CN117580645A

CN117580645A - Analyte detection cartridge and method of using the same

Info

Publication number: CN117580645A
Application number: CN202280044763.9A
Authority: CN
Inventors: R·J·金; A·K·阿加瓦尔; M·A·布茨勒; D·M·凯尔索; S·M·麦克法尔; T·韦斯特伯格; J·L·里德; K·贝兰德
Original assignee: Fine Molecular Diagnostics Inc; NORTHWEST UNIVERSITY
Current assignee: Fine Molecular Diagnostics Inc; NORTHWEST UNIVERSITY
Priority date: 2021-04-27
Filing date: 2022-04-27
Publication date: 2024-02-20

Abstract

Provided herein are devices (e.g., cartridges), apparatuses, systems, and components thereof, and methods of use thereof, for rapid sample processing and analyte detection (e.g., nucleic acid purification, amplification, and/or detection). A cartridge for analyte detection may comprise: a storage section comprising a storage chamber; a processing section comprising a processing chamber; a microfluidic section in fluid communication with the processing section; and a transfer bladder configured to transfer fluid between the storage chamber and the processing chamber.

Description

Analyte detection cartridge and method of using the same

Cross Reference to Related Applications

The application claims the benefits of: U.S. provisional patent application Ser. No. 63/289,481, filed on 4/27 of 2021; U.S. provisional patent application Ser. No. 63/274,332, filed on 1 at 11/2021; U.S. provisional patent application Ser. No. 63/289,481, filed on 12 months and 14 days 2021; and U.S. provisional patent application number 63/304,034 filed on day 28 of month 1 2022, each of which is incorporated herein by reference.

Statement regarding federally sponsored research

The present invention was carried out with government support under grant No. EB027049 awarded by the national institutes of health. The government has certain rights in the invention.

Technical Field

Provided herein are devices (e.g., cartridges), apparatuses, systems and components thereof, and methods of use thereof, for rapid sample processing and analyte detection (e.g., nucleic acid purification, amplification and/or detection).

Technical Field

Nucleic acid testing provides methods for detecting and diagnosing infectious diseases, as well as many other uses. The most widely used and reliable nucleic acid testing methods employ the Polymerase Chain Reaction (PCR). The limitation of PCR is that it takes one hour or more to circulate the reaction solution through multiple temperatures, which may differ by 30 ℃ or more. Quantitative or real-time PCR (qPCR or RT-PCR) takes even longer because fluorescent readings must be taken during or between each thermal cycle. The long processing time and power required to perform qPCR makes it unusable in many situations where a diagnosis must be made quickly and accurately.

A typical qPCR protocol carries out 30 to 50 cycles of the following operations: the test solution was heated to 95 ℃, then cooled to 60 ℃, and then the fluorescence reading was taken. In a typical thermal cycle, the heating and cooling steps are accomplished in a plastic tube with a thermoelectric cooler (TEC) that pumps heat into and out of the test solution through the tube wall. Such thermal cyclers result in inefficiency of the PCR procedure.

Disclosure of Invention

Provided herein are devices (e.g., cartridges), apparatuses, systems, and components thereof, and methods of use thereof, for rapid sample processing and analyte detection (e.g., nucleic acid purification, amplification, and/or detection).

In some aspects, provided herein are cartridge devices and methods for determining analyte levels by specific binding methods (e.g., immunoassays, nucleic acid amplification). The analyte may be present as a bulk liquid solution or absorbed in a porous medium such as a swab. The cartridge contains all the components and chambers necessary for processing and detecting the target analyte. In other words, the cartridge is self-contained. The cartridge is acted upon by a processing instrument having a complementary mechanism to perform operations including, but not limited to, heat transfer, liquid transfer, magnetic transfer, and optical detection.

