of a microbead. eads useful in
coupling to oligonucleotides are known in the art, and include magnetic, magnetizable, and non-magnetic beads.
Microbeads can be labeled with l, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dyes to facilitate coding of the beads and
identification of an aptamer joined thereto. Coding of microbeads can be used to guish at least 10, 50, 100,
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 5000, or more different microbeads in a single assay,
each microbead corresponding to a ent aptamer with specificity for a different analyte.
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In some embodiments, reagents are coupled to the surface of a reaction chamber, such as a tip. For
example, the interior surface of a tip may be coated with an aptamer specific for a single analyte. Alternatively,
the interior surface of a tip may be coated with two or more different aptamers specific for different analytes.
When two or more different aptamers are coupled to the same interior tip surface, each of the different aptamers
may be coupled at different known ons, such as forming distinct ordered rings or bands at ent
positions along the axis of a tip. In this case, multiple different analytes may be analyzed in the same sample by
drawing a sample up a tip and ng analytes contained in the sample to bind with the aptamers coated at
successive positions along the tip. Binding events can then be visualized as described herein, with the location
of each band in a banding pattern corresponding to a specific known analyte.
In some embodiments, binding of one or more aptamers to one or more target analytes is detected
using an optical feature. In some embodiments, the optical feature is fluorescence. In some ments, a
sample containing es to be analyzed is treated with a labeling compound to ate the analytes with a
fluorescent tag. Binding can then be measured by fluorescence to detect ce and optionally ty of one
or more analytes, such as illustrated in Figure 136 in combination with aptamers d to an array, and in
Figure 137 in combination with aptamers coupled to coded beads. In some embodiments, the sample is treated
with a labeling compound to conjugate the analytes with a linker. Upon binding the linker is functionalized with
a fluorescent tag and the positive event is measured by fluorescence. In some ments, the analyte binding
domain of an aptamer is partially hybridized to a complentary probe that is fluorescently labeled. Upon binding
to the analyte, the complementary probe is ed, which s in an optically measurable decrease in
fluorescent signal. In some embodiments, an aptamer is fluorescently labeled and is partially hybridized to a
complementary probe labeled with a quencher that is in proximity to the fluorescent label. Upon binding to the
analyte, the mentary probe is released resulting in a measurable increase in fluorescence of the label
conjugated to the aptamer. In some embodiments, the aptamer is partially hybridized to a complementary probe,
which ization occludes a domain containing a secondary structure. Upon binding to the analyte, the
complementary probe is released, and the secondary structure is made ble to an intercalating dye used to
produce a measurable signal. Labels useful in the detection of binding between an apatamer and an analyte in a
binding pair can include, for example, fluorescein, tetramethylrhodamine, Texas Red, or any other fluorescent
le known in the art. The level of label detected at each address on the biochip will then vary with the
amount of target analyte in the mixture being d.
In some embodiments, the displaced complementary probe is conjugated to one member of an
affinity pair, such as biotin. A detectable molecule is then conjugated to the other member of the affinity pair,
for example avidin. After the test mixture is applied to the biochip, the conjugated able molecule is added.
The amount of detectable molecule at each site on the biochip will vary inversely with the amount of target
molecule present in the test mixture. In another embodiment, the displaced complementary probe will be biotin
labeled, and can be detected by addition of fluorescently d avidin; the avidin itself will then be linked to
another fluorescently labeled, -conjugated compound. The biotin group on the displaced oligonucleotide
can also be used to bind an avidin-linked reporter enzyme; the enzyme will then catalyze a reaction leading to
the deposition of a detectable compound. Alternatively, the er enzyme will catalyze the tion of an
insoluble product that will locally quench the cence of an intrinsically-fluorescent biochip. In another
embodiment of the displacement assay, the displaced complementary probe will be labeled with an
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immunologically-detectable probe, such as digoxigenin. The displaced complementary probe will then be
bound by a first set of antibodies that specifically recognize the probe. These first antibodies will then be
recognized and bound by a second set of antibodies that are fluorescently labeled or ated to a reporter
enzyme. Many variations on these examples are known or will now occur to those skilled in the art. Assays
analogous to “double-sandwich" ELISAs can also be set up using combinations of antibodies and aptamers as
receptors. For example, a capture e can be onalized with an aptamer and the detection reagent can
be an enzyme-labeled antibody. Conversely, the antibody can be on the capture surface and the detection
reagent a labeled aptamer.
In some embodiments, a sample containing an anlalyte to be analyzed is dispersed into a three-
dimensional hydrogel matrix. The hydrogel matrix can be activated to covalenly trap proteins and small
molecules. After a wash of the excess and unbound , fluorescently labeled aptamers can be introduced
for the detection of the specific analytes present, such as illustrated in Figure 138. In some embodiments, the
dimensional hydrogel matrix is divided in small subsets or microwells to which a single aptamer can be
added to undergo a c analysis of the analyte present. In some embodiments, aptamers are labeled with a
set of coded quantum dots or fluorescent tags corresponding to a unique signature. In some ments,
labeled aptamers are added to the three-dimensional matrix simultaneously with the sample.
In some embodiments, an aptamer is used instead of an antibody in an ELISA assay. In general, a
sample is exposed to a e and specifically or non-specifically coupled thereto. In a sandwich ELISA, an
analyte is specifically coupled to a surface by binding to first antibody that is coupled to the surface. In a typical
ELISA, the analyte, whether bound specifically or non-specifically, is then detected by binding to a second
antibody carrying a label. In an aptamer ELISA, the first antibody, second antibody, or both are replaced with
aptamers specific for an analyte.
Imaging Analysis of Samples and Assay Reaction Products
] In some embodiments of the invention, analysis of sample and the assay on products can be
performed using l g. The assay cuvettes can be aligned for measurement and scanned or imaged in a
single operation. In the instrumented system of the ion this is ed automatically by mechanical
components. Assay cuvettes are located at defined locations in a cartridge and moved to the scanner maintaining
the same orientation and spacing. The graph shown in Figure 92 corresponds to the green l response over
the width of the cuvette. As shown, the edges of the cuvettes are well-defined, as is the location corresponding
to the middle of the cuvette.
The images obtained by ng or imaging can be a two-dimensional array of pixels, where each
pixel ses a plurality of intensity values corresponding to a distinct detection spectral region (e. g., red,
blue, green). The images can be interpreted by line-scans, which may correspond to a horizontal portion of a
tip. If the tip is circular-shaped, then an effective absorbance can be determined by deconvoluting the line-scan
over an appropriate function. Example ons include parabolic functions, and functions for circles. In some
embodiments, the images can be data-averaged over multiple images taken of a tip or a sample over a range of
physical locations.
In an ment, a sensor is provided to locate an assay unit relative to a detector when an assay
is detected.
[Annotation] eaa
As shown in Figure 61 and Figure 62, bromophenol blue solutions were aspirated into a set of
conical tips and imaged with front face illumination (light source and detector on the same side of the object).
Small volumes (5 uL) of serial dilutions of a 0.78 mg/mL on were used with the highest concentration at
the top of the image. In Figure 61, tips on the left have the sample located at the widest location in the conical
tip whereas tips on the right have the sample at the narrowest. The image in Figure 61 was taken using a
scanning optical system.
Figure 62 shows tips that were imaged using a back-lit configuration (light source and detector on
opposite sides of the imaged object). The back-lit configuration can be preferred because of the higher image
quality.
As shown in Figure 61 and Figure 62, the effective optical path length of a colored solution can be
varied by changing tip design. In ular, the pathlength can be varied within a single tip to increase
sensitivity of measurement of light absorbance (long pathlength) or to se the dynamic range of the
measurement. The ngth can be changed, for e, by changing the diameter of the tip.
An additional feature of the tip design can be that it enables assays to be read with a very small
volume of assay reaction product ing a very small volume of sample. Typically, assay reaction mixtures
are incubated in a narrow part of a tip which provides a high ratio of liquid/air surface area to volume, thus
minimizing evaporation. The small volume can then be moved to a wide part of the tip for measurement of the
colored product thus zing the optical pathlength available (and thereby increasing the absorbance of
light) for a given reaction mixture volume.
For e in the table below, we compare g an assay reaction mixture of 10 uL in which a
1 uL sample is diluted 1:10. In the tips of the current invention, incubation of an assay mixture can be achieved
in a 13 mm length of tip region having a diameter of 1 mm then be moved to a 3 mm diameter region for color
measurement. In comparison with using a microtiter plate of standard dimensions (typical of 384-well plates) to
incubate and read the same assay, the area of liquid surface exposed to air (allowing evaporation) is about 5
times less and the l pathlength is about twice as great.
Lengthofli uid column‘1 3 m
Tip er 3.00-Pathlength for reading
[Annotation] eaa
Length of liquid column 1.41 Pathlength
] Optimizing opticalpath length
Spectroscopic ements of colored solutes are traditionally measured by recording the
fraction of light transmitted h a cuvette at the absorbance wavelength maximum. The data are then
ormed to give Absorbance (A) or optical density (OD) values. According to Beer’s law, A(7tmax) =
8M*l*Concentration where SM is the molar extinction t (L/Mole.cm), l is the optical pathlength (cm) and
Concentration is in molar units. OD = A for l = 1. This is done to provide a measure, A, which is directly
tional to solute concentration.
There are two significant limitations of absorbance measurements for assaying solute
concentrations. At low concentrations, the change in transmission is small and therefore imprecise because of
variations in the background (or blank) transmission. At high trations transmission is very low (for
example at A = 3, the transmitted light is l/1000 th of the input light. Any “stray" light or other forms of signal
noise have a significant effect on the ement and the response to concentration becomes non-linear and
imprecise. Typically, absorbance measurements are regarded as precise and accurate over a range from about
0.1 to about 2.0 (a 20-fold range).
The method of the present invention mes these problems to a significant degree by enabling
facile measurements of color over a very wide dynamic range (up to lOOO-fold):
1. At different pathlengths: low concentrations can be measured at long pathlengths and
high concentrations at short pathlengths.
2. In different color channels: low concentrations can be measured in the best matching
color channel and high concentrations in color ls mismatched to the color.
This is illustrated by the data shown in Figure 79. Bromphenol blue solutions serially diluted from
a 5 mg/mL stock were ed using the three-color method in tips at two locations, one with a maximum
pathlength (also “path lengt " herein) of about 5
mm (“wide"), the other of about 1 mm (“narrow"). Signals in
the three color ls were normalized to their highest and lowest levels as shown in the graph below. An
algorithm to lly extract the concentration of the analyte (bromphenol blue) was set up as follows:
1. For normalized signals in the range 10% maximum < signal < 90% maximum, compute a
value concentration = a + b*Log(signal) + c*(Log(signal))AZ where a b and c are arbitrary constants. This
operation was performed for each color at both pathlengths.
] 2. Using a well-known optimization e (for example “Solver" in oft Excel),
compute the best-fit values of a, b and c for all colors and pathlengths.
3. Average the computed concentration values for all colors and both pathlengths.
As shown in Figure 80, the method yielded accurate results across a 1000-fold concentration
range. When the algorithm was used to compute concentration values for replicate measurements (N = 3), the
average CV was 3.5 %.
[Annotation] eaa
Measurements can be made at various pathlengths. In some cases, pathlengths are at least partially
dependent on container (e. g., cuvette, tip, vial) geometry. The container geometry and/or features in the
container, such as scattering features, may affect the optical path and path length in the container.
Mum-color analysis
Scanners and cameras have detectors that can measure a plurality of different colors channel
ion spectrum s (e. g., red, green, and blue). Because the al width of each of these channels is
wide and color chemistries produce colored ts with wide band widths, colored reaction products can be
ed using a plurality of channel detection spectrums. For e, Figure 71 shows the response of red
(squares), green (diamonds), and blue (triangles) detection channel spectrums as a function of analyte
concentration. The s produced by each detector correspond to light intensity within each detection
spectrum and are typically expressed as a number from 0 to 255. When white light is transmitted through a
circular section cuvette containing a colored solute as shown above, light is absorbed and the light intensity
reduced so that the detector responses change.
For example, when henol blue dissolved in alkaline buffer at concentrations g from 0
to 5 mg/mL and scanned at the on ted “C3" in Figure 62, signals shown in Figure 66, which are the
detector responses averaged over a zone corresponding to seven pixels along the length of the cuvette. The
signals were ed on an Epson backlit scanner. Figure 66 shows the three color responses for a set of 11
es containing 2-fold serial dilutions of a 5 mg/mL bromophenol blue solution and a “blank" solution
(arranged left to right on the . The image of the scanned tips is shown in Figure 67. The signal in each
channel corresponding to the solution is reduced to an extent related to the optical path. Accordingly, the
maximum change in signal is seen at the center of the cuvette. When signals in the central region of the cuvette
were averaged (over the zone shown by the small rectangles for the fourth cuvette from the left) and plotted
t the bromophenol blue concentration, the dose-responses shown Figure 68 were observed. In each color
“channel" the signal declined smoothly with concentration. The green signal changed most and the blue signal
least. Corresponding optical densities measured in an M5 spectrometer ular Devices) at the wavelength
of maximal absorbance (e. g., 589 nm) are also shown. At the highest concentrations, the spectrophotometer
response becomes not linear and changes very little with concentration. A similar effect was noted in the
scanner green and red channel responses. The blue channel response in contrast, is very slight until the highest
trations.
According to Beer’s law, absorbance of a solution is equal to 8M*Concentration*pathlength.
ance is defined as LoglO(Transmission/Blank Transmission), where blank transmission is that
corresponding to that for the solvent. Strictly Beer’s law s to a parallel beam of monochromatic light (in
practice a band width of a few nm) passing normally through a rectangular cuvette. Spectrophotometers
respond linearly to concentration up to Absorbance values about 1.5. At higher absorbance, instrument response
becomes non-linear due to “stray light" and other effects. Optical density is defined as absorbance for a one cm
optical pathlength.
When the color signal data from the above experiment was ormed according to an expression
that linearizes optical transmission so as to obtain an absorbance value proportional to concentration in
conventional spectrophotometry (-Log(signa1/blank ), the graph shown in Figure 69 was obtained for the
green (squares) and red nds) channels.
[Annotation] eaa
The green channel data followed Beer’s law but the red channel data did not reaching a plateau
level at for a sample having about 2 mg/mL in a fashion similar to that of the OD response of the
spectrophotometer.
Improved assay utilization by three-color analysis and optimization ofopticalpath length
Assay results from on setups that would otherwise provide uninterpretable data can be
salvaged using the present invention. The present invention allows for increased dynamic range and sensitivity
of assays by the combination of optical pathlength optimization and color analysis. The ity to
salvage data plagued by reduced c range is a major problem in assay management, especially in the
context of samples being evaluated for diagnostic or therapy management es is that assays have a limited
dynamic range or limited range of analyte values that can be reported with good confidence. There are two
main s why an assay result may not be available fiom laboratory-based assay s or from distributed
test situations. Namely the analyte value is too high or too low to be reported. This may in some circumstances
be rectified in clinical laboratories by re-analyzing a n of a retained sample using a different dilution. In
distributed g typically there is no recourse but to recall the patient, obtain a new sample and use a different
(laboratory) method. This is because assay systems use fixed ols and fixed levels of sample on. In
either situation, it is very inconvenient and expensive to rectify the problem. Moreover, valuable information
pertinent to proper diagnosis and/or therapy management may be lost with resultant harm to the patient.
In the system of the present invention, these problems are eliminated by monitoring assays during
their execution, recognizing any problem and modifying either the optical pathlength used to measure the assay
product or making use of the different ivity levels of the three color channels to the assay color and in turn
to the analyte sensitivity.
Specifically when the assay reaction product is ed if the measured signal is either too high
or too low, the system can respond by:
1. making the measurement with a different pathlength (moving the optical cuvette relative to the
optical system such that the pathlength is either bigger or smaller). This can be performed by (a) making a
measurement at a standard, first on, (b) reporting the result to the software ng the assay (in
instrument and/or on a remote server), (c) recognizing a m condition, and (d) modifying the read position
and making a second measurement; and/or
2. izing a more or less sensitive color channel in signal analysis. This can be implemented
automatically by le assay analysis algorithms.
Color Calibration
The signal responses can be calibrated to allow for computation of the concentration of the colored
species from imaging data. To obtain a data transform predictive of the concentration of the colored solute, the
following procedure can be used. In other embodiments, other methods may also be used.
] I. For each channel for all concentrations, the transform -Log(signal/blank signal) was computed
and designated “A".
2. For all concentrations, a further transform (“C") was computed as a*A + b*AA2 + c*AA3
(initially values for a, b and c were set at arbitrary values).
3. For all concentrations, C values for the three color channels were summed and designated
Cestimate.
[Annotation] eaa
4. The sum of square differences between the target (known) tration and Cestimate was
computed over all concentrations.
5. Values of a, b and c ters for all channels were derived by a well-known algorithm
which minimized the sum of the square differences.
The results shown in Figure 70 trates accurate calibration of the r response over the
entire concentration range.
] Other automated calibration thms have been developed and found to be equally effective.
For example, the following is an example of calibration for a terol assay performed in a reaction tip.
The ed signal is decomposed into Red (R), Green (G), and Blue (B) color channels.
Calibration equations are computed to optimize the accuracy, ion, and dynamic range according to assay
design requirements.
In this assay example, only Red and Green channels are utilized to compute concentration. These
two signals are transformed to compute an intermediate variable (F) as follows:
umwm FEfifi+pr+pr2+ppR+pra
where p,- are ation parameters.
Finally, the signal F is used to compute the concentration (C) via a linear transformation:
[mam C=G¥p9/,p7
where C is the calculated concentration, and p6 and [17 are calibration parameters, in this case,
representing the intercept and slope parameters of a linear relationship, respectively.
When the same approach was followed for a large set of assays for a variety of analytes which
ed colored products ng the entire visible spectrum (kmax from 400 -700 nm), comparable results
were obtained.
In conventional transmission spectrophotometric measurements, a “blank" value is used to
normalize the measurement. Method (1) Blanks are lly constructed by measuring a sample that is
equivalent to the sample but does not have any of the component to be measured. The measurement is typically
made in the same cuvette as that which will be used for the sample or an optically equivalent cuvette. Thus in a
spectrophotometric assay, one would combine all the reagents in the same concentrations using the same
protocol substituting a zero analyte solution for the sample. Method (2) uses a two step process making
measurements t an absolute reference such as air (which will never vary in absorbance) and measuring
both sample and blank against the absolute reference. The sample absorbance is then calculated by subtraction
of the blank value from that of the sample. Method (3) is to collect spectra of the sample or assay reaction
product and reference the ed absorbance (or ission) at an optimal wavelength (usually that for
maximum absorbance for the measured species) t the absorbance at a wavelength where the species to be
measured is known to have zero absorbance. The absorbance is the difference between those recorded at the
two wavelengths.
