EP1131632A2 - Dispositif et procede d'analyse d'un echantillon biologique - Google Patents

Dispositif et procede d'analyse d'un echantillon biologique

Info

Publication number
EP1131632A2
EP1131632A2 EP99972308A EP99972308A EP1131632A2 EP 1131632 A2 EP1131632 A2 EP 1131632A2 EP 99972308 A EP99972308 A EP 99972308A EP 99972308 A EP99972308 A EP 99972308A EP 1131632 A2 EP1131632 A2 EP 1131632A2
Authority
EP
European Patent Office
Prior art keywords
fluid
sample
microspheres
capillary
fluid component
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99972308A
Other languages
German (de)
English (en)
Inventor
Peter Lea
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Umedik Inc USA
Original Assignee
Umedik Inc USA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CA002254223A external-priority patent/CA2254223A1/fr
Application filed by Umedik Inc USA filed Critical Umedik Inc USA
Publication of EP1131632A2 publication Critical patent/EP1131632A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/491—Blood by separating the blood components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5002—Partitioning blood components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/045—Connecting closures to device or container whereby the whole cover is slidable
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0822—Slides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/168—Specific optical properties, e.g. reflective coatings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis

Definitions

  • This invention relates to a device for separating a fluid component, such as plasma, from a biologic sample, such as blood, using suitable small particles such as microspheres and analyte specific labeling.
  • This invention also relates to a device and method for quantitative determination of an amount of analyte present in biologic fluids.
  • the invention further relates to a quantitative assay method and device for measuring one or more analytes in a biologic fluid sample using a point-of-care assay method and device.
  • the sample could be a suspension which is prepared for the purpose of testing for one or more micro-organisms.
  • the test results can be analyzed using a suitable analyzer and, optionally, the assay test results are transmitted by way of digital transmission systems to permit further evaluation of the data.
  • a common assay is the pregnancy test device which involves contacting a urine sample with a test pad, which urine moves by capillary flow along the bibulous chromatography strips whereby the presence of human chorionic gonadotropin (HCG) will be detected usually as shown by a coloured line because of the reaction between HCG and reagents in the bibulous chromatography strips.
  • HCG human chorionic gonadotropin
  • Nonbibulous lateral flow refers to liquid flow in which all of the dissolved or dispersed components of a liquid, which are not permanently entrapped or filtered out, are carried at substantially equal rates and with relatively unimpaired flow laterally through a stabilized membrane. This is distinguished from preferential retention of one or more components as would occur, for example, in materials capable of absorbing or imbibing one or more components, as occurs in chromato graphic configurations.
  • a sample (which may contain the analyte of interest) is collected on the “sample receiving zone” from which it flows to the “labelling zone” at which point it encounters a specific binding reagent for the analyte coupled to visible moieties (the "assay label"), then flows to a “capture zone” where the analyte bound to visible moieties is captured.
  • Device describes methods and devices for measuring an analyte in a sample mixed with reagents, the devices defining a flow path.
  • the specific binding by agglutination may provide for changes in flow rate, light patterns of a flowing medium, or light absorption or scattering which permit measurement of the analyte of interest.
  • U.S. Patent 5,110,724 issued May 5, 1992, entitled “Multi-Analyte Device” the invention described is an assay device for assaying multiple analytes in a drop-sized blood sample.
  • a dispenser distributes a small volume blood sample to multiple transfer sites by capillary flow of the blood sample through sieving and distributing matrices which separate blood cells from plasma as the sample fluid migrates toward the transfer sites.
  • a test plate in the device carries multiple absorbent pads, each containing reagent components for use in detection of a selected analyte. The test plate is mounted on the dispenser toward and away from a transfer position at which the exposed surface regions of the pads are in contact with associated sample- transfer sites, for simultaneous transfer of sample fluid from such sites to the pads in the support.
  • the invention describes a method for separating plasma from red blood cells.
  • the driving force for the movement of plasma from the filter to the reaction area of a device utilizing the method is capillary force provided by a tubular capillary.
  • a filter is selected from glass microfiber filters of specified characteristics.
  • Apparatus for Carrying Out Specific Binding Assays describes the well known antibody binding to antigen assay.
  • the sample which may contain the analyte (a), (the substance being tested for) is mixed with (b) an antibody which binds to the substance being tested for, which antibody is immobilized on a solid support, and (c) another antibody for the substance being tested for which is conjugated to a detectable marker, to thereby form a complex between (b), the substance being tested for and (c) and causes the marker to be immobilized and detected.
  • the described invention is a method and apparatus for conducting specific binding pair assays, such as immunoassays
  • the test substrate is a porous membrane on which a member of the binding pair is affixed in an- "indicator zone”.
  • the sample is applied and is permitted to flow laterally through the indicator zone and any analyte in the sample is complexed with the affixed specific binding member, and detected.
  • a novel method of detection employs entrapment of observable particle in the complex, for instance, red blood cells of blood can be used as the observable particles for detection of the complex.
  • red blood cells are removed from whole blood samples with a solution containing an acid.
  • the agglutinated red blood cells are then removed from the resulting suspension by procedures of filtration, centrifugation or decantation, leaving an essentially red blood cell-free serum or plasma sample.
  • an analyte is measured along a liquid flow path which includes a number of reaction-containing reaction zones spaced apart along the flow path.
  • Detector means are employed to detect analyte, reactant or predetermined product in the reaction zones, the number of zones in which detection occurs indicating the amount of analyte in the liquid.
  • U.S. Patent 5,536,470 issued July 16, 1986, entitled “Test Carrier for Determining an Analyte in Whole Blood red blood cells cannot gain access from the blood sample application side, to the detection side and on the detection side as a result of an analysis reaction, an optically detectable change occurs.
  • a serious deficiency in current one-step assays for the measurement and/or detection of an analyte is that they provide only qualitative results rather than quantitative results. That is to say that the presence or absence of the analyte may be determined but the actual amount or concentration of analyte present in the sample would still not be known.
  • the assay of the present invention provides quantitative results as the test is performed in a determinable volume. In the prior art methods itjs not possible to consistently identify the exact volume of the test sample in repeated testings since the fluids must wash through the test strips.
  • Prior art methods using chromatographic strips and fiberglass strips require larger initial volumes of the biologic fluid in order to mobilize the proteins and labels in the strips. This is particularly true when the biologic fluid is blood and the plasma must first be separated from the blood sample.
  • An advantage of the device and method of the present application is that very small fluid samples can be used to measure one or more analytes.
  • the assay method and device of the present invention is also advantageous because the test volume can be made constant and therefore repeated testings will yield quantitative data which can be directly compared between samples and within a sample.
  • the assay device and methodology allows for separation of the plasma from the whole blood during the assaying of a fluid sample. In other words it is not necessary to previously separate out the cellular component of the blood before assaying the sample.