In some embodiments, provided herein is a cartridge for analyte detection, the cartridge comprising: a storage section comprising a storage chamber; a processing section comprising a processing chamber; a microfluidic section in fluid communication with the processing section; and a transfer bladder configured to transfer fluid between the storage chamber and the processing chamber. In some embodiments, the processing section is positioned between the storage section and the microfluidic section. In some embodiments, the cartridge further comprises a docking section having an inlet port in fluid communication with the storage chamber and a process inlet port in fluid communication with the process chamber. In some embodiments, the cartridge further comprises a body forming at least a portion of the storage section, at least a portion of the processing section, and at least a portion of the docking section. In some embodiments, a first channel fluidly connects the storage inlet port with the storage chamber, and a second channel fluidly connects the process inlet port with the process chamber. In some embodiments, the storage chamber includes a first end and a second end opposite the first end, the first end positioned closer to the storage access port than the second end, and wherein the first channel is connected to the storage chamber at the second end. In some embodiments, the process chamber includes a first end and a second end opposite the first end, the first end positioned closer to the process access port than the second end, and wherein the second channel is connected to the process chamber at the second end. In some embodiments, the storage chamber is a first storage chamber and the storage section further comprises a second storage chamber. In some embodiments, the processing chamber is a first processing chamber and the processing section further comprises a second processing chamber. In some embodiments, the storage section includes a cavity configured to receive the transfer balloon tube. In some embodiments, the cartridge further comprises a first vent fluidly coupled to the storage chamber, and a second vent fluidly coupled to the processing chamber. In some embodiments, the microfluidic section includes a reaction chamber, a microfluidic vent channel fluidly connected to the reaction chamber, and a microfluidic inlet channel fluidly connecting the processing chamber and the reaction chamber. In some embodiments, the microfluidic section further comprises a wax seal. In some embodiments, the wax seal is a first wax seal and the microfluidic section further comprises a second wax seal, wherein the first wax seal is positioned adjacent the microfluidic inlet channel and the second wax seal is positioned adjacent the microfluidic vent channel. In some embodiments, the second wax seal is positioned at a distance from the reaction chamber, wherein the distance is at least 2mm. In some embodiments, the cartridge further comprises an offset vent channel fluidly connected to the microfluidic inlet channel. In some embodiments, the offset vent channel is a first offset vent channel and the cartridge further comprises a second offset vent channel fluidly connecting the first offset vent channel with the second vent.

In some embodiments, provided herein are microfluidic devices comprising: (i) A reaction chamber, (ii) an inlet channel in fluid communication with the reaction chamber; (iii) A vent passage in fluid communication with the reaction chamber; (iv) A first wax seal positioned adjacent to and in fluid communication with the inlet channel, wherein when the first wax seal is in a first position, the first wax seal does not block the inlet channel and allows fluid to enter the reaction chamber through the inlet channel, and wherein when the first wax seal is in a second position, the first wax seal blocks the inlet channel and prevents fluid from entering or seeping out of the reaction chamber through the inlet channel; (v) A second wax seal positioned adjacent to and in fluid communication with the vent channel, wherein when the second wax seal is in a first position, the second wax seal does not block the vent channel and allows gas to exit the reaction chamber through the vent channel, and wherein when the second wax seal is in a second position, the second wax seal blocks the vent channel and prevents fluid from entering or exiting the reaction chamber through the vent channel; wherein a liquid reagent is introducible into the reaction chamber through the inlet channel; and wherein heating the wax seal above a threshold temperature melts the wax seal, and subsequently cooling the wax seal below the threshold temperature solidifies the wax seal in the second position.

In some embodiments, provided herein are systems comprising a cartridge described herein and an instrument into which the cartridge can be inserted, wherein the instrument comprises components that impart heating, magnetic transfer, fluid transfer, and/or analyte detection functionality into the cartridge. In some embodiments, provided herein are uses of the systems herein for sample processing and analyte detection.