Digital imaging and three-color analysis can be employed, but in some embodiments can be
modified according to the l (pixilated) character of the assay signal. Namely:
[Annotation] eaa
1. For each pixel in the image and for each color a white standard is imaged and the intensities of
the signal adjusted to a value corresponding to no absorbance. This can be done by the following exemplary
procedure:
a. ing the intensity of the light source
b. adjusting the sensitivity of the detector (preferred), or
c. software adjustment (not preferred by itself)
A preferred approach is a combination of (b) and (c) above. First, adjust the detector in the analog
realm, and then fine tune the result in the digital realm.
For the analog adjustment, the gain and offset of the iers between the light sensors and the
-to-digital section are adjusted to ensure maximum resolution of the digitization. The lower end of the
light range of interest will be set to zero and the high end of the range will be set to just below saturation of the
sensor.
Subsequently, the images may be fine-tuned in the digital domain. A preferred approach,
specifically, would be to use what is called the mage calibration" for an m x n image. The mechanism is
to first collect a black image by blocking all light to the detector. We’ll call this image BLACK[m,n]. A second
calibration image is recorded consisting of light at the maximum end of the sensitivity range. We’ll call this
image WHITE[m,n]. Thus a corrected image a[m,n] could be constructed, pixel-wise, as:
c[m, n] — BLA CK[m, 11]
a[m, 11]=m
Note that this l correction does not improve the dynamic range of the digitized data, but
adjusts the values so that the full white and black references are consistent.
2. An image of a physical blank in a tip can be used as a by-pixel and color by color blank.
The blank can be:
a. Air;
b. Water;
c. Blank assay reaction product (no analyte);
d. Sample blank (no assay reagents); or
e. Some combination of the above;
] 3. The signal from a color channel where there is a zero or weak response can be used to
normalize signals from the other channels.
] A further method of controlling and normalizing the optics is to image a set of physical (stable)
standards before or during an assay. For example, an array of d dyes (shown in Figure 104) can be made
corresponding to a set of standard colors with standard intensities (similar to rd color “wheels" used to
calibrate cameras and scanners).
] Such standards may be measured using reflectance from an opaque surface or (preferred) by
transmission through a clear film.
Depending on the ity of the optics, calibration and normalization of the optics may be (1) a
one-time exercise, (2) performed at regular intervals or (3) performed for each assay.
Calibrating a digital imager range
[Annotation] eaa
In some embodiments, methods may be provided for calibrating a digital imager used for imaging
optical ies.
] In g the optical density of an analyte, it may be desirable to make use of as much of the
dynamic range of the imager as possible. Under normal use, the setup may se a relatively homogenous
illuminated white background, the imager and the analyte to be tested in a transparent cuvette between them.
Operationally, the test may comprise placing the cuvette between the imager and the white backlight source and
measure the amount of light absorbed by the analyte in the cuvette. To maximize the full c range of the
sensor, the background may be sensed as the maximum intensity measurable. It may be desirable to take care to
not saturate the sensor because then information could be lost since when the sensor is saturated, and attenuation
may not be tly measured. The system may be red to efficiently maximize the measured values of
the backlight while minimizing number of saturated pixels.
The illuminated ound may emit white light of equal intensity over its entire surface. The
light output may vary somewhat, producing a normal distribution of pixel intensities as detected by the imager.
This is illustrated by the curves shown in Figure 128. For this example, the sensor may return a value from 0 to
256 from each pixel as an tor of the amount of light it receives. Each pixel may saturate at a value of 256.
That is, regardless of further increasing of light intensity or sensor sensitivity, only a value of 256 may be
recorded. Series 1 in Figure 128, the dotted line, shows where the light is too intense, cutting off the normal
curve. Series 3, the dashed line, shows that all pixels are correctly reading intensity, but that the imager
ivity is lower than it might be for maximum dynamic range. The majority of the pixels are at a value of
less than 200. Series 2 represents the desired gs, where the mean of the distribution is as high as possible,
but that a sufficiently small number of pixels are saturated.
In one embodiment, the ity of the backlight may be held constant while the imager’s settings
may be adjusted. For the purpose of imager ivity, two controls may be used: exposure time and gain.
re time may be the amount of time that the sensor pixels are permitted to collect photons before the value
is read out. For a given amount of light, the readout value may be larger when the exposure time is made longer.
This control may be the “coarse" control for the application. Gain may be the control adjusting the amount of
amplification applied to the sensor . Increasing gain may increase the value of the signal from the sensor.
Gain may be the “fine" control.
An exemplary procedure for setting the imager’s sensitivity parameters may include one or more
of the following steps:
1. Set exposure time to value known to be below saturation. Set gain to highest usable value.
2. Binary search starting upwards adjust exposure time to find the setting where not all of the
pixels in the region of interest of the image are saturated. This may be detected by observing
the point at which the mean pixel value becomes less than 256.
3. Back gain down incrementally until there are sufficiently few pixels that are at the saturation
limit. The number of pixels at an acceptable level will be ined by the shape of the
distribution. Wide standard deviation will increase the number of pixels permitted to be
saturated.
Next, the white balance may be corrected. There are three groups of sensors in a digital imager.
Members of each group collect light of a different wavelength, red, green or blue. When detecting white light,
the sensors would preferably see equal values or red, green and blue. The white balance control adjusts the
[Annotation] eaa
relative gains of the red and blue channel. Since the light coming from the backlight is defined as white, the
procedure would be to simply adjust the white balance until the channels read the same values. In ce, the
green channel is typically left unadjusted, and the red and blue ls are changed in opposite directions to
each other as the control is changed. However, in other embodiments, another channel, such as the red channel
or blue channel may be left unadjusted while the other two channels may be changed.
Finally, the images may be uned in the digital domain. A preferable approach, specifically,
would be to use what is called the mage calibration" for an m X n image, as previously described.
Assays making a variety of colored products have been analyzed in the subject invention. Colors
from those with low wavelength absorption maxima (yellow) to high wavelength maxima (blue) have been
successfully measured. Wavelength maxima for some representative assays were: 405, 450, 500, 510, 540,
570, 612 and 620 nm demonstrating the ability to read color over the entire visible spectrum.
Colors may be quantified using average data for many pixels (typically about 1000). A parameter
(f) which produces a good fit (e. g., st R2) to the dose-response data may be selected. The parameter may
be first fitted to the form a1 Ib1*R i c1"‘R2 i b2*G i c2"‘G2 Ib3 *B i c2"‘B2 where a, b, c are constants and R, G and
B are color intensity values for red, green and blue channels tively. The parameter f may then be derived
by forcing it to have a m value of 1 and a minimum value of 0. Parameter f is related to transmission of
light through the colored reaction product. As would be expected, fmay be closely related to the parameter
optical density (OD) used in spectrophotometry to quantify an absorbing species. When 1 - f ed by 3-
color imaging is plotted t OD measured at the absorption maximum for the same assay on products
in a microtiterplate in a spectrophotometer, it may be observed that 1 , f is ially ly related to OD. In
Figure 129, such data for five assays is presented. OD may be normalized as “relative OD" = (OD , OD
min)/(ODmax , OD min). In some cases, there is a somewhat curved relationship but the correlation coefficient
(R) is usually > 0.99.
The parameter fmay be used to calibrate assays measured by r image is. When
plotted against concentration of the analyte, a smooth calibration relationship may be shown in Figure 130 for a
representative cholesterol assay. An equation of the form concentration = a + b*f + c"‘f2 (where a, b and c are
constants) relating concentration to f is derived and as shown in Figure 130, the calculated concentration is
essentially identical to that of the “nomina " (expected, desired) value ssion line slope close to 1.0,
intercept close to 0.0 and R2 = 0.998. Also shown in Figure 130 are graphs of assay accuracy and precision.
Accuracy is close to 100 % (mean 100.2 %) and imprecision (represented by CV %) is low (less than 10 %,
average CV 3.9 %).
Simultaneous imaging ys
As shown in Figure 56, Figure 57, Figure 58, Figure 59, and Figure 60, several assay elements
(tips, wells, blots) can be imaged in parallel. In general, the elements can be placed at known locations in a
cartridge or mounted on a subsystem of the ment, so that a particular element can be associated with a
particular assay. Even if the elements are not perfectly oriented or located, image is can be used to rectify
any such miss-positioning by locating features of the assay elements.
Commercially available assays for albumin (Figure 56) and cholesterol (Figure 57) were used
according to the cturer’s directions. A series of analyte concentrations in the range of clinical interest
was measured using a series of calibrators in which the analyte concentration was reduced two-fold from the
[Annotation] eaa
highest concentration. In Figure 56 and Figure 57, analyte concentration was highest on the right and the
furthest left tip corresponded to zero analyte. The volume of assay reaction mixture aspirated into the tips was
uL.
Figure 58, Figure 59, and Figure 60 show wells that can be imaged in parallel. A set of shallow
hemispherical wells was made by machining a block of white opaque plastic. Three commercially ble
color forming assays were performed in these wells and reaction products imaged. As above, the wells to the far
right have the highest analyte concentration and each adjacent well has a two-fold lower concentration except
the left-most well which has zero analyte. Seven uL of assay reaction product were introduced into each well.
Reaction products can also be imaged after blotting them onto porous membranes or paper and
imaging once the liquid has soaked into the medium. It is also possible to use any of a variety of assay
chemistries impregnated into paper or membranes and to image the resulting reaction products ing
addition of sample.
Analyzing ity
Turbidimetry is performed by measuring the ion in the intensity of the incident light after it
passes h the sample being measured. This technique is used where the result of the assay is a dispersed
precipitate that increases the opacity of the liquid.
Turbidimetry can be measured in latex agglutination assays. As a model of latex agglutination
assay responses, polystyrene latex particles (1 um diameter) were dispersed in buffer at the given (w/v)
concentrations and subject to three-color image analysis. As can be seen in Figure 72, a good se was
found in all three channels and could be used to measure the latex particle concentration and agglutination of
latex.
Analyzing Agglutination
Similarly to turbidity analysis, the system can be used to measure agglutination, lutination,
and the inhibition thereof.
The system can be used to perform blood typing by red blood cell agglutination. Blood was
diluted and mixed with blood typing reagents (anti-A, anti-B, anti-D) from a commercial typing kit. As shown
below for a B+ blood, the appropriate ination responses can easily be seen when the mixtures are imaged.
er, when the images shown in Figure 77 were scanned along the vertical axis of the tips, a tative
measure of agglutination could be obtained by measuring the variance of the three-color s, as shown in
Figure 78. Greater variance indicated agglutination and can be ed in each color channel. It is evident that
the method can be used to measure the extent of such agglutination reactions.
Shape Recognition
Images can be analyzed for shape recognition. Shape ition can be performed at normal
magnification and at very high magnification. Under high magnification image analysis may be used to
recognize the size and shape of cells. These techniques are commonly used in cell counting to determine relative
concentrations of red blood cells, white blood cells and ets. Under normal ication, shape
recognition is used to observe the state of the sample. Bubble and other defect recognition s are used to
ensure that measured liquid s are aspirated and dispensed correctly.
Analyzing samples on solidplzase substrates
[Annotation] eaa
Digital imaging with face illumination can also be used to read out assay responses on solid
phase substrates as shown in Figure 76. Solutions of potassium chloride (0, 2, 4 and 8 mM) were added to
ReflotronTM potassium assay strips (Boehringer-Mannheim/Roche) designed for use in a reflectance assay
system.
Analyzing sample quality
Certain sample characteristics can render assay results invalid. For example, hemolysis causes
ium ions to leak from red cells into plasma g the measured plasma or serum potassium ion
concentrations to be falsely high. rly, icteria and lipemia can interfere with several color-forming
chemistries by altering the measured absorbances. In the t invention, we can detect and fy such
interfering substances using image analysis. Assays which would give false results can then be either (1)
eliminated from the list of s delivered by the analytical system or (2) optical signals can be corrected to
account for the measured level of interferent. An image of different types of serum samples is shown in Figure
99 (from left to right: Hemolyzed, Lipemic, Icteric (yellow) and “norma ").
Digital data analysis
Conventional methods for data generation and calibration in assay methods which generate and/or
change color typically measure an analog signal enting the change in absorbance characteristics of an
assay mixture generated by mixing a sample with reagents. Some portion of the on mixture is illuminated
and the light transmitted through or ted from that portion impinges on a detector and evaluated as an
analog signal. The quality of the assay as determined by the volume and quality of the sample, sample
sing, assembly of the assay into the assay mixture and of the physical element used to present the mixture
to the optical system rely on an assumed quality of the physical system used.
In the present invention, we can image (1) the sample, (2) sample processing processes, and (3) the
assay mixture and collect the data as a set of one or more digital images. Each pixel in the image of the assay
mixture represents a very small fraction of the total but by averaging the 3-color signal from many pixels, we
collect an assay signal at least as good as that obtained by conventional analog s. Where however,
tional methods lose information by averaging, the present invention both aggregates the information and
retains the detail lost by conventional methods. In this context, color-based assays include assays for:
Metabolites, Electrolytes, Enzymes, Biomarkers (using immunoassay), Drugs (using immunoassay), and
c acid targets (using “LAMP" technology). The same principles can be applied to assays using
fluorescence and/or luminescence.
Volume confirmation and correction
The volume of a sample, or any other material, such as a liquid or a solid, can be determined
optically. This can be performed by imaging a ner whose internal dimensions are known and
mathematically determining sample volume from observed segment of the container occupied. Solid
measurements are primarily used to measure solids that are centrifuged down. The most common case is
reading the volume of centrifuged red blood cells to determining hematocrit level. Examples 6-11 and 16
describe the use of imaging analysis to calculate sample volumes and other ements. This can allow for
ed assay results. For example, if the target volume to be used is 10 uL and the technology of the
invention ines that the actual volume is 8 uL, the assay system can correct the results for the volume (in
[Annotation] eaa
this example, the concentration of analytes calculated on the ption of a 10 uL sample would be
multiplied by 10/8).
Knowledge of actual sample and reagent volumes can be performed by g the sample and
reagents and can be used to correct the calculations used to detect and/or quantify analytes in the sample.
As shown in many examples above, the use of imaging allows samples and assay mixtures to be
evaluated for quality and assay se. Additionally, imaging of ‘tips” used as reaction vessels and sample
acquisition methods enables (l) the accurate and e measurement of sample and reagent volumes and (2)
the use of such data to correct any inaccuracies and or imprecision in assay results due to volume errors. To
achieve this, tips can have accurately and precisely known geometry (as is the case for tips made by injection
molding). ate measurements of tips using imaging has demonstrated that their dimensions are precise to
better than about 1 %. It is thus possible to measure the volume of liquid samples and reagents in such tips with
corresponding precision. If the pipetting of samples and reagents is less te and precise, correction of
results knowing the actual volumes (by image measurement) is possible.
For e, consider an assay in which the response is directly proportional to analyte
concentration (as is true for many of the assays discussed herein). A sample volume error of 10 % would lead to
an error of 10 % in the value reported by the analytical system. If however, the inaccurately dispensed sample
volume is measured accurately (say to within 2 % of the actual value), the system se can be corrected so
as to reduce the error from 10 % to 2 %. Corresponding corrections can be made for volume errors in reagent
volumes. The correction thm can depend on the response of the assay system to volume or knowledge of
each assay component (sample, reagents), but this information can easily be determined during assay
development and validation.
Thus, the invention provides a variety of advantages over conventional techniques. In the
generation of the “assay ", the present invention can detect physical defects in the assay cuvette, defects in
the assay mixture (bubbles and the like). Once these defects are identified (image is) the assay result can
be ed so that false results do not occur or (preferred) the effect of the defect can be ated and an
accurate assay signal ed.
In the assembly of the assay mixture, any and all defects can be detected including: incorrect
sample type (e. g. blood versus ), ect sample volume, for a blood sample, failure to separate plasma
from formed elements (red and white cells), sample factors that may compromise the quality of the assay result
(e. g., lipemia, icteria, hemolysis, presence of precipitates, or other unidentified in-homogeneities), defects in
assembly of the assay mixture (e.g., presence of s, failure to mix tely (non-uniformity of color)),
mechanisms for retrospective quality evaluation and preservation of detailed archival information, mechanisms
for measuring sample and reagent volumes (and to correct for inaccuracies and/or imprecision in such volumes).
Assessing Therapeutic Agents
In a separate embodiment, devices and s for monitoring more than one pharmacological
parameter useful for assessing efficacy and/or toxicity of a eutic agent is provided. For example, a
eutic agent can include any substances that have therapeutic utility and/or potential. Such substances
e but are not limited to biological or chemical compounds such as simple or complex organic or inorganic
molecules, peptides, proteins (e. g. antibodies) or a polynucleotides (e.g. anti-sense). A vast array of compounds
can be sized, for example polymers, such as polypeptides and polynucleotides, and synthetic organic
[Annotation] eaa
nds based on various core structures, and these can also be included as therapeutic agents. In addition,
various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. It
should be understood, gh not always itly stated that the agent is used alone or in combination with
another agent, having the same or different biological activity as the agents identified by the inventive .
The agents and methods also are intended to be combined with other therapies. For example, small molecule
drugs are often measured by mass-spectrometry which can be imprecise. ELISA (antibody-based) assays can be
much more accurate and precise.
Physiological parameters according to the present invention include t limitation parameters
such as temperature, heart rate/pulse, blood pressure, and respiratory rate. Pharmacodynamic parameters include
concentrations of biomarkers such as proteins, nucleic acids, cells, and cell markers. Biomarkers could be
indicative of disease or could be a result of the action of a drug. Pharmacokinetic (PK) parameters according to
the t invention include without tion drug and drug metabolite concentration. Identifying and
quantifying the PK parameters in real time from a sample volume is extremely desirable for proper safety and
efficacy of drugs. If the drug and metabolite concentrations are outside a desired range and/or unexpected
metabolites are generated due to an unexpected reaction to the drug, immediate action may be necessary to
ensure the safety of the patient. Similarly, if any of the pharmacodynamic (PD) ters fall outside the
desired range during a treatment regime, immediate action may have to be taken as well.
] Being able to monitor the rate of change of an e concentration or PD or PK parameters over
a period of time in a single subject, or performing trend is on the concentration, PD, or PK parameters,
whether they are concentrations of drugs or their lites, can help prevent potentially dangerous situations.