  • This is a significant advantage as it allows that the assay can be used at the point of patient care, for example, by the patient themself, at the patient's bedside or in a doctor's office.
  • the device and assay methodology of the present invention a generic point-of-care platform suitable for use in one or more diagnostic or prognostic assays performed on one or more fluid samples.
  • a method for separating out the fluid component of a biologic sample is provided.
  • the biologic sample is placed in contact with a group of microspheres and the fluid component separates from the sample as the fluid portion flows through the microspheres, by capillary action.
  • the microspheres are of a defined diameter or size.
  • particles of non-uniform size and/or shape may be used to separate a fluid portion from a biologic sample instead of using microspheres.
  • a quantitative assay method and device for measuring one or more analytes in a fluid sample using a point-of-care assay method and device.
  • the assay and device are designed for use by a patient themself, at the bedside of a patient, or in a doctor's office.
  • the test results are analyzed using a suitable analyzer and, optionally, the assay test results are transmitted by way of digital transmission systems to permit further evaluation of the data by an off-site professional.
  • the microspheres, or other particles act as a dynamic filter to extract or partition a fluid portion away from the non-fluid portion.
  • the channels may be transient since the beads exhibit motion during the separation step. Therefore the rapid, instantaneous capillary extraction is by a dynamic capillary filter created by the transient capillary channels formed by the interstitial spaces between the microspheres or particles.
  • an assay method and portable assay device are provided for testing small volumes of biologic fluids, including blood, in a timely manner.
  • a method and device are provided for testing samples of biologic fluids in which a consistent volume of the biologic fluid sample is tested for one or more analytes and the data generated from the tests are used for collecting and compiling in a database pertaining, for example, to a particular disease condition.
  • the data collected can be used to train neural network algorithms and the algorithms may then be used to provide diagnostic and/or prognostic information based on the individual test results of any given test subject.
  • the cellular components of blood are separated from plasma by . allowing the whole blood to be exposed to microsphere beads which permit the plasma to pass in the spaces formed between the microspheres by capillary action but not the cellular component.
  • the present invention is not limited to the separation of cells from plasma in blood but includes broader applications where microsphere beads may be used to separate a fluid component from a cellular component in a biologic fluid. The microsphere beads are effectively acting as a fluid filter.
  • a device for separating plasma from blood in a sample.
  • the device comprises a plurality of microspheres disposed in abutting relation and forming therebetween a plurality of capillary channels, whereby when the microspheres are disposed in fluid communication with a blood sample cellular and plasma components of the biologic sample are separated by capillary flow of the plasma component through the capillary channels formed by the interstitial spacing between abutting microspheres.
  • the device comprises a plurality of groups of smaller microspheres each impregnated with a different label and interspersed with the larger microspheres in separate zones of the larger microspheres.
  • the microspheres may be of substantially the same diameter, or the microspheres may be of differing diameters.
  • the size of microsphere selected may be based on the viscosity of the sample or the size of the component one wishes to exclude or separate.
  • the microspheres are bundled in a fluid-permeable material or the microspheres are maintained in abutting relation by a surface tension of the fluid which passes through them, for example plasma.
  • the microsphere beads also known simply as microspheres, are dried on a surface of the device.
  • the device comprises a sample shelf adjacent to the fluid entrance and the microspheres are disposed on the sample shelf.
  • the device comprises a plurality of smaller microspheres which are impregnated with at least one label interspersed with a plurality of larger microspheres such that the smaller microspheres occupy the interstitial spacing between the larger microspheres and release a label into the fluid as it flows through the interstitial spacing between the larger microspheres.
  • the smaller microspheres may be mobilized and carried forward by the fluid as it passes along the capillary channels formed by the larger microspheres.
  • the device comprises an indicator containing patient identification information to be associated with results of the assay, for example a bar code which can be read by a bar code reader.
  • a method of separating fluid from a biologic sample is provided.
  • the sample has a fluid component and a non-fluid component and the method comprises the steps of,
  • a method of conducting an assay utilizing a device comprising a capillary chamber defined by first and second opposed surfaces spaced a capillary distance apart having a fluid entrance and at least one reagent disposed within the capillary chamber, comprising the steps of,
  • the method further comprise the step of analyzing the reagent to determine a proportion of the reagent which binds to the sample.
  • the method further comprises a plurality of capillary chambers for conducting a plurality of assays on one or more fluid samples.
  • the results of the tests are recorded in a computer database and may be further applied in a trained neural network algorithm to generate a profile of one or more selected disorders.
  • the assay further comprising the step of applying a receiver operating characteristic analysis to the data to determine a statistical significance of the data.
  • a wick or a capillary is brought into fluid communication with the fluid sample to remove the fluid sample from the capillary chamber.
  • microspheres are used to separate a cellular component from a fluid component in a biologic fluid, for example plasma from whole blood, and the fluid component can be tested in chromatography test strips.
  • a biologic fluid for example plasma from whole blood
  • the microsphere beads of the present invention may be used as a labeling device, in addition to a filtration device, in standard nitrocellulose chromatography assays.
  • the microspheres could be replaced by non-uniform particles of differing sizes and/or shapes as described further below.
  • silica sand could be used to replace the polystyrene microsphere beads.
  • suitable particles would be known to a person skilled in the art having bthe benefit of the present description.
  • Figure 1 is an schematic, exploded, perspective view of an embodiment of the device of the present invention.
  • Figure 2 is a longitudinal cross section of the preferred embodiment illustrated in Figure 1 along line 1 A - 1 A.
  • Figure 2A is an end elevation view of the device illustrated in Figure 2 taken from the perspective of line 2 A - 2 A.
  • Figure 3 is a side view of an embodiment described in Example 1 illustrating the cover slip in relation to the beads when starting to form the curl.
  • Figure 4 is also a side view of an embodiment described in Example 1 illustrating the curl after formation;
  • Figure 5 is another side view of an embodiment described in Example 1 illustrating the position of the cover slip in relationship to the beads on the microscope slide.
  • Figure 6 is a top plan view of an embodiment described in Example 1.
  • Figure 7 is a side view of an embodiment described in Example 2 illustrating the label pad variant.
  • Figure 7A is a side view of another embodiment described in Example 2 illustrating the replacement of the label pad with microsphere beads.
  • Figure 8 illustrates an example ROC curve for the expected test results for a neural network risk analysis test.
  • Figure 9 is a photomicrograph taken at 400x magnification using a light powered microscope showing the appearance of unseparated yogurt as applied to the shelf of the biochip.
  • Figure 10 is a photomicrograph taken at 400x magnification using a light powered microscope showing a fluid portion of the yogurt seen in Figure 9 after separation using microsphere beads having 15 micrometer diameters.