In some embodiments, provided herein is a microfluidic system comprising an inlet channel, a vent channel, and a reaction chamber; wherein the inlet channel is in fluid communication with the reaction chamber, wherein the vent channel is in fluid communication with the reaction chamber; the system further comprises a heating element capable of raising the temperature of the reaction chamber; the system further comprises a first seal positioned adjacent to and in fluid communication with the inlet channel, wherein when the first seal is in a first position, the first seal does not block the inlet channel and allows fluid to enter the reaction chamber through the inlet channel, and wherein when the first seal is in a second position, the first seal blocks the inlet channel and prevents fluid from entering or seeping out of the reaction chamber through the inlet channel; the system further comprises a second seal positioned adjacent to and in fluid communication with the vent channel, wherein when the second seal is in a first position, the second seal does not block the vent channel and allows gas to exit the reaction chamber through the vent channel, and wherein when the second seal is in a second position, the second seal blocks the vent channel and prevents fluid from entering or exiting the reaction chamber through the vent channel; and wherein heating the seal above a threshold temperature melts the seal and allows the seal to flow from the first position to the second position, and wherein subsequently cooling the seal below the threshold temperature solidifies the wax seal on the second position. In some embodiments, the first seal and the second seal comprise wax or a polymeric material.

In some embodiments, provided herein is a heat transfer device for heating and cooling a reaction chamber, the heat transfer device comprising: a heat reservoir comprising a base, a first aperture, a second aperture, and a heat exchanger extending from the base; the heat exchanger includes a planar surface configured to surround the reaction chamber; a heater positioned within the first aperture; and a temperature sensor positioned within the second aperture. In some embodiments, the first and second apertures are formed in the base. In some embodiments, the heat exchanger is cylindrical. In some embodiments, the heat reservoir is aluminum. In some embodiments, the heater is a resistive heater. In some embodiments, the reaction chamber is a PCR reaction chamber. In some embodiments, the apparatus further comprises a processor and a non-transitory memory comprising instructions that when executed by the processor perform closed-loop temperature control of the heat reservoir.

In some embodiments, provided herein are assemblies comprising: a first support; a second support movable relative to the first support; a first heat transfer device having a first planar surface, the first heat transfer device coupled to the first support; a second heat transfer device having a second planar surface positioned opposite the first planar surface, the second heat transfer device coupled to the second support; wherein the first heat transfer device and the second heat transfer device are at a first temperature; a third heat transfer device having a third planar surface, the third heat transfer device coupled to the first support; a fourth heat transfer device having a fourth planar surface positioned opposite the third planar surface, the fourth heat transfer device coupled to the second support; wherein the third heat transfer device and the fourth heat transfer device are at a second temperature different from the first temperature. In some embodiments, the assembly further comprises a fluorometer coupled to the first support and positioned between the first heat transfer device and the third heat transfer device. In some embodiments, the assembly further comprises a fifth heat transfer device having a fifth planar surface positioned opposite the fluorometer, the fifth heat transfer device coupled to the second support and positioned between the second heat transfer device and the fourth heat transfer device. In some embodiments, the assembly is configured to receive a reaction chamber between the first planar surface and the second planar surface to bring the reaction chamber to the first temperature; and receiving the reaction chamber between the third planar surface and the fourth planar surface to bring the reaction chamber to the second temperature. In some embodiments, the reaction chamber is a PCR chamber. In some embodiments, the assembly further comprises an actuator coupled to the second support and configured to move the second support along the clamp axis between a first position in which the first planar surface is spaced apart from the second planar surface by a first distance and a second position in which the first planar surface is spaced apart from the second planar surface by a second distance less than the first distance. In some embodiments, the second distance is in the range of 400 microns to 600 microns. In some embodiments, the actuator is a first actuator, and the assembly further comprises a second actuator coupled to the first support and the second support, wherein the second actuator is configured to move the first support and the second support together along a translation axis. In some embodiments, the translation axis is perpendicular to the clamp axis. In some embodiments, the first temperature is in the range of 80 ℃ and 100 ℃. In some embodiments, the second temperature is in the range of 50 ℃ and 70 ℃.