For example, if glucose were the analyte of interest, the concentration of glucose in a sample at a given time as
well as the rate of change of the glucose concentration over a given period of time could be highly useful in
predicting and avoiding, for example, hypoglycemic events. Such trend analysis has widespread beneficial
ations in drug dosing regimen. When multiple drugs and their metabolites are ned, the y to
spot a trend and take proactive measures is often desirable.
In some ments, the present invention provides a business method of assisting a clinician in
providing an dualized medical treatment. A business method can comprise post prescription monitoring of
drug therapy by monitoring trends in biomarkers over time. The business method can comprise collecting at
least one pharmacological parameter from an dual receiving a medication, said ting step is effected
by subjecting a sample of bodily fluid to reactants contained in a fluidic device, which is provided to said
individual to yield a detectable signal indicative of said at least one pharmacological parameter; and cross
referencing with the aid of a computer medical records of said individual with the at least one pharmacological
parameter of said individual, thereby assisting said clinician in providing individualized medical treatment.
The devices, systems, and methods herein allow for automatic quantification of a pharmacological
parameter of a patient as well as automatic comparison of the parameter with, for example, the patient’s l
records which may include a history of the monitored parameter, or medical records of another group of
subjects. Coupling real-time analyte monitoring with an external device which can store data as well as perform
any type of data processing or algorithm, for example, provides a device that can assist with typical patient care
which can include, for e, comparing current patient data with past patient data. Therefore, also provided
[Annotation] eaa
herein is a business method which effectively performs at least part of the monitoring of a patient that is
currently performed by medical personnel.
Optical Setup for Sample and on Product Imaging
Sample and reaction product analysis can be med using an optical setup. The optical setup
can includes a light source, an aperture, and a sensor or a detector. A schematic for an optical setup is shown in
Figure 100 and Figure 101. In some embodiments, the camera can be a Logitech C600 Webcamera, the camera
sensor can be a 1/3" 2.0 MP 1200) CMOS: (MISOC), the lens can be glass with a standard object
distance webcam lens (Lens-to-Object distance: 35mm). The light source can be a Moritex White Edge
Illuminator MEBL-Cw25 ) operating at 9.4 volts. Camera images can be taken in a sequence where l, 2,
3 4, or more tips are moved by an x-y-z stage into the optical path.
In an embodiment, the detector is a reader assembly housing a detection assembly for detecting a
signal produced by at least one assay on the device. The detection assembly may be above the device or at a
ent orientation in relation to the deVice based on, for example, the type of assay being med and the
detection mechanism being employed. The detection assembly can be moved into communication with the assay
unit or the assay unit can be moved into communication with the detection assembly.
] The sensors can be PMTs, wide range photo diodes, avalanche photodiodes, single frequency
photo diodes, image sensors, CMOS chips, and CCDs. The illumination sources can be , single color
LEDs, broad frequency light from fluorescent lamps or LEDs, LED arrays, mixtures of red, green, and blue light
sources, phosphors activated by an LED, fluorescent tubes, incandescent lights, and arc sources, such as a flash
tube.
In many instances, an l detector is provided and used as the ion device. Non-limiting
examples include a iode, photomultiplier tube (PMT), photon counting detector, che photo diode,
or charge-coupled device (CCD). In some embodiments a pin diode may be used. In some embodiments a pin
diode can be coupled to an amplifier to create a detection device with a sensitivity comparable to a PMT. Some
assays may generate scence as described herein. In some embodiments chemiluminescence is detected. In
some embodiments a detection assembly could include a plurality of fiber optic cables connected as a bundle to
a CCD detector or to a PMT array. The fiber optic bundle could be ucted of discrete fibers or of many
small fibers fused together to form a solid bundle. Such solid bundles are commercially available and easily
interfaced to CCD detectors.
A detector can also comprise a light source, such as a bulb or light emitting diode (LED). The light
source can illuminate an assay in order to detect the results. For example, the assay can be a fluorescence assay
or an absorbance assay, as are commonly used with nucleic acid assays. The detector can also comprise optics to
deliver the light source to the assay, such as a lens or fiber optics.
In some embodiments, the detection system may se non-optical detectors or s for
ing a particular parameter of a subject. Such sensors may include temperature, conductivity,
potentiometric s, and amperometric signals, for compounds that are oxidized or reduced, for example, 02,
H202, and 12, or oxidizable/reducible organic compounds.
The illumination can be back lit, front lit, and oblique (side) lit. Back lighting can be used in
general chemistry for the purpose of detecting either light absorption (colorimetry) or scattering (turbidity). The
arrangement takes two forms, a broad, evenly illuminated rear field, and a specifically shaped beam that is
[Annotation] eaa
interrupted by the subject. Front lit illumination can be used for reflectance and fluorescence excitation. In
reflectance, a subject is lit from the front by a light source are ed by observing the light reflected from the
subject. The colors absorbed produce the same information as a liquid illuminated by a back light. In
reflectance, a t can also be illuminated using oblique lighting. The use of oblique (from the side)
illumination gives the image a 3-dimensional appearance and can highlight otherwise inVisible features. A more
recent technique based on this method is Hoffmann's modulation contrast, a system found on inverted
microscopes for use in cell culture. Oblique illumination suffers from the same tions as bright field
microscopy (low contrast of many biological samples; low apparent resolution due to out of focus objects), but
may highlight otherwise invisible structures.
In fluorescence excitation, subjects can be illuminated from the front for the purpose of
fluorescence illumination. These are usually single color lights, most commonly lasers. The Confocal Laser
Scanning Microscope is a common ment of this. Oblique lighting can also be used in fluorescence
excitation. In fluorescence cytometry, the subjects are often excited at an angle, usually 90 degrees, from which
the decay photons will appear. This form of lighting enables scatter detection directly behind the subject (back
lit) as well as the fluorescence ons exiting from the side.
In some embodiments, fluorescent light is imaged at 90 degrees to the excitation beam. In Figure
102A, a photon source (S), typically a high-intensity LED, passes through a beam er (D) and a shaping
lens (Ll), producing a collimated or slowly diverging excitation beam. The excitation beam passes through a
band-pass filter (F1) and illuminates the sample, ting of a vessel (tube, cuvette, or e tip) containing a
solution with a fluorescently-labeled sample. Isotropically-emitted fluorescence is spectrally separated from
excitation light with a long- or band-pass filter (F2) appropriate to pass Stokes-shifted fluorescence. Light is
then imaged through a lens (L2) onto a digital camera (C) or other or. Fluorescence intensity is extracted
from the resulting images Via image analysis.
Images taken using the optical setup shown in Figure 102A produces single-tube images (as shown
in Figure 103A. Successive experiments show the difference in fluorescence ity from Negative and
Positive LAMP experiments using intercalating dye.
In other embodiments, transmitted light is imaged after optical filtering to remove the light at the
exciting wavelength. In Figure 102B, a photon source (S), typically a high-intensity LED, passes through a
beam diffuser (D) and a shaping lens (Ll), producing slowly ent, elliptical excitation beam. The
excitation beam passes through a band-pass filter (F1) and illuminates the samples, presented as an array of
sample vessels (tube, cuvette, or pipette tip), each containing a solution with a fluorescently-labeled sample.
pically-emitted fluorescence is spectrally separated from tion light with a long- or band-pass filter
(F2) appropriate to pass -shifted fluorescence. Light is then imaged through a camera lens (L2) onto a
digital camera (C). Fluorescence intensity is extracted from the resulting images Via image is. The
optical setup shown in Figure 103 can be used to produces array images of le tubes simultaneously (as
shown in Figure 103B).
For colorimetry, the red embodiment for sensing is backlighting the subject with white light
with the result sensed by an imaging sensor. In this case the transmissive color absorption is measured.
[Annotation] eaa
For Turbidimetry, the preferred embodiment for sensing is backlighting the subject with white
light with the result sensed by an imaging sensor. For turbidimetry, the reduction of the intensity of the
transmitted light is ed.
Luminometry utilizes no illumination method as the subject emits its own s. The emitted
light can be weak and can be detecting using an extremely sensitive sensor such as a photomultiplier tube
(PMT).
In some embodiments, imaging may occur using fluorescence, darkfield illumination, or
brightfield illumination. Such imaging can be used for cytometry or other applications. Epi-fluorescence
nation may be achieved by the use of three illumination sources of differing wavelengths. Further, two
different sources can be used simultaneously, if required. Consequently, the imaging platform can be used to
image a large variety of fluorescent dyes. The ation of illumination sources and emission optics can be
configured to achieve a plurality of spectrally ndent channels of imaging.
Darkfield illumination may be achieved by the use of a ringlight (located either above or below the
), a eld abbe condenser, a darkfield condenser with a toroidal mirror, an epi-darkfield condenser
built within a sleeve around the objective lens, or a combination of ringlight with a stage condenser equipped
with a dark stop. Fundamentally, these Optical ents create a light cone of cal re (NA)
greater than the NA of the ive being used. The choice of the illumination scheme depends upon a number
of considerations such as magnification ed, mechanical design considerations, size of the imaging sensor
etc. A ringlight based illumination scheme generally provides uniform darkfield illumination over a wider area
while at the same time providing sufficient flexibility in mechanical design of the overall system.
Brightfield illumination may be achieved by the use of a white light source along with a stage-
condenser to create Koehler illumination.
In some embodiments, an automatic filter wheel may be employed. The tic filter wheel
allows control of the imaging optical path to enable imaging of multiple fluorophores on the same field of view.
In some embodiments, image based auto-focusing may take place. An image-based algorithm may
be used to control the z-position (e. g., vertical position) of an objective (i.e., its distance from the ) to
achieve auto-focusing. Briefly, a small image (for example, 128x128 pixels) is captured at a fast rate using
darkfield illumination. This image may be ed to derive the auto-focus on which is measure of image
sharpness. Based on a fast search algorithm the next z-location of the objective is calculated. The objective may
be moved to the new z-location and another small image may be captured. This closed-loop system does not
require the use of any other re for focusing. The microscope stage may be connected to computer-
lled stepper motors to allow translation in the X and Y directions (e.g., horizontal directions). At every
location, the desired number of images is captured and the stage is moved to the next XY position.
Imaging or other g may be performed with the aid of a detector. A detector can include a
camera or other sensing apparatus configured to convert electromagnetic radiation to an electronic signal. In an
example, a camera can be a charge-coupled (CCD) or electron-multiplying CCD (EMCCD) camera. A detector
may be a sensor, such as an active pixel sensor or CMOS sensor. A detector may include a photo-multiplier
tube for detecting a signal.
The detector can be in optical ication with a sample container (e. g., e, tip, vial). In
some cases, the detector is in direct line of sight of the sample container. In other cases, the detector is in optical
[Annotation] eaa
communication with the sample ner with the aid of one or more optics, such as , mirrors
collimators, or combinations thereof.
Cell counting can be med using imaging and cytometry. In situations where the subjects
may be -field illuminated, the preferred ment is to illuminate the subjects from the front with a
white light and to sense the cells with an g sensor. Subsequent digital processing will count the cells.
Where the cells are infrequent or are small, the red embodiment is to attach a fluorescent marker, and then
illuminating the subject field with a laser. al scanning imaging is preferred. For flow cytometry, the
subjects are marked with fluorescent markers and flowed past the sensing device. There are two types of
sensors, one is position such that the subject is back lit, measuring beam scatter to determine presence of a cell.
The other sensor, aligned so that the illumination is from the side, measures the fluorescent light emitted from
the marked subjects. Further description is provided below relating to imaging methodology for cytometry.
End-User Systems
A device and system may, after manufacturing, be shipped to the end user, together or
individually. The device or system of the ion can be packaged with a user manual or instructions for use.
In an embodiment, the system of the invention is generic to the type of assays run on ent devices. Because
components of the device can be modular, a user may only need one system and a variety of devices or assay
units or reagent units to run a multitude of assays in a point-of-care or other distributed testing environment. In
this context, a system can be repeatedly used with multiple devices, and it may be necessary to have sensors on
both the device and the system to detect such changes during shipping, for example. During shipping, pressure
or temperature s can impact the performance of a number of components of the present system, and as
such a sensor located on either the device or system can relay these s to, for example, the external device
so that adjustments can be made during calibration or during data processing on the external device. For
example, if the ature of a fluidic device is d to a n level during shipping, a sensor located on
the device could detect this change and convey this information to the system when the device is inserted into
the system by the user. There may be an additional detection device in the system to perform these tasks, or such
a device may be incorporated into another system component. In some embodiments information may be
wirelessly transmitted to either the system or the external device, such as a personal computer or a television.
Likewise, a sensor in the system can detect similar changes. In some embodiments, it may be desirable to have a
sensor in the shipping packaging as well, either instead of in the system components or in addition thereto. For
e, adverse conditions that would render an assay cartridge or system invalid that can be sensed can
include exposure to a temperature higher than the maximum tolerable or breach of the cartridge integrity such
that moisture penetration.
In an embodiment, the system comprises a communication assembly capable of transmitting and
receiving information wirelessly from an external device. Such wireless communication may be Bluetooth or
RTM technology. Various communication s can be utilized, such as a dial-up wired connection with a
modem, a direct link such as a T1, ISDN, or cable line. In some embodiments, a wireless connection is
established using exemplary ss networks such as cellular, satellite, or pager networks, GPRS, or a local
data ort system such as Ethernet or token ring over a local area network. In some embodiments the
information is encrypted before it is transmitted over a wireless network. In some embodiments the
[Annotation] eaa
communication assembly may n a wireless infrared communication component for sending and receiving
information. The system may include integrated graphic cards to facilitate display of ation.
In some embodiments the communication assembly can have a memory or storage device, for
example localized RAM, in which the ation collected can be stored. A storage device may be ed if
information cannot be transmitted at a given time due to, for example, a temporary inability to wirelessly
connect to a network. The information can be associated with the device identifier in the e device. In some
embodiments the communication assembly can retry sending the stored information after a certain amount of
time.
In some embodiments an external device communicates with the communication assembly within
the reader assembly. An external device can wirelessly or physically communicate with a system, but can also
communicate with a third party, including without limitation a patient, medical personnel, clinicians, laboratory
personnel, or others in the health care ry.
In some embodiments the system can se an external device such as a computer system,
server, or other electronic device capable of storing information or processing information. In some
embodiments the external device es one or more computer s, servers, or other electronic devices
capable of storing ation or processing ation. In some embodiments an external device may include
a database of patient information, for example but not limited to, medical records or patient history, clinical trial
records, or preclinical trial records. An external device can store protocols to be run on a system which can be
transmitted to the communication assembly of a system when it has received an identifier indicating which
device has been inserted in the system. In some embodiments a protocol can be dependent on a device identifier.
In some embodiments the external device stores more than one protocol for each device. In other ments
patient information on the external device includes more than one protocol. In some instances, the external
server stores mathematical algorithms to process a photon count sent from a communication assembly and in
some embodiments to calculate the analyte concentration in a bodily fluid sample.
] In some embodiments, the external device can include one or more servers as are known in the art
and commercially available. Such servers can provide load balancing, task management, and backup capacity in
the event of failure of one or more of the servers or other components of the al , to improve the
bility of the server. A server can also be implemented on a distributed network of storage and processor
units, as known in the art, wherein the data processing according to the present invention reside on workstations
such as computers, thereby eliminating the need for a server.
A server can includes a database and system processes. A database can reside within the server, or
it can reside on another server system that is accessible to the server. As the information in a database may
contain sensitive information, a security system can be implemented that ts unauthorized users from
g access to the database.
One advantage of some of the features described herein is that information can be transmitted from
the external device back to not only the reader assembly, but to other parties or other external devices, for
e without tion, a PDA or cell phone. Such ication can be accomplished via a wireless
network as disclosed . In some embodiments a calculated analyte concentration or other patient
ation can be sent to, for example but not limited to, medical personnel or the patient.
[Annotation] eaa
Accordingly, the data generated with the use of the subject devices and systems can be utilized for
performing a trend analysis on the concentration of an analyte in a subject which changes over time.
r age as described herein is that assay results can be ntially immediately
communicated to any third party that may benefit from ing the results. For example, once the analyte
concentration is determined at the external device, it can be itted to a patient or medical personnel who
may need to take further action. The communication step to a third party can be performed wirelessly as
described herein, and by transmitting the data to a third party’s hand held , the third party can be notified
of the assay results virtually anytime and re. Thus, in a time-sensitive scenario, a patient may be
contacted immediately anywhere if urgent medical action may be required.
As described elsewhere , imaging may be used for detection. Imaging can be used to detect
one or more characteristic of a sample. For example, imaging may be used to detect the presence or absence of
a sample. The imaging may be used to detect the location, placement, volume or concentration of a sample.
The imaging may be used to detect the presence, absence, and/or concentration of one or more analytes in the
sample.
In some embodiments, a single measurement may be used to capture various information about a
sample and/or analytes. For example, a single measurement may be used to capture information about the
volume of a sample and the concentration of an analyte within the sample. A single measurement may be used
to capture information about the presence and/or concentration of a plurality of es and/or types of analytes
within the sample. A single image may be used to capture information relating to one, two, or more of the
information or types of information described herein.
Such imaging and detection may provide more precise and accurate assays, which may be
advantageous in situations with small sample volumes, such as those described elsewhere herein. Additional
examples mes of sample may include 500 [LL or less, 250 [LL or less, 200 [LL or less, 175 [LL or less, 150
[LL or less, 100 [LL or less, 80 [LL or less, 70 [LL or less, 60 [LL or less, 50 [LL or less, 30 [LL or less, 20 [LL or
less, 15 [LL or less, 10 [LL or less, 8 [LL or less, 5 [LL or less, 1 [LL or less, 500 nL or less, 300 nL or less, 100 nL
or less, 50 nL or less, 10 nL or less, 1 nL or less, 500 pL or less, 250 pL or less, 100 pL or less, 50 pL or less, 10
pL or less, 5 pL or less, or 1 pL or less. In some embodiments, the sample volume may include less than or
equal to about 3 drops from a fingerstick, less than or equal to about 2 drops from a fingerstick, or less than or
equal to about 1 drop from a fingerstick. Such small volumes may be useful in point of service applications.
Such imaging and/or detection may yield assays with low coefficient of variation. A cient of
variation may be the ratio n the standard deviation and an absolute value of the mean. In an embodiment,
a reaction and/or assay may have a coefficient of variation (CV) (also “relative rd deviation" ) less
than or equal to about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%. A
single reaction and/or assay, or a procedure with a plurality of reactions and/or assays may have a coefficient of
variation ofless than or equal to about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,
0.3%, or 0.1%. In some embodiments, an imaging and/or detection step, or a procedure with a plurality of
imaging and/or detection steps may have a coefficient of variation of less than or equal to about 20%, 15%,
12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.3%, or 0.1%.