  • Figure 11 is a photomicrograph taken at 400x magnification using a light powered microscope showing a fluid portion of the yogurt seen in Figure 9 after separation using microsphere beads having 10 micrometer diameters.
  • Figure 12 is a photomicrograph taken at 400x magnification using a light powered microscope showing the appearance of unseparated E.coli and bread suspension as applied to the shelf of the biochip.
  • Figure 13 is a photomicrograph taken at 400x magnification using a light powered microscope showing a fluid portion of the E.eo///bread suspension seen in Figure 12 after separation using microsphere beads having 15 micrometer diameters.
  • Figure 14 is a photomicrograph taken at 400x magnification using a light powered microscope showing the appearance of unseparated cow feces -as applied to the shelf of the biochip.
  • Figure 15 is a photomicrograph taken at 400x magnification using a light powered microscope showing a fluid portion of the cow feces seen in Figure 14 after separation using microsphere beads having 15 micrometer diameters.
  • Figure 16 is a photomicrograph taken at 400x magnification using a light powered microscope showing a fluid portion of the cow feces seen in Figure 14 after separation using microsphere beads having 10 micrometer diameters.
  • Figure 17 is a photomicrograph taken using a light powered microscope showing silica sand as applied to the shelf of the biochip and showing a 1 mm scale illustrating the size of the silica sand grains.
  • Figure 18 is a photomicrograph taken at 400x magnification using a light powered microscope showing a fluid portion of the E.co/z/bread suspension seen in Figure 12 after separation using silica sand grains.
  • Figure 19 is a photomicrograph taken at 400x magnification using a light powered microscope showing a fluid portion of the cow feces suspension seen in Figure 14 after separation using silica sand grains.
  • the present invention relates to a method of separating a fluid component from a biologic sample using microsphere beads or other suitable particles.
  • the present invention further relates to a device and a method for analyzing the presence or absence of an analyte in a biologic fluid sample.
  • the invention also relates in one aspect to quantifying with precision the amount of one or more analytes present in a biologic fluid sample.
  • the present invention can also provide qualitative, not quantitative results as well.
  • the present invention further relates to an assay which can interpret test results and be used to further identify certain medical conditions from which a person or animal may be suffering or is likely to suffer from in the future.
  • the present invention further relates to a prognostic assay technique in which the results of the test assay defined in the present invention may be used to predict the likelihood of a person or animal developing a certain condition or disease state at a future time.
  • fluid sample as used in this specification is intended to be interpreted broadly tb include suspensions and other samples that have a fluid portion which can be separated by fluid flow and/or capillary action.
  • the fluid component containing an analyte of interest may be used to measure any of the following, alone or in combination: a) the presence of the analyte in the sample b) the absence of the analyte in the sample c) concentration of the analyte in the sample d) total amount of analyte in the sample.
  • Suitable analytes which may be measured by the assay and device of the present invention include soluble analytes: including but not limited to, enzymes, proteins, bacteria, viruses, antigens, antibodies, immunoglobulins, drugs, and hormones. Other suitable analytes would be known to one skilled in the art.
  • the assay and device of the present invention are useful for the detection and measurement of drugs of abuse in human biologic samples such as performance enhancing drugs or other street drugs.
  • biologic samples can be assayed without first separating out cellular components; however, for example, in the case of blood, the cellular component can interfere with the assay.
  • biologic samples where it is necessary, or preferred, to remove the cellular component before assaying it is necessary to first separate the fluid component from any cellular components.
  • the fluid component of a biologic sample can be separated from its non-fluid component by applying the sample to a grouping of microsphere beads.
  • the fluid component will flow in between microsphere beads thereby separating it from the cellular components in a simple and effective way.
  • the beads act as a means of separating the fluid component from the non-fluid component as the fluid component moves by capillary action, through the spaces formed between the beads, when the beads are grouped together. So, in the case of blood, the plasma is separated from the cells in the blood sample. It has been surprisingly recognized in the present invention that microspheres have the ability to separate out the plasma from whole blood quickly and efficiently.
  • interstitial spaces For the purposes of this patent application the spaces between the beads are called “interstitial spaces” or “pores”. It is believed that the fluid flows by capillary action from one interstitial space to the next. In the present application the flow of the fluid passing through the interstitial spaces between the beads is likened to flowing along channels formed by the spaces between the beads. The channels are referred to as “capillary” channels because it appears that the fluid flows between the beads by "capillary" action.
  • the size of the space formed between the microspheres is a function of the radius of curvature of the microspheres.
  • the radius of curvature is, for the purposes of the present invention, the same as the diameter of the microsphere.
  • the ratio of the microsphere diameter to pore diameter is approximately 1 to 0.4.
  • a pore size of 4 ⁇ m is considered optimal. Therefore, the bead size for this particular embodiment should be lO ⁇ m. This permits an easy fluid flow (and therefore faster fluid flow) while still preventing cells from passing through the pores.
  • the small spaces formed between the beads provide a certain capillarity when a fluid is present.
  • the use of microspheres is an effective and inexpensive means for separating plasma from whole blood as the erythrocytes and leukocytes in the blood will stay on one side of the beads while the plasma portion of the blood sample will pass through the beads, by capillary-like action along the interstitial spaces or pores, formed between the beads. It is considered that the capillary action observed in the present invention is related to the surface tension exerted by the microspheres on the fluid so as to draw the fluid forward. As the fluid is drawn forward between the microspheres it provides the additional advantage of mobilizing any reagents present in the region of the microspheres.
  • the microsphere layer could be impregnated with secondary antibodies or another detection molecule.
  • the microsphere beads are effectively acting as a fluid filter and as such can be used at any point in an assay where simple fluid filtration, separation or partitioning, is required. Since it is believed that the microspheres act to filter, separate or partition the fluid component from the non-fluid component by capillary action, the microsphere filter may be termed a capillary filter and this term is used for that purpose herein.
  • the capillary filter is a dynamic filter in the sense that the beads are seen, under a microscope, to move during the partitioning of the fluid component. - Some beads move and others remain still but overall movement of the beads is observed microscopically. It is expected that with the movement of the beads some capillary channels will close and others will open. In this sense, the capillary channels may be transient. The interstitial spaces between the beads or particles also are expected to show the same transience with movement of the beads or particles during fluid partitioning.
  • the microspheres could have analyte specific antibodies bound to them, for example, by adsorption or coupling. As the fluid containing the plasma passes through the capillary channels formed by the microspheres the analyte will mobilize the secondary antibodies contained on the microspheres and then react with the primary antibodies contained in the biochip.
  • the microspheres may act solely to separate the cellular component from the fluid component and the microspheres need not be labeled with antibodies.
  • microspheres of the present invention provide an advantage over the prior art technology because it provides improved fluid flow without restriction by the fiber which is present in the chromatographic paper.