In some embodiments, provided herein are fluidic devices comprising: a reaction chamber; a channel in fluid communication with the reaction chamber; and a wax seal, wherein when the wax seal is in a first position, the wax seal does not block the channel and allows fluid to enter or exit the reaction chamber through the channel, and when the wax seal is in a second position, the wax seal blocks the channel and prevents fluid from entering or exiting the reaction chamber through the channel; wherein heating the wax seal above a threshold temperature melts the wax seal, and subsequently cooling the wax seal below the threshold temperature solidifies the wax seal in the second position. In some embodiments, the reaction chamber is in fluid communication with an inlet channel; and the fluidic device further comprises a vent channel in fluid communication with the reaction chamber. In some embodiments, the inlet channel wax seal is a first wax seal and the fluidic device further comprises a second wax seal, wherein when the second wax seal is in a first position, the second wax seal does not block the vent channel and allow fluid to exit the reaction chamber through the vent channel, and when the second wax seal is in a second position, the second wax seal blocks the vent channel and prevents fluid from entering or exiting the reaction chamber through the vent channel. In some embodiments, the first wax seal is positioned adjacent the inlet channel and the second wax seal is positioned adjacent the vent channel. In some embodiments, the fluid device further comprises a first high temperature movable heater positionable within a range of the wax seal to heat the wax seal above the threshold temperature. In some embodiments, the fluid device further comprises a second cryogenically movable heater positionable within a range of the wax seal to cool the wax seal below the threshold temperature. In some embodiments, the wax seal has a diameter that is less than the diameters of the first heater and the second heater. In some embodiments, the wax seal is coated in an adhesive. In some embodiments, the adhesive is an acrylic adhesive. In some embodiments, the first heater and the second heater can be positioned at selected locations relative to the wax seal. In some embodiments, the second wax seal is positioned at a distance from the reaction chamber, wherein the distance is at least 2mm. In some embodiments, a plurality of liquid reagents may be introduced to the reaction chamber through the inlet channel.

In some embodiments, provided herein are fluorometers comprising: a housing including a measurement aperture; a first light source coupled to the housing along a first light source axis; a second light source coupled to the housing along a second light source axis; a third light source coupled to the housing along a third light source axis; a fourth light source coupled to the housing along a fourth light source axis; a first light detector coupled to the housing along a first detector axis; a second light detector coupled to the housing along a second detector axis; a third light detector coupled to the housing along a third detector axis; a fourth light detector coupled to the housing along a fourth detector axis; wherein the first, second, third, fourth, first, second, third, and fourth light source axes intersect the measurement aperture. In some embodiments, a circular measurement aperture defines a normal axis through its center and perpendicular to the plane of the measurement aperture. In some embodiments, the normal axis, the first light source axis, the second light source axis, the third light source axis, the fourth light source axis, the first detector axis, the second detector axis, the third detector axis, and the fourth detector axis are not coaxial. In some embodiments, the first light source axis, the second light source axis, the third light source axis, the fourth light source axis, the first detector axis, the second detector axis, the third detector axis, and the fourth detector axis are positioned circumferentially about the normal axis. In some embodiments, the first light source is positioned circumferentially adjacent to the first detector. In some embodiments, the fluorometer does not include a dichroic mirror or beam splitter. In some implementations, the fluorometer further includes a processor and a non-transitory memory that includes instructions that when executed by the processor store 400 analog-to-digital readings taken by the first detector over a 100 millisecond period. In some embodiments, the first light source emits first excitation light along the first light source axis; and wherein the first excitation light is reflected away from the first photodetector axis at the measurement aperture. In some embodiments, the measurement aperture is configured to receive a sample; and wherein the first excitation light from the first light source has a first spectrum and the first light detector measures a first fluorescence of the sample in response to the first excitation light. In some embodiments, the second excitation light from the second light source has a second spectrum and the second light detector measures a second fluorescence of the sample in response to the second excitation light. In some embodiments, the measurement aperture is configured to align with a planar surface of the PCR chamber. In some embodiments, the first detector includes a first lens, a filter, a second lens, and a solid state detector.