In some ments, the use of imaging with a device that may be placed at a point of service
location may e the overall performance of the . The accuracy and/or precision may be improved
ation] eaa
and/or the cient of variation may be reduced. The mance of the device may be improved when
handling small samples, such as those volumes described herein. The imaging may be used in combination with
other detection systems, in combination with other processes, or as a standalone system. Improvement in
mance may include a decrease in the coefficient of ion of about 15%, 12%, 10%, 9%, 8%, 7%, 6%,
%54%53%52%51%505%5039@or01%.
Imaging may be useful for various detection types for one or more types of assays or sample
ng ures. Examples of such assays or sample handling procedures may include centrifugation,
separation, cytometry, immunoassay, ELISA, nucleic acid assay, enzymatic assay, colorimetry, or any other
type of assay or reaction described elsewhere herein.
Imaging systems may provide multiple advantages over other methods for data collection, data
processing, and results interpretation. Imaging systems may ze, or increase the efficiency of, the use of
small samples and enhancing system-level performance. g systems may be used for detection as
standalone systems or may be used in combination with other detection systems or mechanisms.
In some systems, sensors and systems may be used (such as photodiodes and photomultiplier tubes
and associated optics/devices) that typically do not provide any spatial information about the sample being
interrogated. Rather, these systems may collect ation about the sample after the information has been
spatially integrated, typically losing l information related to the sample. While integrating the signal in
space from the sample may augment the signal levels being detected by the sensor, advances in sensitivity of
optical and other sensors may negate the need for such integration. g for detection may be used in the
place of such sensors, or may be used in conjunction with such s.
Imaging systems may be used that may advantageously have one or more of the following
features. Imaging sensors may have sensitivity and dynamic range that meet and/or exceed that of conventional
non-imaging sensors. Imaging devices may maintain spatial aspects of the sample being interrogated, providing
significant ability for post processing. Post processing can include QA/QC (e. g., quality control, such as
automated error detection and/or review by pathologist), and/or image is to extract specific sample
features. The imaging device can utilize 3D, 2D, 1D (line sensors), and/or point sensors with a means to
translate the sample relative to the collection optics/sensor to enable the spatial reconstruction of the sample.
Data collected from the imaging device can be processed to extract very specific information, such as
morphological features of the sample (such as cell counts), data from select regions of the image (peak
fluorescence across a sample or in a cell within the . Data collected from the imaging device can be
processed to improve the sensitivity and tion of the measurement. Data collected from the imaging device
can enable the assessment of signal variation across the sample being imaged. The data may be post processed
to calculate mean, standard deviation, maximum, minimum, and/or other applicable statistics across the sample
or within any regions of interest identified in the sample images. Imaging devices enable the exploration of
changes in the sample over time by collecting multiple images and ing changes in the images over time
and space, such as would be evident in an aggregation processes (such as for an assay of prothrombin time) or
other (e. g., al, physical, biologic, electrical, morphological) changes in the sample over time and space.
Imaging devices may enable more rapid data acquisition of , tissue sections, and other sample
configurations.
[Annotation] eaa
Cytometry Application
In some embodiments, any of the embodiments described herein may be adapted to enable the
system to perform cytometry. Cytometry (e. g., enumeration and function analysis of cells) in the system may be
performed using image analysis. Blood can be processed using the pipette and centrifuge as described
previously . Typically, a known measured volume of blood (1 i 50 uL) may first be centrifuged and the
plasma fraction removed. The cell fraction may then be pended into buffer by use of the pipette
repeatedly to dispense and aspirate. A cocktail of fluorescent antibodies may be directed to selected cell markers
(such as CD45, CD4 etc.). Following a brief incubation, a t which may act as a fixative for the white
cells and a lysing agent for red cells can be added. Following another incubation white cells may be collected
by centrifugation and the atant hemolysate removed by aspiration. The stained white cells can be resuspended
in a measured volume of buffer (typically less than the original blood volume (say 1 i 20 uL) and
dispensed into transparent capillary channels for image analysis. Typically up to three or even five or more cell
types can be imaged using antibodies having different fluorescent labels or and/or antibodies labeled with
ent fluor/protein ratios. When more cell types have to be counted or analyzed, more than one reaction
mixture can be used. In some embodiments, a reaction mixture can be used to count or analyze s s
of cell types.
In some embodiments, the capillary channels are typically about 10 , 100 um deep, 0.5 , 2 mm
wide and 0.5 , 5 cm long. The ary channels may have other dimensions, including but limited to other
dimensions described elsewhere herein. The stained cell dispersion may fill the channel usually by capillary
action and the cells may be allowed to settle on the lower channel surface. The channels can be illuminated with
one or more lasers or other light s (e. g., LEDs). The optical train may have one or more optical elements,
such as dichroic mirrors or lenses, and may or may not magnify the field of view. In some embodiments, the
field of view may be magnified 2 , 100 fold. A series of images may be collected typically representing a field
of view of about 1 mm x 0.5 mm and which ns 1 , 10,000 cells (ideally, 300 cells of interest) imaged onto
a sensor having an area of about 1000 x 1000 pixels (1 million .
] A series of images representing nt sections of channel may be collected. A mechanical stage
can be used to move the channels relative to the light source. In some cases, a servo-mechanism may move the
stage in a vertical direction so as to focus the image. In some embodiments, the light source or one or more
optical elements may move relative to the stage to focus the image. Images are usually made using one or more
combinations of light sources and optical filters. The light s may be turned on and off and s moved
into the light path as needed. Preferably up to 1000 cells of any given type may be d. In other
embodiments, various numbers of cells of any given type may be counted, including but not limited to more
than, less than, or equal to about 1 cell, 5 cells, 10 cells, 30 cells, 50 cells, 100 cells, 150 cells, 200 cells, 300
cells, 500 cells, 700 cells, 1000 cells, 1500 cells, 2000 cells, 3000 cells, 5000 cells. Cells may be counted using
available counting algorithms. Cells can be recognized by their characteristic fluorescence, size and shape.
Pattern recognition algorithms may be employed to exclude stained cell debris and in most cases where there are
cells which are aggregated these can either be excluded from the analysis or interpreted as aggregates.
A try platform may be an integrated automated microscopy device capable of the following
tasks in a fully automated, controlled environment. One or more of the following tasks may occur in cytometry
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applications. The following tasks may occur in the order they appear or in alternate orders or other tasks may be
substitute as appropriate.
1. Isolation of blood cells of the desired type
2. Labeling of cells with fluorescent and/0r colored dyes and/0r beads
3. Confinement of cell suspension in an optically compatible cuvette
4. Imaging of cells using fluorescence microscopy, darkfield illumination, and/or brightfield
nation
. Automated analysis of images to extract desired cellular attributes
6. Automated analysis of extracted information using advanced statistical and classification
s to derive clinically reportable information.
In the following sections, each of these tasks is discussed in greater detail; images and sketches are
provided er deemed necessary.
1. Isolation ofblood cells ofthe desired type. Blood cells of a desired type may be isolated in
accordance with one or more embodiments described elsewhere . For example, such isolation may occur
as referred to in previous descriptions relating to the cytometry or the centrifuge.
2. ng ofcells withfluorescent and/or colored dyes and/or beads.
Specific fluorescent dyes may be employed. Cells of interest can be incubated with pre-aliquoted
solutions of fluorescently labeled binders (e. g., antibodies, aptamers, etc.) which are specific to markers on these
cells. A key consideration may be pairing t” or high extinction coefficient and high quantum yield fluors
with s for which cells have a lower binding capacity; and Vice versa. For e, the marker CD22 may
be expressed on B-lymphocytes at about one tenth the level as CD45. Given this relative expression, CD22 may
be labeled with a “bright" dye and CD45 may be labeled with the r" dye. The markers to be d
using this technique can be either intracellular or cell-surface markers. The sensitivity of detection and
quantification can be improved by using a secondary labeling scheme for low expression s. Briefly, a
primary binder may be conjugated with another molecule which can be specifically recognized by a secondary
binder. A secondary binder labeled with a higher number of fluorophores can then bind the primary binder in
situ and enhance fluorescence . One scheme for achieving this may be the use of biotin conjugated anti-
CD22 antibody which may be in turn ized by an anti-biotin antibody that is labeled with fluorescein
isothiocyanate (FITC). The use of can dramatically enhance fluorescence . Figure 123 provides an
example of a fluorescence micrograph showing d leukocytes. The example rates a cence
micrograph of Alexa-Fluor 647-anti-CD45 labeled human leukocytes in a fixed, lysed blood sample. The
pseudocolor scheme is used to enhance perception of the different between 'bright' cells (with high CD45
expression) and 'dim' cells (with low CD45 expression).
Color stains of cell smears may also be ed within the system. For example, the manual
procedure given in StainRITETM Wright-Giemsa Stain (Polysciences Inc.) can be automated and read in the
devices of the t invention.
In some embodiments, non-specific fluorescent dyes can be used. For the purposes of
differentiating leukocyte sub-populations, the platform can also use fluorescent dyes which may bind to nucleic
acids (e.g., SYTO, Hoechst) or lipid membranes (e.g., Dil, DiD, FM64).
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3. Confinement ofcell suspension in an optically compatible cuvette.
In some embodiments, cytometry es may be designed to confine a pre-labeled cell
suspension of fixed volume into a ‘channel’ fabricated so as to provide an optically clear imaging al
above and below the cells. Sample may be introduced into the channel via a sample entry port. At some distance
from the sample entry port, an air vent may allow the release of air pressure and flow of sample into the channel.
The channel dimensions may be designed to hold a pre-defined known volume of fluid, regardless
of the volume sed at the sample entry port. Each cuvette may have multiple channels of same and/or
different volumes, each with at least one sample entry port and at least one air vent.
The concentration of cells of interest in the sample can be adjusted during sample preparation such
that after ement in the cuvette, a desired number of cells per field of view in the imaging system can be
achieved. One method for doing this may be to image a container with the cell dispersion and measure
turbidity. Using a pre-established relationship between turbidity and cell count, the cell density can be
calculated. lly, the cell dispersion will be made in a volume of buffer such that with the lowest likely cell
count, and the cell concentration will be r than optimal for image-based cell ng. More buffer may
then be added to bring the dispersion to the optimal level.
The g area of the cuvette may be designed so as to provide a sufficient number of cells for
the application of interest. For example, counting the abundant RBCs may require counting of only 000
cells and hence a diluted sample and only a small imaging area in the cuvette. However, counting rare
myeloblasts may require in some cases the ability to image more than 100,000 (total) cells. In such a scenario,
the system may concentrate the cell sion so that 100,000 cells may be imaged with a reasonable number
of fields of view. ore, the channel on the cuvette dedicated to RBC imaging will be smaller than the one
dedicated to imaging myeloblasts.
] The cuvette may be designed to be picked up by a standard ing ism in an automated
fashion to allow the transfer of the cuvette to the imaging platform. The pipetting mechanism’s tip ejector can
eject the e from the pipetting mechanism onto the imaging platform. Registration of cuvette to imaging
platform may take place in two steps. Upon transfer of the cuvette to the imaging platform, static registration
features on the cuvette may interface with mating features on the imaging platform to align the cuvette parallel
to the g platform’s optical axis (X,Y registration). ration may then be completed by a mechanism
located on the imaging platform. This mechanism may bias the cuvette against a planar e perpendicular to
the imaging platform’s optical axis (Z registration), y constraining the sample within the imaging
platform’s focal range.
4. Imaging ofcells usingfluorescence, darkfield illumination, brightfield illumination. The
method of imaging the cells may also be applied to other applications of the invention described elsewhere
. The imaging techniques, as previously described, can be used for other imaging uses.
Illumination capabilities: The cytometry platform may be designed to have three types of
illumination schemes: epi-fluorescence, darkf1eld and brightfield. The modular nature of the setup also allows
integration of phase-contrast and differential-interference contrast (DIC).
Epi-fluorescence illumination may be achieved by the use of three laser lines (e.g., 488nm, 532nm
and 640nm), but the modular nature of the system also allows for integration of other light sources, such as other
laser sources, LEDs and standard mps (e. g. Xenon, Mercury and Halogen). Further, two different sources
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can be used simultaneously, if required. uently, the cytometry platform can be used to image a large
variety of fluorescent dyes. The combination of illumination sources and emission optics can be configured to
achieve various numbers (e. g., 3-5) spectrally independent channels of imaging.
Darkfield illumination may be achieved by the use of a ght (located either above or below the
sample), a ld abbe condenser, a darkfield condenser with a toroidal mirror, an epi-darkfield condenser
built within a sleeve around the ive lens, or a combination of ringlight with a stage condenser equipped
with a dark stop. Fundamentally, these optical components can create a light cone of numerical aperture
(NA) greater than the NA of the objective being used. The choice of the illumination scheme depends upon a
number of considerations such as magnification required, ical design considerations, or size of the
imaging sensor. A ringlight based illumination scheme generally provides uniform darkfield illumination over a
wider area while at the same time ing sufficient flexibility in mechanical design of the l system.
Figure 124 provides an example of intracellular patterns using darkfield images. The example shows different
intracellular patterns in darkfield images of human leukocytes. (a) A strong scattering pattern due to presence of
granules in eosinophils, (b) a rphonuclear neutrophil with characteristic nucleolar lobes and (c) cells that
do not scatter light to a significant degree (lymphocytes or basophils)
Brightfield illumination may be achieved by the use of a white light source along with a stage-
condenser to create Koehler illumination. Figure 126 provides an example of brightfield images of human
whole blood. The example shows brightfield images of a human whole blood smear stained with the Wright-
Giemsa staining method. Characteristic patterns of staining of human leukocytes are apparent. The
characteristically shaped red cells can also be identified in these images.
Automaticfilter wheel: An automatic filter wheel may allow control of the imaging optical path to
enable imaging of multiple fluorophores on the same field of view.
Image based auto-focusing: The try platform may use an image-based thm to l
the z-position (e. g., al position) of the objective (i.e., its distance from the sample) to achieve auto-
focusing. Briefly, a small image (for example, 128x128 pixels) may be captured at a fast rate using darkfield
illumination. This image may be analyzed to derive the auto-focus on which may be used to measure of
image sharpness. Based on a fast search algorithm the next z-location of the objective may be calculated. The
sample may be moved to the new z-location and another small image may be ed. In some embodiments,
this closed-loop system does not require the use of any other hardware for ng.
Translation ofstage: The microscope stage may be connected to er-controlled stepper
motors to allow translation in the X and Y directions (e. g., ntal directions). At every location, the desired
number of images may be captured and the stage may be moved to the next XY position.
Imaging sensor: A camera with a CCD, EMCCD, CMOS or in some cases a photo-multiplier tube
can be used to detect the signal.
5. Analysis ofimages to extract desired ar attributes.
The cytometry platform may use ent illumination techniques to acquire images that reveal
diverse properties and es of the cells. Labeling with cell-marker specific binders may reveal the degree of
expression of that particular marker on the cell e or in the cell. Darkfield image may reveal the light
scattering properties of the cell. The internal and external es of the cell which scatter more light appear
brighter and the features which scatter lesser amounts of light appear darker in a darkfield image. Cells such as
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granulocytes have internal es of size range (100-500nm) which can scatter significant amount of light and
generally appear brighter in darkf1eld images. Furthermore, the outer boundary of any cell may scatter light and
may appear as a ring of bright light. The diameter of this ring may ly give the size of the cell. Brightfield
images of cells can reveal cell size, phase-dense material within the cells and colored features in the cell if the
cells have been usly stained.
An image sing library may extract one or more of the following information for each cell
(but is not d to the following):
1. Cell size
2. Quantitative measure of cell granularity (also rly called side scatter, based on flow cytometry
3. Quantitative measure of fluorescence in the each spectral channel of g, after compensating for
cross-talk between spectral channels
4. Shape of the cell, as quantified by standard and custom shape attributes such as aspect ratio, Feret
diameters, Kurtosis, moment of inertia, circularity, solidity etc.
. Color, color distribution and shape of the cell, in cases where the cells have been stained with dyes (not
attached to dies or other types of receptor).
6. Intracellular patterns of staining or scattering or color that are defined as quantitative s of a
biological feature, for example density of granules within cells in a darkfield image, or the number and
size of nucleolar lobes in a Giemsa-Wright stained image of polymorphonuclear
neutrophils etc.
7. Co-localization of features of the cell revealed in separate images
The image processing thms utilized in this step may use combinations of image filtering,
edge detection, template ng, automatic thresholding, morphological operations and shape analysis of
objects.
6. is ofextracted information using advanced statistical and classification methods to
derive clinically reportable information.
Any number of measured attributed may be extracted from images of cells. For example,
measured attributes of each cell extracted from the images can range from 7-15, thus creating a 7 to 15
dimensional space within which each cell is a point. If n measured attributes are ted from the images, an n
dimensional space may be provided, within which each cell is a point.
Based on data acquired for a large number of cells (e.g., 100-100,000 cells) a complex n-
dimensional scattered data set may be ted.
Statistical s may be used for clustering cells into individual separate populations in this ndimensional
space. These methods may also use state-of-the-art knowledge from cell biology and hematology to
aid in clustering and cell population identification.
Figure 125 provides an example of multi-parameter acquisition of data from labeled cell samples.
Human leukocytes were labeled with the pan-leukocyte marker anti-CD45-Alexa Fluor 700 (shown here in
green) and the B-cell marker anti-CD22-APC (shown here in red). The dual channels show different
patterns of CD45, CD22 expression and side scatter. Cells which are positive for CD22 and CD45 (B-
lymphocytes) show the characteristically low side scatter. On the other hand cells such as neutrophils and
eosinophils which have high side scatter do not show labeling for CD22.
Figure 127 provides an e of quantitative multi-parametric data ition and analysis.
For example, a histrogram may be provided which may show distribution of CD45 intensity on human
leukocytes. Any other graphical data distribution techniques may be employed to shows the distribution. In
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some embodiments, a scatter plot of side r may be provided. The side scatter may be determined by dark-
field image analysis versus CD45 fluorescence intensity for a human leukocyte sample. The side r plot
may show two main populations of granulocytes (top left) and lymphocytes (bottom right).