  • the microspheres provide a further advantage in that they provide an excellent surface for binding of proteins such as antibodies or other suitable labels.
  • the size of the microsphere beads used to separate the fluid component can be varied based on the viscosity of the sample. Larger beads should be used for more viscous samples for faster fluid flow between the beads. Also, beads of different colours may be used to facilitate visualization of the beads when they are used as labels and bind to the analyte.
  • the bound beads also serve to increase the density of any bound analyte for subsequent detection by a spectrometer.
  • the regular pattern -of the beads also means that diffraction difference could be used for detecting and - measuring bound analyte.
  • the biologic sample may be applied to the top of the beads or at the side of the beads.
  • latex microsphere beads are used such as those sold under the trademark Bang'sTM.
  • the beads are supplied in a liquid suspension.
  • the beads can either be kept moist or dried when used for other types of beads could be used in the invention, including glass, so long as the beads separating out the fluid component.
  • microsphere beads to quickly separate out a fluid component from a biologic sample can be incorporated into assays for detecting and quantifying analytes present in the sample.
  • this method of separating out a fluid sample from a biologic sample using microsphere beads is incorporated into a one-step assay for analyzing one or more analytes which may be present in the fluid sample is provided.
  • the assay is performed in association with a chamber of defined volume.
  • the chamber comprises microsphere beads for separating out the fluid sample and detection means for detecting and/or measuring an analyte in the sample.
  • the detection means may be drawn from any of several known methods for detecting an analyte in a sample.
  • the analyte may be recognized using detection protein, such as an antibody or antigen, which is specific to the analyte.
  • the detection protein When the analyte binds to the detection protein it changes density and may be measured.
  • the detection protein may be bound to another label, which can be detected.
  • the detection protein may be attached to a small bead so that when the detection molecule binds to the analyte the density will increase and this can be detected or measured:
  • suitable labels would include metals such as gold, fluorescent labels, chemical labels, or colorimetric labels.
  • thisr, invention pertains to a point-of-care diagnostic or prognostic test in the form of a small chip or cassette for use in assaying biologic samples such as blood.
  • the present invention teaches a small, compact assay device referred to as a "biochip” for a simple assay taught in accordance with the present invention.
  • the device of the present invention there is a pairing together of two carrier surfaces in order to define a specific volume in which a quantitative measurement of analyte(s) present in a drop of blood, urine, saliva or other biologic fluid may be measured.
  • the surfaces in question are a coverslip and a microscope slide but the present invention is not intended to be limited to only these specific embodiments.
  • An important aspect of the present invention is the fact that a fluid sample enters a space of defined volume by capillary action.
  • the defined space is therefore referred to herein as a capillary chamber.
  • the capillary chamber is that volume of space between the bottom of the cover slip and the top of the slide.
  • the amount of fluid which is present between the plates or slides is determined by the volume of space between the slides. Therefore small test systems can be designed which allow for precision testing of very small volumes, in some cases, as small as a few microliters.
  • analyte measurement is assessed directly without having to adjust for varying volumes.
  • the concentration or quantity of analyte can be assessed directly without difficulty and with consistency from test to test.
  • the chamber of the biochip of the present invention provides that defined volume.
  • the fluid volume in which the measurement of an analyte is performed is standardized.
  • a method is provided for separating the plasma from the blood cells in a very small blood volume since it is most practical to be able to perform these tests with only a droplet of blood, for example from a finger prick, rather than requiring a larger volume only available by taking a tube of blood through a needle.
  • the biochip test devices comprises a chamber of a determinable volume.
  • the chamber is defined by first and second opposed carrier surfaces. The surfaces are positioned so that they are separated by a distance which is sufficiently narrow to permit fluid to flow between the two surfaces by capillary action.
  • the chamber has a defined volume as it forms a defined space.
  • the chamber has one or more points of fluid entrance which allow a fluid sample to enter.
  • the chamber is also referred to as a capillary chamber since the fluid enters by capillary action.
  • this arrangement of the two carrier surfaces joined together is referred to as a "biochip" but may also be known as a cassette or cartridge.
  • the intention is to provide a compact, portable test system which may be standardized.
  • the bottom surface is, for example, a microscope slide and the top surface is a microscope coverslip.
  • Microscope slides and coverslips are readily available and therefore are useful carrier surfaces.
  • two microscope slides could be mounted one on top of the other, or any two plates, so long as there is a defined space between the plates of a determinable volume into which a fluid sample flows by capillary action.
  • the fluid sample is retained by way of surface tension at the ends and edges of the two surfaces.
  • the device is of a small size which makes it portable and it can be inserted into an analyzer and reaction products between the analyte and detection molecules are measured using the analyzer.
  • the carrier surfaces will be referred to as plates; however, the invention is not to be limited only to flat plates.
  • all types of surfaces which are able to bind proteins, antigens and other detection molecules are contemplated with the scope of the present invention.
  • the composition of the carrier surface includes, but is not limited to, glass, plastic and metal.
  • a drop of biologic sample is placed on the top surface of the microscope slide and, before entering the capillary chamber, the cellular component of the sample is removed by movement of the fluid component through a grouping of microsphere beads.
  • the plasma is separated from the cellular component of blood by movement through capillary channels formed by interstitial spaces between the beads and then the fluid enters the testing chamber in which the analyte reacts with reagents in the chamber and the reaction product is a measure of the analyte present in the sample.
  • the fluid Once the fluid has entered the defined space it is exposed to one or more reagents present on an interior face of a carrier surface.
  • the reagents are therefore exposed in the capillary chamber and available for reacting with one or more analytes which may be present in the fluid sample which ultimately fills the capillary chamber.
  • the reagents are labelled and the quantity of analyte present in a fluid sample is measured based on a reaction product which results from the interaction of the analyte in the sample with the reagent in the chamber.
  • the test results are then compared to standard calibrations to determine the quantity of analyte present in the sample.
  • the reagent is one or more analyte specific antibodies which are adhered to the carrier surface, preferably by protein printing.
  • an antigen is present on an interior face of the carrier surface and the amount of antigen specific antibody in the sample is measured.
  • the protein or other detection molecule When bound to the carrier surface the protein or other detection molecule will project into the defined space where it can react with the analyte in the sample.
  • the detection molecule which is present on the interior face of the carrier surface may be bound to the surface by any one of several means known to a person skilled in the art.
  • Detection molecules are either coated, printed or otherwise bound to one plate or the other using one of several techniques well known in the art. Numerous techniques for immunoassays are known to persons in the art and are described, for example, in “Principles and Practise of Immunology” (1997), C.P. Price and D.J. Newman eds. (Stockton Press) and this document is hereby incorporated by reference into the instant patent application and made a part hereof as if set out in full herein.