In some embodiments, provided herein are methods of nucleic acid quantification comprising: (a) Subjecting a sample suspected of containing a target nucleic acid to a multicycle amplification reaction in the presence of a detectable reporter to produce an amplified product; (b) Detecting a signal from the detectable reporter, the signal being related to the amount of detectable reporter incorporated into the amplification product after each cycle of the amplification reaction; (c) Identifying an earliest cycle in which the normalized increase of the signal is greater than the cutoff value; (d) Fitting a linear equation to the plurality of signals from loops earlier than the earliest loop in which the normalized increase of the signals is greater than the threshold; (e) Fitting a curve to a plurality of signals from loops later than the earliest loop in which the normalized increase in signal is greater than the threshold; (f) -identifying a cycle (Cq) in which the normalized difference of the signals of the linear equation and the curve is equal to a threshold; wherein Cq is inversely proportional to the amount of target nucleic acid present in the sample. In some embodiments, step (c) comprises: (i) Identifying a cycle having a maximum normalized increase in signal; (ii) If the maximum normalized increase of the signal is greater than the cutoff value, determining an earliest cycle of the signal before the cycle having the maximum normalized increase of the signal that is greater than a lower cutoff value. In some embodiments, the method further comprises the steps of: calculating a moving average of the detected signal for each cycle of the amplification reaction and using the moving average for each cycle of steps (c) - (f). In some embodiments, the moving average is calculated as an average of the signal at each cycle, calculated with the signal at the two immediately earlier cycles and the two immediately later cycles. In some embodiments, the curve is a conic. In some embodiments, the multicycle amplification reaction is a 30-50 (e.g., 35, 40, 45) cycle amplification reaction. In some embodiments, the multicycle amplification reaction is a 40-cycle amplification reaction. In some embodiments, the multicycle amplification reaction is a quantitative polymerase chain reaction (qPCR). In some embodiments, the detectable reporter is a fluorophore and the signal is fluorescence. In some embodiments, each cycle includes a nucleic acid denaturation step, an annealing/extension step, and a detection step. In some embodiments, each cycle includes a nucleic acid denaturation step, an annealing step, an extension step, and a detection step. In some embodiments, the sample is a biological sample. In some embodiments, the target nucleic acid is a viral nucleic acid. In some embodiments, the amount of target nucleic acid present in the sample is proportional to the viral load in the sample.

In some embodiments, provided herein are kits and methods for rapid sorting of target nucleic acids from biological samples. In particular, reagents and method steps are provided for releasing and capturing target nucleic acids from whole cells, separating the target nucleic acids from cellular contaminants, and amplifying/detecting the target nucleic acids.

In some embodiments, provided herein are methods and kits for preparing samples for PCR analysis that reduce the time required to lyse cells, extract nucleic acids, capture target nucleic acids, and separate them from contaminating/interfering substances. The methods/kits herein can be used on a variety of different platforms including, but not limited to, manual manipulation using pipettes and tubes, automation using self-contained single use cartridges and external handling instruments, or automation using robotic high throughput platforms and microplates.

The methods/kits herein reduce the complexity of nucleic acid purification and detection. In some embodiments, the methods/kits herein comprise liquid reagents (e.g., single buffer compositions employed at multiple steps of the processes herein). In some embodiments, the methods/kits herein comprise two additional liquid reagents. In some embodiments, the liquid reagent is used to bring the biological sample into suspension in a volume sufficient for manipulation in the process steps herein, and to resuspend a plurality of dried reagents (e.g., lyophilized reagents). In some embodiments, the dry reagents include a lysis reagent (e.g., protease K, SDS and salts), a capture reagent comprising a nucleic acid probe (e.g., a nucleic acid comprising a hybridization sequence tethered to a capture moiety (e.g., biotin), and a capture agent coated magnetic bead (e.g., streptavidin coated paramagnetic bead). In some embodiments, analytical reagents are provided that contain, for example, components required for amplification and detection/quantification of a target nucleic acid.