Foregoing sections describe the main components and lities of the try platform and
applications. Based on these capabilities, a wide gamut of cell-based assays can be designed to work on this
platform. For example, an assay for performing a 5-part leukocyte differential may be provided. The reportables
in this case may be number of cells per iter of blood for the following types of leukocytes: tes,
lymphocytes, neutrophils, basophils and eosinophils. The basic strategy for development of this assay on the
try platform may be to convert this into a problem where some utes of leukocytes are measured
such as side scatter, CD45 fluorescence intensity, or CD20 fluorescence intensity so that leukocytes can be
segregated into (e.g., 5) different populations in this n-dimensional space. The regions made around a cluster of
cells can be positioned on a scatter plot in 2-dimensional space are called “gates" after flow cytometry parlance.
An example labeling and “gating" strategy is as follows:
CD2/CRTH2/CDl9/CD3 cocktail PE-Cy7 Identification of lymphocytes, labeling of
basophils and eosinophils
CD45 Alexa-Fluor 647 Pan-leukocyte marker to label all leukocytes
CDl4/CD36 cocktail FITC Identification of monocytes
] The cytometry platform and analysis system described herein may advantageously permit
automated sample preparation and execution based on ordered . The s and methods described
may also enable specific identification of cells as opposed to VCS (volume, conductivity and scatter), which can
increase confidence in identification and reduce ces for confirmatory testing. The image analysis
described herein may also permit preservation of cell images for later confirmation, analysis as required. There
may also advantageously be availability of logical features of the cell. In some embodiments, dynamic
ment of sample prep and imaging parameters to deal with cell samples of wide range of concentrations
may be provided.
In some embodiments, the centrifuge may be used to prepare and concentrate cell populations. A
method may include the use of the centrifuge for cell preparation and the imaging and analysis system described
elsewhere herein.
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In some embodiments, a combination of dark-field imaging and imaging of cells stained with
multiple fluorescent antibodies may be used. Such a ation may give the equivalent of FACS analysis in
a much simpler and less expensive device than other techniques.
] In ance with some embodiments of the invention, the systems and methods described herein
may enable one or more of the following features. Such features may be advantageous for various applications.
In some embodiments, ted sample inspection and processing may be enabled. Such sample inspection
and processing may include one or more of the following: sample quality, sample volume measurement, dilution
(and measurement of dilution factors), and separation of red and white cells from plasma.
An automated chemical/assay related process may also be employed. This may include
precipitation, mixing or ntation.
In some embodiments, there may be automated measurement of any and all assays that produce
luminescence or change light (e.g., color chemistry). These may include one or more of the following:
spectrophotometry, fluorimetry, luminometry, turbidimetry, nephelometry, refractometry, 3-color image
analysis, polarimetry, measurement of agglutination, image analysis (which may employ one or more of the
following: camera, digital camera, scanner, lens-less photography, 3-D photography, video photography), or
copy.
Automated quality control and/or calibration of assays may also be provided within the systems
and methods described herein.
In some embodiments, y communication may be ed. Such communication may
enable record keeping of all assay steps. The y communication may also enable changes in assay
protocols to optimize or increase completion of multiple assays.
Quality Control/Complementary Applications
In some ments, imaging may be used in conjunction with one or more other measurements
or ion steps. The imaging may be complementary to other techniques, procedures, reactions, and/or
assays. For example, imaging may be used to perform one or more quality control check or step for any other
action, such as a sample preparation, assay, or detection step. Imaging may be used for the facilitation of other
detections. Imaging may be used to improve the accuracy and/or precision of collected data. The imaging may
be a quality control aspect to verify data, results, and/or any measurements. The g may be a control
mechanism or improvement mechanism. Imaging may be used to detect one or more condition that may affect
collected data and/or the accuracy and/or precision of the data. Thus, imaging may improve sample preparation,
assay, and/or detection procedures. This may be particularly advantageous in situations where there are small
sample volumes, such as volumes described elsewhere herein.
In an e, a detection step may occur to determine the ce and/or tration of an
analyte. ion may occur of one or more signal that may be representative of data that may be useful for
subsequent qualitative and/or quantitative evaluation. ion may or may not include the detection of visible
light. Detection may include the measurement of energy from anywhere along the electromagnetic spectrum
(e. g., infra-red, microwave, ultraviolet, gamma ray, x-ray, visible light). Detection may occur using any type of
sensor, which may include an l sensor, temperature sensor, motion sensor, pressure sensor, icity
sensor, acoustic sensor, chemical sensor, spectrometer, or any other sensor described elsewhere herein, or any
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combination thereof. In some embodiments, detection may or may not e a spatial distribution of light
and/or energy. In some instances, detection may or may not include an energy density distribution.
Imaging may be capable of detecting one or more condition under which the detection takes place.
Imaging may be used to detect the condition of a sample, reagent, container, portion of the device that may be
used in the detection. In some embodiments, the g may be visible imaging. For example, imaging may
include capturing a snapshot, photo, and/or picture. Imaging may include capturing a spatial distribution of
energy along the electromagnetic um. The energy along the electromagnetic spectrum may include visible
light, or may include other ranges (e.g., infra-red, ultraviolet, or any other described ). For example, a
spatial distribution of visible light may include a two-dimensional image. In some embodiments, imaging may
include the use of an image capture , which is described in greater detail elsewhere herein. Some
examples of image capture devices may include a camera, such as a lens-less (computational) camera (e.g.,
Frankencamera) or open-source . An image capture device may be capable of capturing signals that may
be capable of ting a one-dimensional, two-dimensional, or three-dimensional representation of the item
that is imaged. In some cases, an image e device may be a motion-sensing input device configured to
provide a three-dimensional or pseudo three-dimensional representation of an object.
The imaging technique may be the same or may be different from the detection mechanism
utilized. In some instances, different types of detection mechanisms are used between the detection step and the
quality control imaging step. In some instances, detection may include an energy band assessment or energy
density bution, such as from a spectrometer, while y control imaging may e a spatial
distribution of visible light, such as from a camera.
Sensitive detection may be achieved by imaging. For example, an imaging device may be able to
capture an image to within 1 mm, 500 micrometer (um), 200 um, 100 um, 75 um, 50 um, 25 um, 15 um, 10 um,
7 um, 5 um, I um, 800 nanometer (nm), 700 nm, 500 nm, 300 nm, 100 nm, 50 nm, 30 nm, 10 nm, 5 nm, 1 nm,
500 picometer (pm), 300 pm, or 100 pm. In an example, the imaging may be achieved by a camera which may
have a resolution of greater than or equal to about 2 xels, 4 megapixels, 6 megapixels, 8 megapixels, 10
megapixels, 12 megapixels, 15 megapixels, 20 xels, 25 xels, 30 megapixels, 40 xels, or 50
megapixels, or more.
Imaging may be used to detect an error or other fault state. Imaging may be used to ine a
condition that may increase the likelihood of an error and/or result in inaccuracies and/or ision. For
example, g may be used to determine the presence and/or absence of one or more undesirable materials.
Examples of undesirable materials may include bubbles, particles, fibers, particulates, debris, precipitates, or
other material that may affect a measurement. In another example imaging may be used to ine if a
volume of sample, reagent, or other material falls within a desired range, or whether a sample, reagent, or other
material is located in a desired location. The imaging may be used to determine the concentration of a sample,
reagent or other material, or whether the sample, reagent, or other material falls into a desired concentration
range.
In one example, an enzymatic assay may be performed on a small volume of . Examples of
volume values may be provided elsewhere herein. A spectrometer or other detection method or ism
described herein may be used to perform a detection step for the enzymatic assay. An imaging step may occur
to ine the conditions under which the detection is occurring. For e, the imaging step may
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determine whether there are undesired particulates, such as bubbles, or any other undesired ions. The
imaging step may verify whether the assay is operating as it . The imaging step may confirm whether the
operating conditions under which the assay is occurring and/or detection is being performed falls within desired
tolerances or optimized conditions. In some examples, the imaging may include taking a snapshot of a reaction
occurring in a container. The captured image may be analyzed for any rable and/or desirable conditions.
In some instances, the captured image may be analyzed tically in a computer assisted method. One or
more processor may aid with the analysis of the captured image, in some cases using one or more routines
implemented by way of machine-executable code stored in a memory location. The imaging may be used for
quality control without requiring the intervention of a human.
The imaging may provide igence for a system. The imaging step may provide intelligence on
the conditions under which sample preparation, assay, and/or detection occurs. The detection s may
provide more reliable, te, and/or precise measurements from a point of service device or component of the
device, when utilizing the imaging in a quality control procedure. The quality control may be beneficial when
small volumes are utilized.
Dynamic Feedback
In some ments, dynamic feedback may be provided during a sample processing step. For
example, dynamic feedback may occur during a sample preparation step, assay step, and/or detection step. In
some embodiments, dynamic feedback may be provided via imaging. Alternatively, dynamic feedback may
occur via any other detection mechanism, such as those described elsewhere herein. In some embodiments, a
dynamic feedback mechanism may utilize optical detection, electromechanics, impedance, electrochemistry,
microfluidics, or any other mechanism or combination thereof
Dynamic feedback may ally utilize imaging or other detection mechanisms. The dynamic
feedback may be involved in automated decision making for a . For example, an image may be captured,
and data may be captured that may be considered in the determination of a step. A sensor, such as an imaging
sensor, may e physical information which may be utilized in the determination of a uent step or
ure. Such subsequent steps or procedures may be determined on the fly in an automated fashion.
In an example, dynamic dilution may occur. A container, such as a cuvette or any other container
described herein, may have a sample therein. A dynamic feedback mechanism (e.g., imaging,
spectrophotometer, or other ion mechanism) may determine the concentration of a sample. In some
embodiments, the determination may be a rough or crude determination. The initial determination may be a
ballpark determination that may provide feedback that may put the sample into a condition for more precise or
fine-tuned ion and/or analysis. In an example, the c ck mechanism may be an imaging
method that may use an initial fluorescence detection to do the initial estimate for concentration.
The dynamic feedback mechanism may determine whether the sample concentration falls within
an able range. In one example, the concentration may be a cell concentration. A rough cell count may be
performed to determine cell tration. One or more signal from the dynamic feedback ism may be
used for the cell count. In some embodiments, cells may be provided in a wide range of concentrations. In
some instances, the concentrations may vary on over 1, 2, 3, 4, 5, 6, 7 or more orders of magnitude. In some
ments, depending on the cell or analyte to be measured and/or analyzed, different concentrations may be
provided within the same sample. Based on the determined concentration, the sample may be diluted or
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concentrated and/or ed. For example, if the tration is higher than a desired range, the sample may
be diluted. If the concentration is lower than a desired range, the sample may be concentrated and/or amplified.
The degree of dilution and/or concentration/amplification may be ined on the fly, based on the estimated
concentration.
] The degree of dilution and/or concentration/amplification may be determined in an automated
fashion. Dynamic feedback may be automated. The dynamic ck mechanism (e.g., imaging or other
detection mechanism) may e data which may be analyzed to determine an operational condition. For
example, a sample concentration may be determined based on the dynamic feedback mechanism. A processor
may be provided, capable of receiving and/or processing one or more signals from the dynamic feedback
mechanism. Based on the received signals the processor may ine the tration and whether the
concentration falls within a desired range. If the concentration falls within the desired range, the processor may
determine that no further dilution or concentration/amplif1cation is . If the concentration is higher than
the desired range, the processor may determine that dilution is needed. The processor may determine the degree
of dilution needed based on how far the concentration falls outside the desired range. If the concentration is
lower than the desired range, the processor may determine that concentration (or amplification) is needed. The
processor may determine the degree of ication needed based on how far the concentration falls below the
desired range. Such determinations may be based on tangible computer readable media which may e
code, logic, or instructions for ming one or more steps. Such determinations may be automated and thus
made without requiring the intervention of a human. This may apply to any ional condition, and need not
be limited to sample concentration, such as cell concentration.
In some embodiments, after an initial feedback measurement and dilution or
concentration/amplification step, a more precise measurement may be taken. For example, a more precise
ement of cell counting may occur after the sample is determined to be in a desirable range. In some
embodiments, a sample may reach a desirable range after a single dilution and/or concentration/amplif1cation
step. In other embodiments, additional feedback steps may occur and additional dilution and/or
concentration/amplification steps may be provided, as necessary. For example, if an initial determination yields
that a sample has a high tration, a dilution step may occur. Following the dilution step, an additional
feedback step may optionally occur. If the sample concentration does not fall into the desired (or otherwise
predetermined) range, an additional dilution or tration/amplification step may occur, depending on
whether the measured concentration is above or below the desired range, respectively. This may be repeated as
many times as necessary for the sample to fall into the desired range. Alternatively, feedback steps may or may
not be ed, or may be repeated a fixed number of times. In some embodiments, each ck step may
occur with a greater degree of precision. Alternatively, the same degree of precision may be utilized in each of
the feedback steps.
In some ments, when a sample concentration (e.g., cell concentration, analyte
concentration) falls into a desired range, the sample may be analyzed effectively. For example, the sample cell
concentration may have a desired range that may be beneficial for imaging. A desired number of cells per field
of view may be provided.
Cell quantification and enumeration by imaging can ed by controlling the cells density
during imaging, thus limiting crowding and clustering of cells. Consequently the range of analyte concentration
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over which the assay is linear can be maximized or increased. In order to extend the assay linear range, the
dynamic system may perform a prior, non-destructive measurement on the sample using a method which has a
high dynamic range to provide determine a rough cell concentration in the sample. An algorithm may then
calculate the dilution ratio required to bring the cell concentration in the acceptable range for the main
measurement. Dilution and/or tration/amplification may be ed accordingly, thereby providing
dynamic dilution and/or concentration.
Such dynamic feedback, such as dynamic dilution, may be advantageous in systems utilizing small
volumes. In some embodiments, a total sample volume may include any of the volumes described elsewhere
herein. In some instances, the volumes for a ular portion of a sample to be analyzed may have any of the
volumes described elsewhere . Dynamic dilution may assist with ing low coefficient of variation.
For example, a coefficient of variation for a sample preparation, assay, and/or ion step may have a
coefficient of variation value as described elsewhere herein. This may be advantageous in point of service
devices, which may utilize small volumes, and/or have low coefficients of variation.
Dynamic feedback may advantageously permit structive testing of a sample. This may be
advantageous in systems using small volumes. The same sample may be used for the initial feedback detection
and for subsequent detections. The same sample may under initial feedback detection and subsequent detections
within the same container (e.g., cuvette, vial, tip). A vessel may be provided with a sample that is outside a
desired and/or able range in its initial state. For example, a concentration of one or more analytes and/or
cells may fall outside a desired and/or detectable concentration range initially. The same sample may be
measured within the range in the same vessel. In some embodiments, the concentration of the one or more
analytes and/or cells may later fall within a desired and/or detectable range in the same vessel. In some
embodiments, one or more intervening steps, such as on and/or concentration/amplification may be
med on the sample in order to get the sample into the desired and/or detectable range. Such intervening
steps may be med in an automated n.
In some embodiments, dilution may be provided to the sample in an automated fashion. For
example, a diluent may be dispensed into a container holding the sample and mixed with the sample to effect a
new sample volume. In some cases, the diluent includes a single diluent. In other cases, the diluent includes a
plurality of diluents. The diluent can be dispensed into the container with the aid of a pumping system, valves
and/or fluid flow channels for facilitating the flow, such as a uidic system having one or more
microfluidic channels and/or one or more microfluidic pumps. The microfluidic system may e one or
more ical and/or electromechanical ents, such as a mechanical pumping system having one or
more actuated (e. g., pneumatically actuated) valves for facilitating the flow of a fluid. The pumping system in
some cases includes a ical pump configured to facilitate fluid flow. The pumping system can include
one or more sensors for measuring and relaying operating ters, such as fluid flow rate, concentration,
temperature and/or pressure, to a control system. In an example, the diluent is dispensed into the container with
the aid of a microfluidic system having a mechanical pump coupled to a microfluidic channel bringing the
container in fluid communication with a diluent reservoir.
In some cases, a pumping system is provided to release a diluent based on a measured sample
dilution. The sample dilution can be measured with the aid of a , such as, for example, a light sensor. In
an example, the light sensor is d with a light source for directing a beam of light through the sample, and
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subsequently measuring sample dilution based at least in part on the scattering of light through the sample. If
the measured sample (e.g., cell, tissue) concentration is above a predetermined limit (or threshold), then the
pumping system directs a diluent (e.g., water) from a diluent oir to a container holding the sample.
] In some embodiments, dynamic dilution is electronically ted with the aid of a fluid flow
system having a pump (e. g., microfluidic pump) in fluid communication with a fluid flow channel (e. g.,
microfluidic channel), and further including one or more valves for regulating fluid flow. The automation of
dilution can be used to test and/or adjust calibration settings, such as preset dilution fluid s used to effect
a d concentration.
In some situations, the pump ses one or more valves, such as pneumatically-actuated
valves. The pump, fluid flow channel and one or more valves bring a diluent reservoir in fluid communication
with a container configured to hold a sample. The one or more valves and/or the pump can be in electrical
communication with a control system having a processor for ting the flow of diluent from the diluent
reservoir to the to regulate the concentration of the sample.
Dynamic feedback advantageously enables the ted regulation of sample concentration
while minimizing, if not eliminating, user involvement. In some cases, the concentration of a sample is
automatically ted (e.g., d or amplified) without any user involvement. Such minimal user
involvement can provide low coefficient of variation in imaging and overall system use, as described elsewhere
In an example, dynamic feedback system is used to regulate the concentration of cells in a fluid
sample using imaging. With the sample provided in a sample container, such as cuvette, the imaging is used to
measure the concentration of cells in the fluid sample. The measured concentration can be a rough (or ballpark)
ement of concentration. The dynamic feedback system then dilutes the fluid sample by providing a
diluent into the sample container. This may minimize, if not eliminate, any disturbance to (or destruction of) the
cells upon dilution. An optional ement of the tration of cells in the fluid sample can then be made
to measure the concentration following dilution. In some ions, following dilution a reaction can take place
in the same sample container that was used to dilute the sample. In some situations, the reaction may take place
in cases in which the dilution is not optimal.
In some cases, during dynamic feedback a rough measurement of sample tration is made
with the aid of a spectrometer, and a more precise measurement of sample concentration is made with the aid of
an imaging device. The imaging device can include a light source (e.g., coherent light, such as a laser, or
incoherent light) and a camera, such as a charge-coupled device (CCD) camera. In an example, following the
rough ement, the dynamic feedback system coarse adjusts the concentration of the sample by providing
the diluent, and subsequently makes the more precise measurement. The sample tration can be further
adjusted by providing smaller volumes of a t (i.e., fine adjustment) in relation to the volume of the diluent
provided during coarse adjustment. Alternatively, the rough measurement of sample concentration is made with
the aid of an imaging device, and the more precise measurement is made with the aid of a spectrometer. Coarse
and fine adjustment
c feedback systems provided herein can be configured to concentrate/amplify (i.e.,
increase the concentration of) a sample, such as cells in a fluid sample. In some cases, this is accomplished with
the aid of centrifugation or induced separation (e. g., electric field separation, magnetic separation).