  • the distance between the two plates is limited only by the ability of the plates to effectively draw a fluid such as plasma between the two plates by capillary action and to retain the fluid in the defined volume.
  • the size of the plates used would also be dictated by practical considerations such as the desired volume for testing.
  • a fluid sample such as a drop of blood is placed at one edge of the two plates and is drawn into the space defined between the plates.
  • a fluid sample could be drawn against the edge of the two plates by any number of means which would be known to a person skilled in the art.
  • the sample could be brought directly to touch the edges such that a portion of the fluid sample is drawn into the space so as to completely fill the defined volume of the space. For example by touching the patient's finger to the plate. It is important in the present assay that the sample always fill the defined volume entirely so that suitable quantitative analysis may be performed. In a standardized model the volume would be consistent from one biochip to another.
  • the plates are joined together such- that the fluid sample may be readily removed.
  • the fluid sample may be readily removed.
  • the end opposite the point of fluid entry is a preferred embodiment the plates.
  • the space between the two plates could be divided into lanes and the volume of each lane would similarly be known. This approach would allow multiple tests to be done on a single sample.
  • test results be made available in a short time frame, preferably on the order of 1 to 30 minutes, from beginning to end.
  • An advantage of the present invention is that the fluid sample enters the test chamber in a shorter time than prior art assays since the use of microsphere beads to separate the plasma from the blood sample, for example, eliminates the delay which would occur using fiberglass or chromatographic strips. Cumbersome equipment such as a centrifuge is not required for cell separation. All of which facilitates the test being performed at the point-of-care.
  • the present invention has further advantages over the prior art since the biochip device of the present invention permits several assays to be performed on one sample. This facilitates the speed with which test results can be obtained and minimizes the amount of sample required for testing.
  • Analyte-specific antibodies themselves may be labeled with anyone of several labels known to persons skilled in the art of such assays.
  • preferred labels include fluorescent labels, colorimetric labels, another microsphere, gold particles or any high contrast molecule. Other labels would be suitable so long as the presence of the label can be detected.
  • microsphere beads having a diameter which is smaller than the test beads can be used so that the smaller beads are mobilized through the larger beads with the movement of the fluid sample (e.g. plasma). The smaller beads can be labeled accordingly.
  • the upper plate for example a coverslip
  • the upper plate has analyte-specific reagents bound on the surface which comes in contact with the fluid.
  • the analyte-specific reagents are printed on the interior surface of the carrier plate using a protein printer. Suitable protein printing devices are well known in the marketplace. These include ink jet, spray, piezoelectric and bubble jet protein printers. The piezo-electric printer is preferred.
  • the analyte-specific reagent acts as a detection molecule, typically proteins. These molecules adhere to glass, metal and plastic surfaces.
  • Preferred surfaces include polystyrene or polypropylene.
  • the use of such printing devices is advantageous in the present invention to allow several different analyte-specific detection molecules to be printed onto the plate or coverslip such that different "lanes" are defined and different analytes may be assessed simultaneously using a single fluid sample. Additional background and calibration lanes can be provided in the same test chamber.
  • the biochip carrier surfaces be colorless or transparent such that a colorimetric, or fluorescent or other reaction products can be read using a suitable spectrometer or other appropriate detection coupled to a reader.
  • the change in density is measured to determine the amount of analyte present in the sample.
  • a mask is provided in accordance with a preferred embodiment of the present invention.
  • the mask 32 is made of an opaque material except for the openings 36, 38 and 40 which correspond to lanes 26, 28 and 30 on the plate.
  • the mask is designed to fit neatly over the upper plate 10 so that only the lanes themselves are available to be read.
  • the use of the mask has the advantage of reducing the amount of background noise and setting- baseline values when reading the density change in the lanes.
  • the biochip is designed to be read by a portable spectrometer which reads for example, the change in color after the analyte has reacted with the labeled antibody.
  • the spectrometer could also read changes in density, film thickness, mass absorption or diffraction depending on the test reagents used.
  • the analyzer e.g. spectrometer
  • the results are transmissible by digital transmission over the telephone lines, by cell phone, or other computer network system.
  • changes occurring during an antibody/analyte reaction may be detected or measured by changes in radio frequency if a radio frequency sensor is inco ⁇ orated into the biochip detection system.
  • FIG. 1 a preferred embodiment of the biochip of the present application is illustrated in a schematic exploded perspective view.
  • Two carrier plates 10 and 12 are provided.
  • the two plates define a fixed volume therebetween as indicated by reference number 14.
  • Lower plate 12 may be longer than upper plate 10 to provide a shelf which acts as an application zone 16 upon which a biologic sample 18 may be applied.
  • a shelf is not essential to the invention but provides a place to allow the sample to be separated by the microsphere beads. It is possible that the beads could be placed at the entrance of the capillary chamber 14 within the confines of the plates and the sample would be applied to the edge of the biochip where it would enter the chamber by capillary action.
  • microsphere beads 20 which may or may not also include a label zone 22.
  • the microsphere beads 20 may be grouped or bundled using a fluid-permeable material.
  • the microsphere beads 20 and label zone 22 are illustrated as separately defined regions; however the microsphere beads may also bear the label themselves and in this embodiment the two zones would converge into one with the microsphere beads playing two roles: separation of the fluid and displaying a label to which the fluid is exposed.
  • microsphere beads More than one size of microsphere beads may be present.
  • smaller microspheres could nestle in the interstitial spaces formed by the larger beads.
  • the smaller beads could carry secondary labels which would bind to the analyte as it passes through the beads. Either the label would bind to the analyte in the fluid or the label attached to the small bead would attach to the analyte in the fluid and the small beads would then travel with the fluid into the capillary chamber. At the same time any cellular component in the fluid sample would not pass through the microsphere bead filter.
  • a patient ID may be affixed to either plate 10 or 12 so long as it does not interfere with the test detection areas on the biochip or with reading the biochip after analyte has reacted with the substance bound to the carrier plate surface.
  • the plates 10 and 12 are preferably colorless and/or transparent.
  • Three detection areas 26, 28, 30 are printed on the inner surface of carrier plate 10: a calibration print zone 26, a detector print zone 28 and a baseline print zone 30.
  • Three detection areas, or zones, are depicted for example only to illustrate how one test biochip may be set up; however, several lanes may be present and the number of lanes dedicated to calibration and/or background can vary depending on what is being tested. The test need not be limited to only three lanes. Several lanes could be defined. In a preferred embodiment of the present invention three lanes are printed on the one plate to permit assessment of background readings as well as calibration of the biochip. it is understood that the background and calibration detection zones need not all be placed on the same biochip. It is advantageous to have the background and calibration readings made on the sample carrier plate in the same assay as the test analyte thereby reducing the variance in test results.