Features of embodiments of the present technology include, but are not limited to, one or more of the following: the chemical formulation of the active suspension is modified by the addition of reagents only, without removing the fraction by precipitation, centrifugation or filtration (before the magnetic separation step); the same aqueous buffer is used for multiple steps (e.g., lysis, washing, resuspension, and/or amplification/detection); actively mixing the solution and suspension by sufficiently transferring reactants into and out of the reaction vessel; transferring the solution and suspension between temperature controlled chambers having an optimized temperature that increases the reaction rate and efficiency; rapidly heating the sample solution to obtain temperatures for proteolysis, cell lysis and PK denaturation; no pauses are required at temperatures optimal for PK activity; delaying the addition of streptavidin coated magnetic beads to increase the efficiency of binding to the solid phase by allowing time for the biotinylated probe to hybridize to the target in solution; agglomerating/capturing magnetic beads by placing a magnet near the entrance of the channel or pipette; resuspending the agglomerated beads by lifting them off a membrane having an oscillating air-water interface; amplifying the target nucleic acid while binding the target nucleic acid to the magnetic beads via the capture probes; rehydrating the dried/concentrated amplification reagents directly with a magnetic bead suspension; the target-probe complex is bound to the bead by biotin-avidin, so the complex can be released at a lower temperature where avidin is denatured, etc. The oscillating air-water interface is created by pumping a given volume of buffer into and out of the chamber.

Various devices, apparatuses, systems, components thereof, reagents, and methods are described herein and may be used together or independently in embodiments. For example, the reagents described herein can be used to perform the methods described herein using the systems (e.g., cartridges and instruments) described herein. Alternatively, the methods herein may be performed independently of the cartridges and instruments herein. Similarly, the components of the cartridges and instruments described herein may be used in other devices, methods performed without the cartridges or instruments described herein, and/or in applications not specifically set forth herein. Various combinations of the method steps, compositions, and/or components described herein are contemplated and such combinations are within the scope of the present disclosure. Similarly, the method steps, compositions, and/or components described herein may be used independently of the methods, systems, devices, and systems described herein.

Drawings

The drawings and examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the exemplary figures ("fig.") associated with one or more embodiments.

The patent or application document contains at least one drawing which is presented in color. Copies of this patent or patent application publication with color drawings will be provided by the office upon request and payment of the necessary fee.

Fig. 1 is a perspective view of a cartridge according to one embodiment.

Fig. 2 is a perspective view of the cartridge of fig. 1 with portions removed for clarity and the transfer chamber in a storage configuration.

Fig. 3 is an exploded front view of the cartridge of fig. 1.

Fig. 4 is an exploded rear view of the cartridge of fig. 1.

Fig. 5 is a perspective view of the body of the cartridge of fig. 1.

Fig. 6 is a partial cross-sectional view of the storage section of the cartridge of fig. 1.

Fig. 7 is a partial cross-sectional view of a processing section of the cartridge of fig. 1.

Fig. 8 is a cross-sectional view of the cartridge of fig. 1 with the transfer chamber fluidly coupled to the processing section.

Fig. 9 is a partial cross-sectional view of a microfluidic section of the cartridge of fig. 1.

Fig. 10A is a side view of a microfluidic segment with a wax seal in an initial configuration.

Fig. 10B is a side view of a microfluidic section with a wax seal in a sealed configuration.

Fig. 11 is a partial perspective view of a microfluidic segment.

Fig. 12 is a cross-sectional view of a microfluidic segment.

Fig. 13 is a side view of a microfluidic segment according to another embodiment.

Fig. 14 is a side view of a microfluidic segment according to another embodiment.

Fig. 15A and 15B are views of the cassette, with each access port for a respective chamber depicted with an overmolded seal (blue).

Fig. 16 contains a view of a cartridge including an energy director (green) for the heat seal components of the cartridge.

Fig. 17 is a perspective view of a heating and detection assembly.

Fig. 18 is a side view of a system including a cartridge and the heating and detection assembly of fig. 17.

Fig. 19 is a perspective view of a portion of the heating and detection assembly of fig. 17.

Fig. 20 is a side view of fig. 19.

Fig. 21 is a perspective view of a heat transfer device.

Fig. 22 is a perspective view of a heat transfer device.

Fig. 23 is a perspective cross-sectional view of the heat transfer device of fig. 22.

FIG. 24 is a graph of temperature versus time for the reaction chambers of the cartridge subjected to the heating and detection assembly of FIG. 18.

Fig. 25 is an enlarged portion of the graph of fig. 24.

Fig. 26 is a partial cross-sectional view of a microfluidic section of the cartridge of fig. 18.