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In some situations, the concentration of a sample is made using an imaging device, with the
location of the imaging device selected to select a desired path length and/or focal point. In some cases, the
on of one or more optics associated with the imaging device are adjusted to provide a desired path length
and/or focal point. In some cases, a lens-less camera is used for image capture, which can computationally
provide image analysis and various focal points.
c dilution can be performed on various sample volumes. In some cases, if a sample
volume is above a predetermined limit, the sample can be distributed in multiple sample ners (e. g.,
cuvettes) for sequential or parallel processing and/or imaging.
Self-Learning
The c feedback ism may result in self-learning by the system. For example, for a
dynamic dilution/concentration system, an initial feedback measurement may be made. Based on the feedback
measurement, the sample may have no action, may be d, or may be concentrated/amplified. Subsequent
measurements and/or detection may occur. The subsequent measurements and/or detection may or may not be
additional feedback measurements. Based on the subsequent measurements, a determination may be made
whether the action taken (e.g., no action, dilution, concentration/amplification) was correct and/or whether the
t degree of action was taken (e. g., enough dilution or concentration/amplification). For example, an initial
ck mechanism may determine that the sample concentration is high and needs to be diluted. The sample
may be diluted by a ular . A subsequent measurement may be taken (e.g., image of the sample may
be taken). If the degree of dilution does not bring the sample into the desired range (e. g., dilution was too much
or too little), the system may receive an indication that for subsequent dynamic dilutions/concentrations with the
same or similar initial feedback mechanisms, a different degree of dilution may be used. If the degree of
dilution does bring the sample into the desired range, the system may receive a mation that the amount of
on should be used for subsequent dilutions for the same or similar type of initial feedback measurement.
Data points may be gathered based on initial conditions and uent actions, which may assist
with determining appropriate actions to take in subsequent dynamic feedback situations. This may cause the
system to self-learn over time on steps to take in particular dynamic situations. The self-learning may apply to
individualized situations. For example, the self-learning system may learn that a ular individual from
whom the sample is drawn, may require different degrees of dilution/concentration than another individual. The
self-learning may apply to groups of individuals having one or more characteristic. For example, the self-
learning system may learn that an individual using a particular type of drug may require different degrees of
dilution/concentration than another individual. The self-learning system may also be generalized. For example,
the system may become aware of a pattern that people of a particular demographic or having particular
characteristics may or may not required different degrees of dilution and/or concentration. The system may
draw on past data points, individuals’ records, other duals’ records, l health information, public
information, medical data and statistics, insurance ation, or other information. Some of the information
may be publicly available on the Internet (e. g., web sites, articles, journals, databases, medical statistics). The
system may optionally crawl web sites or databases for updates to information. In some embodiments, self-
learning may occur on the device, the cloud or an external device. As additional data is ed, it may be
uploaded to the cloud or external device, and may be accessible by the earning system.
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Image capture and/or manipulation devices
In some embodiments, sample preparation, sing and/or analysis is performed with the aid of
image capture and/or manipulation devices, including electromagnetic radiation (or light) capture and/or
manipulation devices, such as imaging devices or spectrometers. In some cases, an imaging device can be used
in ation with a ometer. A ometer can be used to measure properties of light over a select
portion of the electromagnetic spectrum, which may be used for oscopic analysis, such as materials
analysis. An imaging (or image capture) device can be used to measure sample concentration, composition,
temperature, turbidity, flow rate, and/or viscosity.
] In an example, an image capture device may be a digital camera. Image capture devices may also
include charge coupled devices (CCDs) or photomultipliers and phototubes, or photodetector or other detection
device such as a scanning microscope, whether back-lit or d-lit. In some instances, cameras may use
CCDs, CMOS, may be ess (computational) cameras (e.g., Frankencamera), open-source cameras, or may
use any other visual detection technology known in the art. In some instances, an imaging device may include
an optical t that may be a lens. For e, the optical element is a lens which captures light from the
focal plane of a lens on the detector. Cameras may include one or more optical elements that may focus light
during use, or may capture images that can be later focused. In some embodiments, imaging devices may
employ 2-d imaging, 3-d imaging, and/or 4-d imaging (incorporating changes over time). Imaging devices may
capture static images or dynamic images (e. g., video). The static images may be captured at one or more points
in time. The imaging devices may also capture video and/or dynamic images. The video images may be
captured continuously over one or more periods of time.
] In some cases, an image capture device is a computational camera that is used to measure the
tration of a ity of s within a vely short period of time, such as at once. In some
embodiments, the computational camera may have an optical which may be different from a lens. In an
example, the computation cameral is a lens-less camera that takes a photograph of a plurality of samples in
staggered sample containers (e. g., cuvettes). The concentration of a sample in a particular sample container can
then be calculated by, for example, mathematically rendering the image to select a focal point at or adjacent to a
portion of the image having the particular sample container, and deriving the sample concentration from the
rendered image. Such mathematical manipulation of an image, as may be acquired with the aid of a lens-less
camera, can e other information at various points in space within the field of view of the lens-less camera,
which may include points in space that may be extrapolated from scattered light. In some embodiments, the
final signal may be analyzed by complex thms. One example of such a setup is a computational camera
with optical elements which may produce a Fourier-transformed image on the detector. The resulting “image"
can be analyzed to extract required information. Such a detector would enable one to obtain rich information
from the imaged subject. ing ent features from the image, for example, information at a different
focal length could be done purely through software, simplifying the g hardware and providing more rapid
and informative data acquisition.
Electromagnetic radiation capture and/or manipulation devices can be used in various applications
provided herein, such as measuring sample concentration, including dynamic dilution. In an example, a light
capture and/or manipulation device includes a light source, such as a nt light source (e.g., laser), coupled
with a light sensor, such as a CCD camera, for capturing scattered light, as may emanate from a sample upon the
ation] eaa
light source being directed through the sample. This can be used to measure the concentration of the sample.
The light sensor can be configured to capture (or sense) various wavelengths of light, such as red, green and
blue, or other color ations, such as combinations of red, orange, yellow, green, blue, indigo and violet, to
name a few examples. In some situations, the light sensor is configured to sense light having ngths at or
greater than infrared or near ed, or less than or equal to ultraviolet, in addition to the visible spectrum of
light.
Light e and/or manipulation devices can be used to collect information at particular points in
time, or at s points in time, which may be used to uct videos having a plurality of still images and/or
sound (or other data, such as textual data) associated with the images.
Light capture and/or manipulation devices, including computational (or lens-less) cameras, can be
used to capture two-dimensional images or three-dimensional (or pseudo three-dimensional) images and/or
video.
In some embodiments, an image capture and/or manipulation device perturbs an object and
measures a response in view of the perturbation. The perturbation can be by way of light (e. g., x-rays,
ultraviolet light), sound, an electromagnetic field, an electrostatic field, or combinations thereof For example,
perturbation by sound can be used in acoustic imaging. Acoustic imaging may use similar principles to
diagnostic ultrasound used in medicine. Acoustic imaging may function similarly to a regular microscope but
may use acoustic waves. A source of ultrasound may e waves that can travel through the sample and get
reflected/scattered due to heterogeneities in the elastic properties of the . The reflected waves may be
“imaged" by a sensor. A variant of this method may include “photo-acoustic imaging" where the acoustic waves
ing through the sample may cause local compression and extension of the . This
compression/extension may cause a change in the refractive index of the sample material which can be detected
by measuring/imaging the reflection of a laser beam by the sample.
In some situations, an imaging device can be used to measuring the volume of a cell. In an
example, the combination of a light source and CCD camera is used to capture a still image of a cell. A
computer system digitizes the still image and draws a line across the cell, such as through the center of the cell.
The computer system then measures the distance between the points at which the line intersects the boundaries
of the cell (or cell wall or ne) to provide an estimate of the diameter of the cell, which may be used to
estimate the volume of the cell.
The imaging device may utilize line scanning microscopy to enable sample illumination with a
thin line or spot of nt laser light, so that power from the source can be concentrated in a small area giving
high power densities. The detector geometry may be matched with the line or spot. Then the line/spot may be
scanned across the sample so that different parts of it can be imaged. Each d line can then be
concatenated to form the whole image (e. g., in a similar manner like a document r). This method may be
advantageous as an analytical/imaging method for one or more of the following reasons: (1) high power y
of illumination, (2) relatively high speeds can be obtained from line scanning as opposed to spot scanning,
(though both may be slower than full-frame or classical g), (3) high precision and/or accuracy of
analytical measurements on the sample such as cence, absorbance, luminescence, (4) combination with
spectral or hyper-spectral imaging such that a complete spectrum of the sample can be acquired for each pixel,
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(5) on-the-fly adjustment of resolution, (i.e. without changing any elements, a sample can be d at low or
high lateral resolution as desired), or (6) can provide high depth of field to allow imaging of tissue samples.
In some embodiments, an imaging device is configured to detect light emanating from an
ionization scence or luminescence) event, such as via llation. In an example, a scintillator is coated
on or embedded in a material comprising a sample container. Upon a sample g to (or ise
interacting with the scintillator), the scintillator emanates light (e.g., fluorescent light) that is detected by a
detector of the g device. This may be used to e the radioactive decay (e.g., alpha and/or beta
decay) of certain samples.
In some situations, an imaging device is a field effect transistor for detecting charged particles,
such as ions. Alternatively, the imaging device may be a thermal detector for measuring a heat change, which
may be used to construct a heat map, for example.
In some situations, a sample container comprises one or more wells for lizing a sample.
The sample ner may be coupled with an imaging device for imaging a sample immobilized in the one or
more wells. Sample immobilization can be facilitated with the aid of beads having surface g agents (e. g.,
antibodies) or e binding agents, which may be disposed, for example, at bottom portions of wells. The
wells can have diameters on the order of nanometers to micrometers or r.
In some embodiments, sample detection and/or analysis is facilitated with the aid of image
ement species, such as dyes. A dye may bind to a sample provide an optical, electrical or optoelectronic
signal that can be ed by a detector of an imaging device. In an example, a dye binds to a cell and
fluoresces, which is recorded by a detector. By measuring fluorescence, the spatial distribution and/or
tration of cells can be measured. Image enhancement species can aid in achieving improved signal-to-
noise during image acquisition (or capture). A dye can bind to a cell with the aid of surface receptors and/or
antibodies.
In some cases, the use of dyes can generate background fluorescence, which may distort an
imageithe fluorescence sample may be difficult to resolve from the fluorescing background. In such a case,
image acquisition can be enhanced by contacting a sample in a fluid with a fluorescent dye. d dye is
removed with the aid of a centrifuge (or magnetic or electric separation), which separates the sample from the
unbound dye. The centrifuge may be integrated in a point of service device having the imaging device. The
sample can then be re-suspended in a fluid and subsequently imaged with the aid of the imaging .
In some cases, image acquisition can be enhanced by using dynamic feedback in addition to, or in
place of, the use of image enhancement species. In an example, the concentration of the sample is zed
with the aid of dilution and/or amplification prior to image acquisition.
Sample separation can be facilitated with the aid of a centrifuge. As an alternative, sample
separation can be performed with the aid of a magnetic or electric field. For instance, a magnetic particle can
bind to a cell, which in the presence of a magnetic field can be used to t the cell towards the source of
magnetic attraction.
Systems and methods provided herein can be applied to s types of samples, such as cells as
may be derived from tissue (e. g., skin, blood), saliva or urine. In an example, dynamic feedback and/or imaging
can be applied to tissue samples or cell samples derived from such tissue samples.
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Exam le 1: Nucleic acid am lificationb L00 -mediated isothermal am lification LAMP
To evaluate the ability of the three-color image analysis method for both fluorescence and
absorption to read LAMP assays the following experiments were performed.
Lamp Reaction Conditions
The LAMP reaction was carried out in a total volume of 25 uL in 500 uL PCR tubes (VWR, West
Chester, PA). The reaction mixture included 0.8 uM of primer 1 and primer 2, 0.2 uM of primer 3 and primer 4,
400 uM each dNTP (Invitrogen, Carlsbad, CA), lM betaine (Sigma, St. Louis, MO), lX Thermopol Buffer
(New England Biolabs, Ipswitch, MA), 2 mM MgSO4 (Rockland Immunochemicals, Gilbertsville, PA), 8U Bst
DNA polymerase large nt (New England Biolabs, Ipswitch, MA), and a given amount of template DNA
(varied n ~10 and ~10A9 copies). In the case of negative control approximately 10A9 copies of irrelevant
DNA was added.
Reaction Conditions
The reaction was ted at 65 0C for 1 hour in sealed tubes. The polymerase was then
inactivated by heating the reaction product to 80 °C for 5 minutes.
Product Detection and Visualization
SYBR Green I stain (Invitrogen, Carlsbad, CA) stock was diluted 100 fold, 5 uL was mixed with
uL of the completed LAMP reaction product mixed, and incubated for 5 minutes at room temperature. The
reaction products were then read out in the following way:
Fluorescence readout: PCR tubes or pipette tips containing the mixture, were illuminated with 302
nm UV light and fluorescent on (kmax ~ 524 nm) imaged by a digital camera (Canon EOS Tli, 18-55
mm, Canon, Lake s, NY).
Color readout: on products were aspirated into tips and imaged using a digital camera
] A fluorescence image of assay products in tubes is shown in Figure 81.
A fluorescence image of the assay product in tips is shown in Figure 89.
Color images of the assay products in tips are shown in Figure 82, Figure 83, Figure 84, Figure 85,
Figure 86, and Figure 87. Figure 88 shows a background color image obtained for calibration.
Figure 90 shows a comparison of LAMP dose-responses obtained by measurement of “bulk"
fluorescence (conventional fluorometry) and responses for two color channels obtained by . As is
evident, the color method shows a response comparable to that of fluorimetry.
When the color images were analyzed and calibrated according to methods described herein using
all thee color channels, the close correspondence of the calibrated color signal with the cence signal is
t as shown in Figure 91.
Example 2: System maximizing sample utilization
A system for maximizing sample utilization can have the following characteristics:
1. Efficient tion of blood into plasma and efficient recovery of the plasma
a. Separation is ed by fugation in a capillary tube
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2. Dilution of the plasma to a few tablished levels appropriate to both high and low
sensitivity assays
3. zing the volume of each assay reaction mixture ed for each assay
a. Using an open-ended low volume cuvette le for assay incubations while precluding
evaporation
i. Cuvette is long relative to width
b. Within said low volume cuvettes enabling increase in assay signal sensitivity by
modifying the optical pathlength
] i. Cuvette is conical or has features where the width is wide and narrow
c. When needed, achieving said increase in assay signal sensitivity by moving the reaction
product (which does not fill the cuvette) to selected locations having greater pathlength at the time of optical
measurement
] i. Cuvette internal volume is much larger than the volume of the assay mixture
4. Use of either or both variable pathlength and 3-color channel analysis to increase the useful
dynamic range of assays
Example 3: Point-of-care assay device
A point of care assay device can e of single-use disposable cartridges an instrument which
processes samples and operates the assays and a server remote from the instrument, the measurement and
detection system comprising:
- A able cartridge containing
- Sample-acquisition and metering methods (such as a sample tip)
' An instrument housing containing
- A light imaging sensor (such as a cell-phone camera having a light source (e.g., a flash)
and a CCD image ting device)
- A mechanism for moving said tip to a location where said light imaging sensor can
acquire images
- ing said images wirelessly (or by other methods) to a server remote from the
instrument
- Image reting software capable of:
- Measuring volumes from the two-dimensional images
- Distinguishing sample types
- Using said sample type and/or volume data as part of an ing algorithm to:
- Provide prompts to system users as to sample integrity
- Provide any needed prompts to provide an augmented or replacement sample
- Interpret signal data from said instrument in terms of assay results making allowance for
sample type and/or sample volume
The system can optionally include additional mechanisms for processing and/or imaging of
samples acquired by users into a “sample acquisition device (capillary)" comprising:
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- isms for ing the capillary in a defined location and moving said capillary to
another defined location where an image can be acquired
' Mechanisms for ejecting substantially all the sample into the said cartridge at a defined
location
Example 4: Analysis of a agy containing a blood sample
s are acquired by users by touching the distal end of the capillary to a drop of blood
(typically as a fingerstick). The capillary usually fills by capillary action provided there is sufficient blood. In
one version of the invention, the user places the ary at a latching location on the cartridge then inserts the
cartridge onto a slide in the instrument then activates an assay by pressing a screen button on the instrument
GUI. All uent assay steps are automated. The instrument moves the cartridge inside its housing and
closes the door through which the cartridge was inserted. In this version of the invention, the instrument moves
a component for grasping the ary and moving it to a location in front of a digital camera equipped with a
flash light source a CCD. An image of the capillary is taken using flash nation and sent ssly to the
server which interprets the image in terms of type, location and quantity of sample. If preset criteria are met the
server instructs the instrument to continue the assay. If the sample is not appropriate the capillary is returned to
the cartridge which is ejected and the server causes the GUI to display an appropriate prompt. The user may
then (1) add more sample or (2) obtain a fresh sample and use a new capillary. Once the user indicates by the
GUI that corrective action has been taken and the capillary/cartridge has been re-inserted into the instrument, the
server instructs the instrument to resume assay processing. The criterion for appropriate volume of sample is
usually that the volume is more than the minimum required for the assay. Thus in some assays for example, 10
uL of sample can be used, so typically the sample is regarded as adequate if the measured volume is > 12 uL.
In a second version of the invention which may be implemented alone or in combination with the
first, image acquisition is used to measure the volume of sample taken from the al sample by the
instrument. In the assay ce, sample is ejected from the capillary into a sample well in the cartridge either
(1) by the user, or (2) by the instrument. Then an exact volume is taken from sample well using a second tip
either by capillary action or (preferred) pneumatic s. At this stage the type of the sub-sample and the
sub-sample volume is ed (as above) by imaging the tip. If the sample type and volume is acceptable
(target +/- < 5%), the assay proceeds. If not, the assay may be aborted and the user prompted to take remedial
action. Sample types that may be discriminated are blood and plasma or serum and others. The imaging system
makes the distinction by observing the much greater st between blood e) and the tip parent)
than is the case for plasma and serum. In the event that the sample volume while not at the target level is still
sufficient for the assay to give satisfactory results (in the above example, a volume > 5 uL would be acceptable
if the target volume is 10 uL). The assay algorithm that calculates the analyte concentration then uses a
correction function Conc. (true) = Conc. (observed assuming target volume) *Target volume/Measured volume.