  • a background mask 32 is optionally provided.
  • the mask is designed to cover the outer surface of the carrier plate 10 without blocking the coated or printed detection zones/lanes. Therefore, openings 36, 38 and 40 are, for example, present in the mark to reduce background interference when reading test results.
  • the background mask is made of an opaque material with openings 36, 38 and 40 which correspond to the detection zones 26, 28 and 30 identified on the inner surface of the upper plate.
  • the opening 40 in the mask need not have a corresponding test zone 30 as illustrated so long as the opening 40 is exposed to a part the plate 10 where reagents are not present.
  • Figure 1 illustrates both an antibody/label zone 22 and a microsphere zone 20, both of these zones are optional depending on the type of test one chooses to conduct.
  • fluid sample 18 When fluid sample 18 is applied to application zone 16 it flows through antibody/label zone 22 (if present) and microsphere bead zone 20 (if present) before it reaches the edge 34 where the two plates 10 and 12 first meet.
  • edge 34 In the schematic illustration of Figure 1 there is a gap between the zone of microsphere beads and the fluid entry point identified by edge 34.
  • this arrangement of the invention will work, it would be most preferred if the microsphere bead zone 20 and/or label zone abutted against the edge 34 of the carrier plate 10.
  • Figures 5 and 6 One example of such a configuration is illustrated in Figures 5 and 6.
  • the fluid sample is drawn under edge 34 into the chamber 14 which defines a known volume.
  • the fluid sample should be of sufficient volume to pass along the application zone 16, through the microsphere and label zone(s) and to completely fill the chamber 14.
  • the biochip of the present invention can be scaled to a small size such that a single drop of blood could be a sufficient sample size for testing. Many dimensions are possible to construct based on the principles taught herein. Although dimensions of 1 cm x 3 cm make a device of convenient size, the nature of the testing to be done would dictate the optimum chip size. As illustrated in Figure 1 a shelf portion 16 extends on the bottom plate.
  • the biologic sample can be applied.
  • the portion of the test which i_r held for example the microscope slide, may be large but the test assay itself which sits on the slide may be very small.
  • the assay may be miniaturized to accommodate sample fluid volumes as small as about 1 microlitre.
  • Figure 2 is a sectional view taken along lines 2 - 2 illustrating the same elements as referenced in Figure 1.
  • Figure 2 A is an end elevation view of Figure 2 along lines 2A - 2 A illustrating that the end of the device may be open, to allow the fluid to be removed from the chamber.
  • a suitable wicking material would be applied to the open end and the fluid would be drawn through thereby allowing additional fluid to enter the chamber. This could be either a continuous or a discontinuous process.
  • FIG. 1 Illustrated in all of Figures 1, 2 and 2 A is a spot of glue 58 which is one way to hold the plates 10 and 12 together.
  • the glue 58 also illustrated in Figure 6, another embodiment of the invention.
  • FIGs 7 and 7A are illustrations of another use of the microsphere method of separation in a one-step assay.
  • the microspheres are used in conjunction with chromatography paper.
  • the biologic sample 18 is placed on a surface such as a microscope slide 52'. It may be placed directly on the microsphere beads 50 (as illustrated) or beside them.
  • the fluid component of the sample then flows through the beads 50 separating from a non-fluid component present in the sample 18.
  • the beads abut against or sit close to a fiberglass filter pad 60 which abuts with a label pad 62.
  • the label pad 62 is usually a fiberglass pad impregnated with the label of interest for labeling analyte in the fluid sample.
  • the fluid flows through the filter 60 and label pad 62.
  • any analyte present in the fluid will be labeled as it flows through the label pad.
  • the fluid then flows into the nitrocellulose chromatography strip 64 where the test results are read, usually as a color change or band on the nitrocellulose strip.
  • the fiberglass filter 62 may be eliminated entirely (not illustrated).
  • the fiberglass label pad 62 may be replaced by microsphere beads 66.
  • the beads 66 are acting as a source of label, not as a filter and the fiberglass filter 60' serves as a spacer between the two sets of beads 50 and 66, respectively.
  • the microspheres 66 can be used to label an analyte present in the fluid directly, without requiring the microsphere filter 50 or the fiberglass spacer 60'.
  • Figures 7 and 7A are illustrative of how current assay methodologies may be modified using the microsphere bead technology of the present invention as taught herein.
  • the assay device and techniques of the present invention are very useful in that they can be used for small volumes of many kinds of fluid samples.
  • the description refers specifically to proteins any number of other marker would be suitable so long as a labeling system can be devised for the detection and measurement of the marker in the system.
  • the present invention could be used to measure and/or detect the presence of microorganisms such as bacteria, viruses, fungi or other infectious organisms.
  • the biochip device of the present invention can be calibrated for the type of assay and the type of analyte so that a table of standard values may be constructed.
  • the assay system or the present invention can detect the levels of a particular hormone or even the amount of a drug in a patient's system and this standardized data can be used to make diagnostic and/or prognostic determinations for a given individual.
  • data is collected on a regular basis and databases constructed based on the patient's medical history, current health and the test results.
  • the data can be transmitted by digital transmission systems over a computer network via modem, the internet, cable lines, telephone lines, satellite or other similar technology.
  • These databases can be. used in the development of neural network algorithms, for assessment of current patient test results and diagnoses as well as for predicting certain health outcomes for a given, individual.
  • a neural network algorithm is found in Example 3 below and a sample Receiver Operator Curve (ROC) is illustrated in Figure 8.
  • the development of the algorithms for the applied neural network will be a function of the medical condition being assessed. Large amounts of patient data will first have to be accumulated in order to have reliable predictive outcomes.
  • the neural network can be trained to recognize the concentration of analyte which is diagnostic or prognostic, using the standardized assays of the present invention.
  • the data and algorithms are encoded in an electronic chip which is placed in the reader, for example a spectrometer, such that the printout from the reader will also identify a particular diagnosis or prognosis simultaneously with providing the test result.
  • the diagnostic or prognostic test result will be optimized as the number of data points increases. With more patient data the predictive and/or diagnostic result will be made with greater certainty.
  • the percent certainty can be calculated and provided to the physician or technician based on analysis of the measured data in comparison to a database contained in an electronic memory chip installed in the analyzer provided.
  • Present technology makes it possible to display the actual standard curve on the reader itself at the time of printing out the test results.
  • the biochip has a radiofrequency sensor inco ⁇ orated into the carrier plate 10.
  • a radiofrequency sensor inco ⁇ orated into the carrier plate 10.
  • more than one test can be run simultaneously on the same biochip and therefore the certainty of the diagnosis or prognosis can be improved. As the number of markers increases so does the certainty of measurement.
  • One of the many examples of uses of the biochip/cassette of the present invention is to measure blood proteins indicating peripheral vascular disease using a drop of the patient's blood.
  • microspheres were observed under a light microscope during the separation/filtration step as a fluid portion of the sample is separated away from the non-fluid portion. It was observed that some of the microspheres were seen to be in motion while others remained static. The separation is dynamic and the action of the beads or particles in separation is a dynamic capillary action. Separation or extraction of the fluid portion is instantaneous.