Fig. 27A is a side view of a microfluidic segment with a wax seal in an initial configuration.

Fig. 27B is a side view of a microfluidic section with a wax seal in a sealed configuration.

Fig. 28 is a top view of a detector portion of the heating and detection assembly of fig. 17.

Fig. 29 is a partial cross-sectional view of the heating and detection assembly of fig. 17.

Fig. 30 is a partial cross-sectional view of the heating and detection assembly of fig. 17.

FIG. 31 is a perspective view of a portion of a fluorometer with portions removed for clarity.

FIG. 32 is a partial cross-sectional view of the fluorometer of FIG. 31.

FIG. 33 is an exemplary plot of fluorescence readings of positive SARS-CoV-2 samples.

Fig. 34 is a view of an area surrounding a breakpoint of the graph shown in fig. 33.

Detailed Description

In some embodiments, provided herein is a cartridge device useful for detecting and/or quantifying analyte levels (e.g., via an immunoassay, nucleic acid amplification). The analyte may be present in the liquid sample or absorbed in a porous medium such as a swab. The cartridge contains all the components (e.g., reagents, buffers, beads, etc.) and chambers necessary to process and detect the target analyte. In other words, the cartridge is self-contained. The cartridge is acted upon by a processing instrument having a complementary mechanism to perform operations including, but not limited to, heat transfer, liquid transfer, magnetic transfer, and optical detection. In some embodiments, the cassette is acted upon by an adjustable assembly on which a plurality of heat transfer devices are mounted. In some embodiments, the adjustable assembly further comprises a fluorometer integrated in and between the plurality of heat transfer devices. In some embodiments, reagents and method steps are provided for releasing and capturing target nucleic acids from whole cells, separating the target nucleic acids from cellular contaminants, and amplifying/detecting the target nucleic acids.

I. Definition of the definition

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, some preferred methods, compositions, and materials are described herein. Before describing the present materials and methods, however, it is to be understood that this invention is not limited to the particular molecules, compositions, methods, or protocols described herein as such may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the present specification is for the purpose of describing particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, however, the present specification, including definitions, will control. Thus, in the context of the embodiments described herein, the following definitions will apply.

The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, but it may. In addition, the phrase "in another embodiment" as used herein does not necessarily refer to a different embodiment, but it may. Accordingly, as described below, various embodiments of the present invention may be readily combined without departing from the scope or spirit of the present invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide amphiphile" is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the terms "comprises," "comprising," and variations thereof, mean that there are enumerated features, elements, method steps, etc., without excluding the existence of additional features, elements, method steps, etc. Conversely, the term "consisting of … …" and language variants thereof mean that the recited features, elements, method steps, etc., are present and that any non-recited features, elements, method steps, etc., are excluded, except for commonly associated impurities. The phrase "consisting essentially of …" means the recited features, elements, method steps, etc., as well as any additional features, elements, method steps, etc., that do not materially affect the basic properties of the composition, system, or method. Many embodiments herein are described using the open-ended "comprising" language. Such embodiments encompass a plurality of closed "consisting of … …" and/or "consisting essentially of … …" embodiments that may alternatively be claimed or described using such language.

As used herein, the terms "subject" and "patient" refer to any animal, such as dogs, cats, birds, livestock, and especially mammals, preferably humans.

As used herein, the terms "sample" and "specimen" are used interchangeably and are used in the broadest sense. In a sense, a sample is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples are available from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, stool, urine, and the like. Environmental samples include environmental materials such as surface materials, soil, mud, sludge, biological films, water, and industrial samples. However, such examples should not be construed as limiting the type of sample suitable for use in the present invention.

As used herein, the term "lysate" refers to a solution and/or suspension resulting from the lysis (cleavage) of cells and/or viruses to release the nucleic acids contained therein. The term "whole lysate" refers to a lysate that contains all the components of the original sample without removing any of the components. The term "partial lysate" or "purified lysate" refers to a lysate from which one or more components or fractions have been removed.