Blood can easily be detected and its volume measured by creating a pixel map of the tip and
counting the dark pixels then comparing with a previously established number for the target volume. Even
though sample types serum and plasma (and other s non-blood samples) are transparent, the imaging
system can still detect the presence of sample due to the change in refraction that occurs over the sample
meniscus and the difference in refractive index between the tip material and the sample. Alternatively a dye
[Annotation] eaa
may be added to the sample by providing a dried dye formulation coated within the capillary which is dissolved
by the sample
Other s for measuring the volume of sample include locating the top and bottom of each
meniscus and using simple geometric techniques (as described herein). Bubbles within the sample liquid
column can be recognized and measured by the methods discussed above and the appropriate volume subtracted
from the total volume ed by the sample.
The methods given above measure the sample within the sample capillary or pendent from the end
of the capillary (as described herein). After the sample is measured and accepted by the system it is ejected by
pipetting/pneumatic methods within the ment. Once this has happened, the tip can be imaged again and
any residual sample measured. The volume that actually is used in the assay is the difference between the total
volume and the residual volume.
Another particular problem in FCC assay systems especially when used by non-technically trained
users is the ce of sample on the outside of the sample ary.
This can be imaged and measured using the invention and the user prompted to remove excess
blood.
The iveness of sample ition and delivery in assay devices depends on the liquid
handling techniques used. Automated s may use (1) pneumatic tion and ejection (as in many
laboratory single and multi-channel pipetting devices that use disposable tips; pneumatic s may use
positive or negative pressure (vacuum)), (2) positive displacement (as in a syringe), ink jet-like technology and
the like. Samples and other liquids such as reagents can be (3) drawn out of oirs by capillary action or (4)
wicking into porous media. Liquids (samples and/or reagents) may be ejected with or without contacting other
liquids. For example, if the sample is to be d, the sample tip can be dipped into the diluent or displaced
into air so as to drop into a dry well or a well containing diluent. The performance of all of the above systems
and methods may be verified and/or measured using the invention.
In other embodiments, the ary can be imaged by a user outside the instrument with an
external camera. Volume measurements can be scaled to the size of the capillary. Such an externally oriented
camera can be used for recognition of the user/patient so that s can more ly be attributed to the
correct patient. The method may also be used to verify appropriate medication is being used (image the pill
container or pill or alternatively the bar code reader in the instrument may be used for this purpose).
The invention may also be used to measure location and volumes of reagent aspirated into assay
tips. In some cases dyes may be added to reagents to make them more easily imaged (improved contrast).
In assays where plasma is separated from blood, the invention can be used to verify the
effectiveness of red cell removal and the available volume of plasma. An alternative to moving the sample
containing tip is to move the camera
Such a system can have the following advantages:
1. Quantitative measurement of sample
2. Ability to identify the sample type
3. Creation of an objective, tative record of sample volume
4. Enables assays to give results when sample volume is not correct
5. es reliability of assay system
[Annotation] eaa
Example 5: Tips
Figure 18 shows diagrams of tips used to aspirate samples and reagents (dimensions in mm).
Example 6: Geometry measurements of a cylindrical capillary
Figure 19 shows dimensions of a cylindrical capillary containing a sample.
R = radius
L1 = distance from lower end of cylinder to lower sample meniscus
L2 = distance from lower end of to lower upper meniscus
Volume introduced = 71*(RA2)*(L2 - Ll)
Example 7: ry measurements of a conical capillapy
Figure 20 and Figure 21 shows dimensions of a conical capillary.
Rb = radius at base of cone
L = length
L1 = ce from (projected) top of the cone to lower sample meniscus
L2 = distance from (projected) top of the cone to lower upper meniscus
Volume introduced = 71* (Rb/L)A2*[(L1)A3 - (L2)A3]/3
Tan 6 = Rb/L
e 8: Effects of liquid meniscus
As is well known, liquids in aries typically have a curved meniscus. Depending on the
t angle the meniscus may be curved inward or outward relative to the liquid. When no net external
pressure is applied, if the capillary surface is hydrophilic (contact angle < 71/2) the meniscus is inward directed
or outward directed if the surface is hydrophobic (contact angle > 71/2). When net pressure is applied to the
liquid column lary oriented vertically or pneumatic pressure applied by the instrument) the lower meniscus
can project below the lower end of the capillary. In small diameter capillaries, surface tension forces are strong
relative to the small gravitational force across a meniscus. Surface tension pressure across a meniscus in a
vertically oriented ar capillary is 27t*R*y*cos0 where y is the surface tension and 0 is the contact angle.
Pressure across a meniscus caused by gravity is pgAL/(71*RA2) where p is liquid density and AL is the distance
across the us and g is the gravitational constant. Accordingly the meniscus surface is spherical. The
volume of liquid in the segment(s) occupied by the us (menisci) can be ated as s and used to
obtain a more accurate estimate of volume.
Distances defined from the bottom of the sample capillary
L1 = ce to the bottom of the lower meniscus
L2 = distance to the top of the lower meniscus
L3 = distance to the bottom of the upper meniscus
L4 = distance to the top of the upper meniscus
Volume of a spherical cap
] I‘m-{3&2 +8335“
Dimensions of a spherical cap are shown in Figure 22.
Several different situations can arise defined by the number and location of the menisci. Note that
the formulae given below deal with both inward and dly curved menisci.
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Case 1: Upper meniscus is curved, lower is horizontal (as shown in Figure 23)
Substituting: a = R, h = L47 L3
Volume between L3 and L4 = n*( L47 L3)*(3*(R) 2 + (L47 L3) 2)/6
Total volume = 2)*L3 + (L47 L3)*(3*(R) 2 + (L47 L3) 2)/6)
] Case 2: Both menisci are within the capillary and curved (as shown in Figure 24)
Total volume = 7T*((R2)*L3 — (L2 , L1)*(3*(R) 2 + (L2 , L1) 2)/6 + (L47 L3)*(3 *(R) 2 + (L47 L3) 2)/6
Case 3: There are two curved menisci. The lower being curved and below the lower end of the
ary (as shown in Figure 25)
Total volume = 7T*((R2)*L3 + (L1)*(3*(R) 2 + (L1) 2)/6 + ( L47 L3)*(3*(R) 2 + (L47 L3) 2)/6)
e 9: Bubbles
Bubbles in liquid samples or reagents cause variable reductions in volume of liquid metered. In
small capillaries bubbles when smaller than the capillary cross section, are cal. When they are bigger they
occupy a cylindrical space (in a cylindrical capillary) and have hemispherical ends.
Case 1: Bubble is not big enough to span the width of the capillary (as shown in Figure 26)
Subtract bubble volume = ‘7T"‘r3
Case 2: Bubble occludes the entire width of the capillary (as shown in Figure 27)
Subtract bubble volume = 471"“R3 + L
Example 10: Blood outside the capillapy tip
Case 1: Pendant blood or reagents outside a vertical capillary can cause major problems in assays
since it represents an -control situation. As shown in Figure 28, imaging can easily ize this
situation.
Case 2: Blood outside the capillary other than pendant
Residual blood outside the capillary also is problematic since it is a potential source of
contamination of reagents and of extra volume. Again imaging can recognize the situation.
Example 11: Residual blood inside the capillagy once sample dispensing has occurred
This can be dealt with by ting the residual volume and subtracting from the total sample
volume. Figure 29 shows an example of a capillary with residual blood.
Residual volume = 71*R2*L
e 12: Evaluation of red cell separation from blood samples
In many assays it is desirable to remove red cells from the sample thus making plasma. When this
is done, it is desirable, especially in POC devices, to know that the separation was effective and to determine
that there is sufficient plasma to perform the assay.
Figure 30 - Figure 39 show a schematic of one preferred embodiment of red cell removal suited to
POC devices of the present ion. Magnetizable particles have antibody to red cells mixed with free
antibody to red cells are provided as a dried preparation in the well that will receive a blood sample, as shown in
Figure 30. When a blood sample is added to the well (shown in Figure 3 l) and mixed with the magnetic reagent
(shown in Figure 32, Figure 33, and Figure 34), the red cells agglutinate with the magnetic particles and can be
d by g the blood-containing well in proximity to a strong magnet (shown in Figure 35). By
appropriately moving the well relative to the magnet, the red cells are separated from plasma (shown in Figure
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36) which can then be ejected into a receiving well for use in an assay (shown in Figure 37, Figure 38, and
Figure 39). It is evident that the imaging analysis can determine how ively the separation was effected and
estimate the volume of plasma available for assay.
Example 13: Images of liquid samples in capillaries
Figure 40 shows a high contrast image of a cylindrical tip containing a liquid with low absorbance.
Figure 41 shows an image of a l tip containing a liquid with high absorbance.
Figure 42 shows a tip with a high absorbance liquid showing two i within the tip.
Figure 43 shows a tip with a sample liquid and large bubbles that span the diameter of the tip.
Figure 44 shows a tip containing water showing a clear upper meniscus in a transparent tip or
capillary.
Figure 41 was analyzed for the length of the liquid column (corresponding to 5 uL) and the
resolution of the upper liquid meniscus (Definition of the position of the meniscus with > 90% confidence). The
precision of the meniscus location corresponded to < 1% of the length of the liquid column.
tion of meniscus
Example 14: Effect of insufficient sample volume an assay result
The system was used to measure Protein-C in blood. The sample volume inserted into the system
was designed to be 20 uL when the sample transfer device was used properly. The instrument was set up to use
lOuL of blood from this sample. The analyte concentration calculated by the system is shown in Figure 45 as
the sample volume was deliberately sed from the target level. The result was essentially constant until the
sample volume was less than the volume required.
Example 15: Sample transfer device
Figure 46 shows an e of a sample transfer device. The device consists of (a) a capillary
(made of glass or plastic) optionally coated with an anticoagulant or other reagent le for pre-analytical
treatment of samples, (b) a g which holds the capillary fitted with (c) a plunger (piston) which can slide
within the housing and has a raised feature which slides within a groove in the housing, (d) a groove in the
housing which engages the piston feature and limits the axial motion of the plunger so that its motion stops once
the sample has been displaced and (e) a vent in the g normally open which is blocked when the plunger is
activated (moved towards the distal end of the device) so as to ce any liquid in the capillary.
Figure 47 shows a sample transfer device with its capillary filled with sample. The “fill to"
location is indicated.
Figure 48 shows a sample transfer device with sample displaced by movement of the plunger.
Figure 49 shows a sample transfer device after a sample has been letely ejected.
Example 16: Measurement of volume by image analysis
Known volumes of a liquid sample were aspirated into sample tips using a pipetting device.
Images of the tips were collected using a cial flatbed scanner (Dell) and the distances (a) from the distal
end of the tip to the meniscus and (b) from the distal end of the tip to the feature marked on the image below
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measured. The orientation of the tip and its position relative to the scanner platen were not controlled since the
image was analyzed by measuring the ratio of distance (a) to distance (b) as a measure of sample volume.
ons of the tip, meniscus and feature were measured using commercially imaging software (Jasc). The
image was oriented horizontally using the software before the locations were recorded and could be read
directly on a scale ed by the software. Figure 50 shows an exemplary image.
] Distance Ll was the on of the tip
Distance L2 was the location of the meniscus
Distance L3 was the location of the arrow indicated in Figure 50.
V01.——Ratio Calc. V01.
uL ALZ-l AL3-1 uL
.0 120 374 590 254.00 470 0.540426 9.7
12.5 112 400 584 288.00 472 69 12.9
.0 156 470 636 314.00 480 0.654167 15.2
17.5 171 505 654 334.00 483 0.691511 17.3
.0 114 469 596 355.00 482 0.736515 20.0
.0 214 m 694 386.00 480 0.804167 24.5
.0 165 585 640 420.00 475 0.884211 30.3
As shown in Figure 51 the volume was simply d to the ratio of distances and could be
calculated. The volume estimate was within less than 2% of the actual volume on average.
Exam le 17: Ima es of blood centrifu ed in a ti
Hematocrits were determined from digital images by measuring the ratio of length of the column
of packed red cells and the total liquid column (tip to meniscus). This is easily achieved by feature recognition
re which orients the tip to a known direction and counts pixels between the features. For the tips above,
the distances corresponded to several hundred pixels permitting a precise measurement.
Figure 10 shows an empty capped sample tip.
Figure 11 shows a capped sample tip containing a sample of blood.
Figure 12 shows a capped sample tip containing a sample of 23% hematocrit blood after
centrifugation.
Figure 13 shows a capped sample tip containing a sample of 31% hematocrit blood after
centrifugation.
Figure 14 shows a capped sample tip containing a sample of 40% hematocrit blood after
centrifugation.
Figure 15 shows a capped sample tip containing a sample of 52% hematocrit blood after
centrifugation.
Figure 16 shows a capped sample tip containing a sample of 68% hematocrit blood after
fugation.
Figure 17 shows a comparison of hematocrit measured using by the lly imaging system a
fuged sample (hematocrit, % reported) and hematocrit measured by rd techniques (hematocrit, %
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target). An example of a standard technique of hematocrit measurement may include ematocrit
measurement in a glass capillary using a rd laboratory centrifuge and measuring the length of the packed
red cell bed and the total length of capillary occupied by the sample.
Example 18: System including components for blood separation
A system designed for separation of blood can include the following features:
1. Design of the tip shape.
a. Aspect ratio about 20:1 lengthzdiameter provides convenient s for measurement of
sample, packed cell and plasma volumes
b. Shaped tips enable tight sealing with a vinyl cap and easy removal of cap if needed.
c. Slight draft angle conical shape of main part of tip and wider conical upper section of tip
" design (tip is
are optimal for insertion of the plasma recovery means. Note that the “counter radia narrower at
the end distal to the axis of rotation) is unusual.
d. Wider conical upper section of tip is configured to be odated on an automated
pipetting and x-y-z movement stage. Connection to form a fluid tight connection and easy removal when
needed are facilitated.
2. Use of a very e and te x-y-Z stage to move the plasma recovery tip.
3. Use of imaging technology automatically to control the operations of centrifugation and
plasma ry. Movement of the plasma recovery tip to within less than a millimeter of the packed cellplasma
interface.
Example 19: Use of image ement of liquid volumes to improve assay calibration
Automatic pipetting devices are generally accurate and e to about 5 % or better in the range
(say) 5 , 50 uL. In many assays, volume cy and precision have both to be very good (say < 2 %) to
obtain the required cy and precision of analyte measurement. ng and delivery of (l) liquids at
volumes less than 5 uL (highly desirable when maximum use of a small volume sample is required), and (2)
liquids having “problematic" physical properties (such as viscous solutions, solutions containing detergents etc.)
can often have poor precision and accuracy which compromises the cy of assay results. One inventive
solution to these issues is to use image analysis of the liquids to measure the volume of liquids es,
diluents, reagents, calibrators and controls) and then to correct the assay calibration function to allow for
deviations (up to [say] 20%) from the intended volume. We have shown that in pipette tips which have very
precise dimensions, volumes as small as 5 uL can be measured with very good accuracy and precision (< 2%).
Below we document (1) volume measurement accuracy and precision and (2) use of known relationships
n the volumes of solutions used in assays es, reagents etc.) and assay response to correct the
calibration of .
(1) Accuracy of volume measurement by image analysis:
In the table below, known volumes of a solution of bromphenol blue were aspirated into conical
tips.
The solutions were positioned in the middle portion of the tip and imaged using a scanner. Tip
orientation and position were determined by standard methods. The tip orientation was adjusted by an algorithm
and the length of the liquid column measured. Using the known internal dimensions of the tip, the liquid
[Annotation] eaa
volume was calculated. Four replicate images were taken for four aliquots in four tips. The error given below
therefore reflects image ucibility and tip dimensional accuracy and precision.
Target Measured Total
volume volume Error
Note that the volume measurement does not rely on accurately positioning the liquid in the tip.
Image analysis provides information as to the location of the liquid in the tip. Knowing the ions of the
tip one can always compute the volume from the portion of the tip occupied by the liquid wherever it is.
] (2) Correction of assay calibration by incorporation of liquid volume measurement
] This is achieved as illustrated in the following simulation. Consider an assay in which a sample is
combined with two reagents (called 1 and 2). The target volume for sample, reagent 1 and reagent 2 is 10 uL.
The assay result is calculated from a standard curve relating measured signal to analyte concentration. As part
of the assay calibration process the experiments can be med in which volumes used for sample and
ts are changed to 8 and 12 uL in addition to 10 uL and assay results calculated based on the ation
appropriate for 10 uL volumes. In Figure 105, Figure 106, and Figure 107, the results were plotted against
actual volumes. For the sample volume, the assay result is essentially directly tional to the volume used
(shown in Figure 105). For the reagents, somewhat non-rectilinear responses were seen (shown in Figure 106
and Figure 107). These responses are based on “typical" assays nown in the field and the magnitude of the
changes with volume are representative.
We then simulate an evaluation in which we have imposed a degree of random error (about 5 %, to
reflect a “real world situation") on assay results in addition to the effects of the use of inappropriate volumes.
We include results in which sample, reagent 1 and reagent 2 s are set at 8, 10 and 12 uL in all
combinations. When the results from this exercise are plotted as shown in Figure 108 without correction for
volume errors, as would be expected there is a significant error in the reported result due to ignoring the fact that
the actual volumes used were different from those used for assay calibration.
When we allow for the volume variances from target values using the known assay response to
volumes, and plot ted analyte values we obtain the much improved result shown in Figure 109, however.
This was achieved by multiple regression analysis of the data.
Summary statistics comparing results calculated with and t volume correction are presented
in the table below and reflect a significant improvement in all metrics by the use of volume correction. SEE is
the standard error of the estimate.
[Annotation] eaa
Regression ation
Volume correction coefficient Analyte CV SEE/Mean
—————
Exam le 20: Hema lutination inhibition assa read usin microsco and ima eanal sis:
In phosphate buffered saline (100 uL), containing 0.3 % w/V glutaraldehyde stabilized turkey red
blood cells, and 0.5 mg/mL bovine serum albumin and, where indicated, 2 hemagglutination units of
inactivated za Virus and 15 ug goat polyclonal anti-influenza B antibody were ted for 15 s
in a conical bottom PCR tube at RT. The reaction product from the bottom of the tube was transferred to a
transparent slide illuminated with white light and imaged with a 4-fold cation using a digital camera. As
may be seen in Figure 131, ination caused by reaction of the red cells with the hemeagglutinin of the Virus
(Figure 131 sample 3) is easily observable by comparison with an unagglutinated control (Figure 131 sample 1).