  • the present invention is also applicable to small particles other than the microsphere beads described herein.
  • the separation technology of the present invention also works using non-uniform particles including silica-based particles, for example sand grains, even though these particles are not necessarily spherical in shape nor uniform in size, as shown in Example 7 below.
  • suitable separation/filtration using non-uniform and/or non-spherical particles or beads can be achieved.
  • Non-uniformity makes the separation less efficient because it is somewhat slower but effective separation is still achieved at least for qualitative assays.
  • Example 7 using sand grains, the separation does not appear to happen as efficiently since fewer organisms are separated from the sample in the same time period as the separation using microsphere beads as described in Examples 4, 5 and 6. Still, the organisms are successfully separated and can be further tested or assayed accordingly.
  • the use of silica and other similar particles is advantageous over the microsphere beads because they are less expensive and may be more readily available in less developed and developing countries.
  • the assays and devices of the present invention can be particularly helpful in identifying the presence of harmful and pathogenic bacteria in certain biologic samples, such as E. coli strain O157:H7, salmonella, listeria, clamydia and other bacteria and microorganisms such as viruses.
  • the assays of the present invention could be used to test food samples for certain pathogens. They could also be used in human or veterinary medicine for diagnosis of infectious diseases.
  • the microsphere beads of the present invention can be generalized to a phenomenon of particles in general and the invention is not restricted to spherical beads. Rather, it includes particles of non- uniform size and shape as illustrated in Example 7 using sand grains to the filter on the Biochip. It is expected that silica sand would be a considerably less expensive option than the commercially purchased microsphere beads and would allow a wider use of this technology. Although the separation of bacteria using the silica sand was not as efficient, i.e. fewer bacteria are separated in the same time frame, it is sufficient for many pu ⁇ oses.
  • silica-type particles Another advantage of the use of silica-type particles is that silica is known in the art to selectively bind proteins and nucleic acids. Silica-based separation particles could be used to devise certain protein and/or nucleic acid positioning mechanisms. In all of Examples 4 to 7 the separation was almost instantaneous and the limit on the size of the particles was limited by the type of bacteria, microorganism or other analyte which one wished to isolate. It is expected that one skilled in the art would know which size of particle to select based on the type of bacteria present in the sample. If the bacteria was unknown, then a person skilled in the art would pick a particle size based on the expected size anticipated and select a bead size generally in accordance with the 0.4 to 1 ratio as described above.
  • Example 1 Verification of Plasma Flow and Separation from Whole Human Blood
  • the coverslip was fixed squarely in place on the slide with one edge aligned parallel to the edge of the curl of dried beads and this edge was left open to allow fluid to pass through the beads and into the capillary chamber formed between the cover slip and the glass slide.
  • the coverslip was attached with nail polish at the corners 58 of the coverslip to secure it to the microscope slide.
  • the coverslip was secured at a spot where no capillary action was intended to take place to permit fluid to flow freely under the coverslip.
  • a 20 microliter drop of whole human blood 18 was placed on the remaining 5 to 10 microliter microsphere beads.
  • the sample of whole human blood was placed on the remaining portion of the beads which did not form part of the curl leaving the plasma component free to move by capillary action through the curl portion of the microsphere beads and into the space defined between the coverslip and the slide (i.e. the capillary chamber).
  • the effect was observed under a binocular light microscope.
  • the plasma immediately began to separate from the whole blood.
  • capillary action between the coverslip and the slide drew the pure, clean, cell-free plasma under the coverslip into the chamber defined between the coverslip and the slide.
  • This chamber defines a known space, the volume of which can be calculated and predetermined.
  • microsphere beads are able to readily and effectively separate plasma from whole blood and to pass, via the capillary channels formed between the microsphere beads, into the capillary chamber.
  • this example (schematically illustrated in Figure 7) demonstrated the use of microsphere separation of plasma from a blood sample of human whole blood.
  • the plasma was separated using latex microsphere beads (Bang'sTM) 50 and then drawn into a standard nitrocellulose chromatography strip.
  • the fiberglass pads which are usually used to retain red blood cells in the prior art, were replaced with about 20 microliters of 10 micrometer latex beads. A drop of human blood (about 60 microliters) was placed on a surface 52', in contact with the latex microspheres.
  • the fiberglass pad 60 effectively functions as a spacer between the beads 50 and the label pad 64 although it could also be used as a second filter.
  • the fiberglass filter 60 may be eliminated entirely and the microsphere beads 50 abut directly with the label pad 64 (not illustrated).
  • FIG. 7A Illustrated in Figure 7A is another embodiment where, instead of a fiberglass label pad 62, microsphere beads 66 are used as the label region of the test device.
  • the fiberglass filter pad 60' is used as a spacer between the two sets of beads, 50 and 66.
  • T would be 1 for coagulation, and 0 for a non-coagulation.
  • TST(C,b,T) be the test result for a testing vector a, given cutoff C, and target output T.
  • a neural network has 3 layers; the first INPUT layer, the second HIDDEN layer, and the third OUTPUT layer:
  • the neurons are connected by a set of weights ⁇ w(i,j,k) ⁇ .
  • w( 1,2,4) connects the second neuron of the first layer with the fourth neuron of the second layer.
  • the weights are adjusted to minimize ERMS, while maintaining good performance on new data.
  • microsphere beads having a diameter of 15 ⁇ m shows a good separation of bacteria from the sample. None of the solid particles seen in Figure 9 appear. After separation, only bacteria are seen in the separated fluid portion ( Figure 10). Separation occurred almost instantaneously.
  • the microsphere beads provided a quick and ready separation step for isolation of the bacteria away from the rest of the solid particles in the yogurt thereby permitting further testing on the separated bacteria. This would permit a determination of the type of bacteria present in a sample. For example, the type of bacteria, or other microorganism, could be determined by a specific antigenic test to determine the type of bacteria or microorganism present.
  • Figure 11 shows a fewer number of bacteria per field but is still shows an effective separation of bacteria from the yogurt.
  • Example 5 Separation of E. Coli from a Bread Suspension
  • Escheria coli E. coli was successfully separated from a bread suspension using the methodology and apparatus of the present invention.
  • bread and NaCl mixture was prepared. 200 mg of bread was weighed.
  • a bread suspension was prepared by repeatedly mixing 500 ⁇ l 150 mM NaCl with the bread.
  • E. coli strain: DH5 ⁇
  • the suspension was mixed again to create an E. co/z ' /bread suspension.
  • lOO ⁇ l of the E. co/z ' /bread suspension was placed on the "Biochip" and almost instantaneously the microsphere beads acted to partition a fluid component containing bacteria from the sample but none of the solid particles from the suspension.
  • bacteria from cow feces were separated using the technology of the present invention.
  • 500mg of cow feces were combined with 500 ⁇ l 150mM NaCl and mixed to form a suspension.
  • a 5 ⁇ l sample of the suspension was used for separation and placed on the Biochip.
  • Photomicrographs before separation ( Figure 14) and after separation ( Figures 15 and 16) are illustrated. Separation in this example was achieved using 15 ⁇ m microsphere beads. The sample was placed on the Biochip and almost instantaneously the microsphere beads partitioned out a fluid component containing bacteria. The separated bacteria can now be stained to further identify them.
  • a good separation was achieved using 15 ⁇ m beads ( Figure 15) and also with 10 ⁇ m diameter beads (Figure 16).

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  • Ecology (AREA)
  • Biophysics (AREA)
  • Dispersion Chemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

L'invention concerne un dispositif permettant de séparer d'un fluide dans un échantillon biologique qui comprend un composant fluide et un composant non fluide. Ce dispositif est un dispositif convenant pour les centres de soins, et permettant de collecter et de transmettre électroniquement des données en vue d'une évaluation complémentaire. L'invention concerne également un procédé permettant de séparer le fluide contenu dans un échantillon biologique, et comprenant une étape consistant à mettre en contact l'échantillon de fluide avec des microsphères, de manière que le composant fluide avance par capillarité entre les microsphères, le long de canaux capillaires formés par les espaces entre les sphères, en laissant par exemple une fraction cellulaire derrière lui. Cette étape de séparation du fluide peut être combinée à d'autres techniques servant à détecter et/ou à mesurer un ou plusieurs analytes qui peuvent être présents dans l'échantillon de fluide, ces techniques pouvant être par exemple des dosages immunologiques et des analyses chromatographiques. Ces dernières peuvent en outre être appliquées en combinaison avec des groupes de microsphères utilisées dans l'étape de détection de l'analyte et dans l'étape de séparation du fluide, ces microsphères agissant alors comme marqueurs de l'analyte ou comme source de marqueur de l'analyte.
EP99972308A 1998-11-16 1999-11-12 Dispositif et procede d'analyse d'un echantillon biologique Withdrawn EP1131632A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
CA002254223A CA2254223A1 (fr) 1998-11-16 1998-11-16 Dispositif et methode pour l'analyse d'un echantillon biologique
CA2254223 1998-11-16
US09/335,732 US6403384B1 (en) 1998-11-16 1999-06-18 Device and method for analyzing a biologic sample
US335732 1999-06-18
PCT/CA1999/001079 WO2000029847A2 (fr) 1998-11-16 1999-11-12 Dispositif et procede d'analyse d'un echantillon biologique

Publications (1)

Publication Number Publication Date
EP1131632A2 true EP1131632A2 (fr) 2001-09-12

Family

ID=25680640

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Application Number Title Priority Date Filing Date
EP99972308A Withdrawn EP1131632A2 (fr) 1998-11-16 1999-11-12 Dispositif et procede d'analyse d'un echantillon biologique

Country Status (6)

Country Link
EP (1) EP1131632A2 (fr)
JP (1) JP4544498B2 (fr)
AU (1) AU1144000A (fr)
BR (1) BR9915406A (fr)
TW (1) TW468046B (fr)
WO (1) WO2000029847A2 (fr)

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WO2011011462A1 (fr) 2009-07-20 2011-01-27 Optiscan Biomedical Corporation Connecteur réglable à espace mort réduit
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Also Published As

Publication number Publication date
TW468046B (en) 2001-12-11
BR9915406A (pt) 2001-07-24
WO2000029847A2 (fr) 2000-05-25
AU1144000A (en) 2000-06-05
WO2000029847A3 (fr) 2000-09-08
JP2002530648A (ja) 2002-09-17
JP4544498B2 (ja) 2010-09-15

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