When excess antibody to the virus was also present agglutination was completely inhibited. Two effects of the
agglutination reaction are notable: (1) in inated samples, there are more red cells due to the more rapid
sedimentation of the agglutinates compared with the control and (2) each red cell is on average more clustered
with other red cells. The ination reaction can be quantified following image recognition software to
identify, locate and count red cells and agglutinates.
Figure 131 sample 1 shows a non-agglutinated control (no Virus, no antibody). Figure 131 sample
2 shows non agglutinated sample (Virus plus antibody). Figure 131 sample 3 shows an agglutinated (Virus, no
antibody) .
Example 21: Sample preparation, ation of supernatant Quality and tion of the Quality of the
LDL precipitate
] Plasma was diluted (1:10) into a mixture of dextran sulfate (25mg/dL) and magnesium sulfate
(100mM) then incubated for 1 minute to precipitate LDL-cholesterol. The reaction product was aspirated into
the tube of a centrifuge, capped then and spun at 3000 rpm for three minutes. Images were taken of the original
reaction e prior to centrifugation (showing the white precipitate), following centrifugation (showing a
clear supernatant) and of the LDL-cholesterol pellet (after removal of the cap). For example, Figures 132, 134
illustrate examples of images taken of reaction product.
Images of the LDL-precipitation reaction product were ed as follows. The pixel color levels
were d as a function of their vertical position. The variance of the values was measured and values for the
three colors . Because of the particles of precipitated LDL, which strongly scatter light, the precipitate
value (1154) was much greater than (672) of the clear supernatant. Comparison of the supernatant value with
that of a l no exposed to the precipitation reagent allows the quality of the centrifugation to be evaluated
(data not shown). Figure 133 provides examples of images that were analyzed before spinning in the centrifuge,
and after spinning in the centrifuge.
After removal of the black Vinyl cap, an image of the LDL precipitate was taken. Its volume can
be measured quite accurately, knowing the geometry of the tip and the size of a pixel in the image. In this
experiment, the volume of the precipitate was estimated as 0.155 +/- 0.015 uL.
[Annotation] eaa
e 22: Improving the performance of an assay for alanine aminotransferase (ALT) by use of 3-
color image blanking of optical signals due to the sample
ALT in serum can be measured in an assay in which the enzyme ts alanine to pymvate
which is in turn used to make hydrogen peroxide with oxygen and pymvate oxidase. The peroxide is then used
to make a colored t by the enzyme horseradish peroxidase, aminoantipyrene and l-N- (3-
sulfopropy1)ani1ine. The colored product absorbs maximally at 560 nm.
It was found that some serum samples have cant ance at this wavelength as shown in
Figure 135 and accordingly interfered with the assay. In particular when using relatively high sample
concentrations (such as a final dilution in the assay of 1:10), 3-color image analysis of the ALT assay gave poor
results with clinical samples.
Figure 135 illustrates spectra of several serum samples diluted 1:10 into buffer; OD is plotted
against wavelength (nm). Great variation of OD is i11ustratedin Figure 135.
In conventional spectroscopy, this issue is dealt with by taking a blank reading of the sample
t the assay ingredients added and subtracting the blank from the signal generated by the assay. In 3-color
image analysis as in the t invention, it has been found that an analogous method can be used. The diluted
sample may be imaged and three-color values extracted. The assay calibration thm may then be changed to
include the signals from the unreacted . Specifically in this case, the original algorithm (not including
sample blank ) was ALT concentration a b*R c*G d*B e"‘R2 where a, b, c, d and e are
empirically derived nts and R, G, B are the signal values in red, green and blue channels respectively. The
improved algorithm was: ALT tration a b*R c*G d*B e*Rs, i f“‘Rs2 where Rs is the signal
from the sample blank in the red channel (note that the empirically derived values of a, b, c, d and e were
different from those of the original algorithm).
When 21 serum samples ranging in ALT activity from 0 to 250 U/L were measured in triplicate
and results of 3-color image analysis compared with those provided by a clinical laboratory method (Teco) the
following regression statistics were obtained indicating much-improved results.
Calibration method .pam
Original 0922 0922
Improved 0.972 0972
Exam le 23: S eedin -u and rovidin ob'ective anal sis ofahema lutination inhibition assa for
anti-influenza antibodies
Reaction mixtures prepared as described in Example 20, were incubated for only one minute
then introduced into three separate micro channels (as described for the cytometry examples above) and
imaged. About 5-10 images are taken for each sample, in order to get te statistics. On average,
each image ts of around 800-900 cells. The agglutination process can be objectively ted
by ing the radial distribution of cells around a representative selection of individual cells using the
function.
The images were processed to obtain centroid positions of the individual cells in 2D space. The 2D
positions were used to compute a radial distribution function (RDF), also known as a pair correlation fimction.
[Annotation] eaa
The radial bution function, g(r) quantifies the probability of finding a cell at a distance r from the selected
cell. Mathematically,
g(r)= p(r)/ pa
where p(r)27:rdr is the number of cells found at a distance of r from a particular cell and p0 is the
average cell density over the entire image window. The value g(r) is calculated as an average over all
particles in the image and over multiple images, to ensure a statistically meaningful result.
Results
The value of the first peak of g(r) quantifies the number of doublets in the sample. Hence, the g(r)
for the inated sample should be higher in magnitude compared to the other two samples. The value ofg
rises quickly from 0 to a maximum over a distance of about 20 pixels (corresponding to about 12 um about
twice the diameter of a red cell) then declines to about 1.0. As shown below, the agglutination due to Virus was
distinguishable from no ination when Virus is absent or antibody inhibits the Virus-induced agglutination.
Ol’lC
—._—
Example 24: Preparation of e detection systems using aptamers
Two oligo DNA aptamers were designed to selectively capture proteins (thrombin and insulin).
The oligo DNA aptamer was composed of a binding site having a sequence selected from published data, an
inert portion to extend the binding site from the surface of the bead or microarray, and a reactive group to
chemically lize the aptamer to the surface. Aptamer 1 (specific for thrombin) had the following
sequence: 5’7Am-(T)45GGTTGGTGTGGTTGG73’. Aptamer 2 (specific for insulin) had the ing
sequence: 5’7Am-(T)32ACAGGGGTGTGGGGACAGGGGTGTGGGG73’. The “Am" at the beginning of
each sequence ents an amino group.
The two aptamers were immobilized on polystyrene beads (Sum) functionalized with carboxyl
groups. The beads were then washed and the excess t removed. The beads were then mixed with oligo
DNA probes fluorescently labeled and complementary to the binding sites of the aptamers. Hybridization of the
probes with the aptamers was detected by fluorescence emission. Only the complementary probe showed a
positive hybridization event, as measured by mean fluorescence emission. Hybridization specificity is
illustrated by comparison of Figure 139, which shows beads after hybridization with mentary probe, to
Figure 140, which shows beads after hybridization with non-complementary probe. Detection was performed
with a laser excitation at 635nm, and on filtered at 650nm m) on a CCD camera, after deposition of
the beads on the analysis substrate. A similar procedure was used on a glass e coated with epoxysilane to
immobilize rs 1 and 2. The array was hybridzed with the cent probe and the specific recognition
of the aptamer binding site measured by fluorescence emission detection with a CCD camera set-up and an
Array Scanner ys). Figure 141 illustrates the binding specificity of the aptamers on the array, with more
detail illustrated in Figure 142. Figure 143 shows an example array scan.
[Annotation] eaa
Example 25: ion of e using aptamers
An array comprising Aptamer 1 hybridized with fluorescently labeled, complementary probe was
prepared as in e 24. Thrombin was introduced to the array at a concentration of about 100nM and
allowed to react with the Aptamer 1-probe complex. The fluorescent emission signal from Aptamer 1 on the
array was d by 2.5 fold, indicating displacement of the probe by binding of Aptamer 1 and thrombin.
Example 26: binders
Two types of binder are biotinylated and used to create capture surfaces on an assay unit solid-
phase coated with avidin. Assay reagent production and luminescence-readout assay results are obtained using
(1) aptamers and (2) single-chain Fv antibody fragments ) on microtiter plates. Aptamer and SCFVS as
binders for luminescence-based assays are adaptable to tips and imaging systems and devices ed herein.
Analytes can be assayed and read using cameras to measure color by changing the signal-generating reagent
from ne phosphatase to, e. g., horse-radish peroxidase with a chromogenic substrate or using ne
phosphatase with a chromogenic substrate. Tips in microtiter plate (or other formats) can be read in any
cartridge (assay unit) format.
Example 27: Vitamin D Assay Using DNA Aptamers on Microtiter Plate
In this example, an assay for vitamin D is performed using single-stranded DNA aptamers.
Biotinylated DNA aptamers are coated on a idin coated polystyrene surface of a microtiter plate having a
plurality of wells. Before coating, the aptamers are quickly denatured and renatured by heating at about 95
degrees Celsius, then immediately cooled on ice. About 15 microliters of the refolded biotinylated vitamin D
DNA aptamers in 25 mM Tris, containing NaCl, MgCl2, 10% Ethanol, pH 7.5, are then added into each well to
form the capture e. After coating, the wells are washed and blocked with about 100 uL of a blocking
reagent to reduce nonspecific g.
The analyte for the assay (vitamin D) is diluted in Tris, NaCl, MgC12, 10% Ethanol, pH 7.5, and is
mixed with a solution of vitamin D-A1ka1ine Phosphatase conjugate at different concentrations in the desired
assay range, and provided to the assay unit for 10 minutes tion at room temperature. The assay unit is
then washed three times with 100 uL of wash buffer. About 40 uL of substrate for Alkaline phosphatase is
added to each assay well and chemiluminescence data (table below) is ted after about 10 minutes. Figure
144 is a plot of chemiluminescence against the tration (ng/ml) of vitamin D.
Vitamin D (ng/ml) 0 l 100 200
Chemluminescence (RLU) 1556741 1138243 49346.13 27
159471 1107942 49699.04 35794.18
1626503 1016557 53158.25 36655.66
159920.8 99266.41 50195.63 35166.41
1594291 1 50599.76 35360.13
1.80 6.60 3.44 3.37
100% 67% 32%
[Annotation] eaa
Example 28: iol Assay 0n Microtiter Plate
In this e, an assay is performed for a steroid hormone (estradiol) using single-chain variable
fragments (scfv). In this assay, the inner surface of the assay unit is coated with biotinylated scFv on ultravidin
coated polystyrene surface of a microtiter plate having a plurality of wells. About 15 microliters of 1 ug/ml
biotinylated scFv in Tris buffered , pH 8, 0.03% BSA, 0.05% Thimerasol were added to each assay unit.
After washing, each assay unit is fixed with 100 uL Fixative reagent ed by an overnight dry with dry air
and stored dessicated.
The analyte for the assay (free estradiol) is diluted in Tris buffered Saline, pH 8, BSA, Thimerasol,
mixed with an estradiol-Alkaline atase conjugate, in stabilizer from Biostab, and is provided to the assay
unit coated with the scfv for about 10 minutes at room temperature.
The assay wells are then washed 5 times with 100 uL of wash buffer. After the washes, 40 uL of
luminogenic ate for Alkaline phosphatase (KPL aGlo) is added to each assay unit and
chemiluminescence data (table below) is collected after about 10 minutes. Figure 145 is a plot of
uminescence against the concentration (pg/ml) of estradiol.
5505.454 85 493.864 389.863
Chemluminescence 5505.454 2005.112 496.932 374.317
(RLU) 5659.613 1739.771 503.25 417.021
Example 29: White blood cell count and ential assay
The concentration of white blood cells (WBCs) in the peripheral blood of human subjects can
range from about 1000 ul to 100,000 cells/ul. However, in some cases the range of the imaging system is
more limited, such as from about 4000 cells/ul to 7000 cells/ul. If the cell concentration is less than 4000
cells/ul, the system may not be able to enumerate a target of 10,000 cells, as may be required by the assay. If the
cell concentration in the sample is more than 7000 cells/ul, each field of view may be too crowded to perform
accurate image segmentation and cell enumeration. An exemplary approach for imaging WBCs is provided
below.
In an example, an imaging system (e.g., cytometer) is provided configured for fluorescence
spectrophotometry. The system uses fluorescence spectrophotometry to measure the cell concentration in the
sample. The sample is labeled with fluorescently conjugated antibodies for imaging (e.g., AF647-CD45) and
also with a fluorescent nucleic acid marker (e.g., DRAQ5). A quantitative fluorescence readout on the
spectrophotometer module provides a measurement of the concentration of WBCs at low sensitivity (LLOQ of
about 5000 cells/ul) but high c range (e. g., 5000-100,000 cells/ul). A concentration measured on the
spectrophotometer allows the calculation of the l dilution ratio such that the final concentration of the cell
suspension is between 4000-7000 cells/ul.
—104—
[Annotation] eaa
] Figure 146 shows the high dynamic range of fluorescence in the spectrophotometric measurement
ofWBC concentration. WBCs tagged with fluorescently d anti-CD45 and other antibodies were excited
with red light having a wavelength of about 640 nm and the quantitative fluorescence emission spectrum was
ted. Integrated fluorescence is plotted on the y-axis.
Example 30: Streptococcus Group A Detection by Isothermal Amplification
] Isothermal amplification of specific genomic samples can be detected by turbidity. In this
example, a genomic sample extracted from ococcus group A (StrepA) cells (stock concentration = 2x108
org/ml from My biosource) was amplified by isothermal amplification and the progress of the reaction measured
by Turbidity. About 5 ul of stock bacterial cells and 45 ul RT PCR grade Water (10X dilution of stock) were
heat treated at about 95 degrees Celsius from about 8 to 10 minutes (Cell ruptures and releases the DNA). The
genomic sample was diluted and introduced in a sample volume of about 25 ul in a PCR tube containing
reagents for amplification (e.g., DNA rase, Primers, Buffer). The PCR tube was incubated at about 61
degrees Celsius for about 60 minutes while the progress of the reaction was ed by turbidity. The results
are as follows, and Figure 147 shows plots of turbidity as a function of time:
Conc. St. dev
(copies/uL) T(min) (min)
Three separate experiments were ted at 800 copies/uL and 80 copies/uL. Experiment A
was performed using StrepA having a synthetic c DNA template (from Genescript). Experiment B was
performed by diluting stock StrepA 10-fold followed by heat inactivation at 95 degrees Celsius from about 8 to
min, and followed by serial on of heat inactivated ten-fold diluted stock . Experiment C was
performed using a variable concentration of stock StrepA (inactivated bacterial cells) followed by heat
inactivation at 95 degrees Celsius for about 10 min. The inflections points for each experiment are shown in
Figure 148. For each of 800 copies/uL and 80 copies/uL, a grouping of three plots includes Experiment A at the
left, Experiment B in the middle and Experiment C at the right. The average inflections points are provided in
the following table:
-———
-Ave Ave Ave
-800 cp/uL---“m
Example 31: use of magnetic beads
] In this example, magnetic beads are used for the is of proteins and small molecules via
ELISA assays. Figure 110 schematically illustrates an exemplary method for the ELISA assay. The assays
include two proteins, Protein 1 and Protein 2. Protein 1 has a sample on of about l50-fold (tip protocol =
[Annotation] eaa
-fold), a sample volume of about 0.007 uL, a diluted sample volume of about 1 uL, a reaction volume of
about 3 uL, and a reaction time of about 10 minutes (min) (sample incubation and substrate incubation). Results
for Protein 1 are shown in the ing table. The results of Test 2 for Protein 1 are shown in Figure 149.
Protein 2 has a sample dilution of about 667-fold, a sample volume of about 0.0015 uL, a d
sample volume of about 1 uL, a on volume of about 3 uL, and a reaction time of about 10 min (sample
incubation and substrate tion). Results for Protein 2 are shown in the ing table. The results of Test
1 for Protein 2 are shown in Figure 150.
C0nc(nghnD Test 1 'RMZ CMI
200000 322161
100000 232455 117056 117 E
50000 133290 192460 43286 52415 105
25000 89282 21908 --n
12500 49856 59574 12576 12041 101 “-
4000 15926 18350 4117 4059 103 101
1000 4547 4722 1140 1124 114 112
200 1238 1229 163
504 458 22 21 109 106
0 302 292
It should be understood from the foregoing that, while ular implementations have been
illustrated and described, s modifications can be made thereto and are contemplated herein. It is also not
intended that the invention be limited by the specific examples provided within the specification. While the
invention has been described with reference to the aforementioned specification, the descriptions and
illustrations of the preferable embodiments herein are not meant to be construed in a ng sense.
Furthermore, it shall be understood that all aspects of the invention are not limited to the specific
depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and
variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a
[Annotation] eaa
person d in the art. It is therefore contemplated that the invention shall also cover any such modifications,
variations and equivalents.
I
Claims (7)
1. A method of detecting the presence or concentration of an analyte in a sample fluid contained in a container comprising: (a) illuminating the container along a first region having a first path length to yield a first measurement of light intensity transmitted across the first path length; (b) moving the sample fluid to another region in the container having another path length if the first ement falls outside a predetermined dynamic range of transmitted light intensity; (c) illuminating the container along the another region to yield r ement of light intensity transmitted across the r path ; and optionally (d) repeating steps (b) and (c) until a measurement of light intensity falls within the predetermined dynamic range, thereby detecting the presence or concentration of the analyte.
2. The method of claim 1, further comprising deconvoluting a line scan of the image, thereby detecting the presence or concentration of an analyte.
3. The method of claim 1, wherein the sample is moved from a first region of the container having a first path length to a second region of the container having r path length by aspirating the sample.
4. The method of claim 3 wherein an end of the container is ed to a pipette which is configured to aspirate the sample.
5. The method of claim 3 wherein the sample is moved up or down the length of the container.
6. The method of claim 1, wherein the container is a pipette tip.
7. The method of claim 1, wherein the container is conically shaped. 9862739
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ706352A NZ706352B2 (en) | 2011-01-21 | 2012-01-20 | Systems and methods for sample use maximization |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161435250P | 2011-01-21 | 2011-01-21 | |
US61/435,250 | 2011-01-21 | ||
PCT/US2012/022130 WO2012100235A2 (en) | 2011-01-21 | 2012-01-20 | Systems and methods for sample use maximization |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ613457A NZ613457A (en) | 2015-04-24 |
NZ613457B2 true NZ613457B2 (en) | 2015-07-28 |
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