The term "system" as used herein refers to a collection of compositions, devices, articles, materials, etc. (e.g., cartridges and instruments) grouped together in any suitable manner (e.g., physically associated; in fluid communication, electronic or data communication; packaged together; etc.) for a particular purpose.

The term "cartridge" refers to such a device as follows: including, for example, a plurality of chambers, compartments, microfluidics, channels, etc., for containing/mixing reagents and samples, but without all of the necessary fluid handling mechanisms, magnetic particle handling mechanisms, heaters, etc., to function independently of the individual instruments containing such mechanisms. When the cartridge is engaged with the instrument (e.g., when the cartridge is placed/inserted into the instrument), mechanisms within the instrument are properly aligned with the cartridge to provide the necessary functions. In certain embodiments, the cartridge may be designed for single use, after which it is discarded. In other embodiments, the cartridge is provided for multiple uses. In certain embodiments, one or more of the compartments in the cartridge contains a reagent.

As used herein, the term "preparing" and its language equivalents refer to any step taken to alter a sample or one or more components thereof, for example, for use in a subsequent analysis or detection step. Exemplary sample preparation steps include, for example, diluting or concentrating a sample, sorting or purifying a sample component, heating or cooling a sample, amplifying a sample component (e.g., a nucleic acid), labeling a sample component, and the like.

As used herein, the term "analysis" and its language equivalents refer to any step taken to characterize a sample or one or more components thereof. Exemplary analytical steps include, for example, quantifying a sample component (e.g., a target nucleic acid), sequencing a sample component, and the like.

As used herein, the term "processor" (e.g., microprocessor, microcontroller, processing unit, or other suitable programmable device) may include, among other things, a control unit, an arithmetic logic unit ("ALC"), and a plurality of registers, and may be implemented using known computer architectures (e.g., modified harvard architecture, von neumann architecture (von Neumann architecture), etc.). In some embodiments, the processor is a microprocessor that may be configured to communicate in a stand-alone and/or distributed environment, and may be configured to communicate with other processors via wired or wireless communication, wherein such one or more processors may be configured to operate on one or more processor control devices, which may be similar or different devices.

As used herein, the term "memory" is any memory storage device and is a non-transitory computer-readable medium. The memory may include, for example, a program storage area and a data storage area. The program storage area and the data storage area may include different types of memory, such as a combination of ROM, RAM (e.g., DRAM, SDRAM, etc.), EEPROM, flash memory, hard disk, SD card, or other suitable magnetic, optical, physical, or electronic memory device. The processor may be connected to a memory and execute software instructions that can be stored in RAM of the memory (e.g., during execution), ROM of the memory (e.g., on a substantially permanent basis), or another non-transitory computer-readable medium, such as another memory or disk. In some embodiments, the memory includes one or more processor readable and accessible memory elements and/or components that are internal to the processor control device, external to the processor control device, and accessible via a wired or wireless network. Software included in implementations of the methods disclosed herein may be stored in memory. Software includes, for example, firmware, one or more application programs, program data, filters, rules, one or more program modules, and other executable instructions. For example, the processor may be configured to retrieve from memory and execute, among other things, instructions related to the processes and methods described herein.

II box

In some embodiments, provided herein are devices for rapid sample processing and analyte detection. In some embodiments, the device can be used to process any suitable sample (e.g., a biological sample (e.g., a solid sample (e.g., a tissue biopsy), a liquid sample (e.g., blood, saliva, urine, etc)), an environmental sample, etc.). In particular embodiments, the devices herein can be used to process a sample containing cells and detect/quantify cellular analytes (e.g., nucleic acids, antigens, etc.).

In some embodiments, the devices herein comprise a cartridge that engages with (e.g., is inserted into) a complementary instrument. In some embodiments, the cartridge devices herein include chambers, channels, vents, apertures, reagents (e.g., lysis reagents, digestion reagents, capture probes, primers, paramagnetic particles (PMPs)) required for sample processing and analyte detection. However, the cartridge lacks the heating elements, pressure sources, magnetic transfer mechanisms, fluorescent detection mechanisms, etc. required for sample processing and analyte detection. Complementary instruments include the mechanisms and functions required for sample processing and analyte detection that are lacking in the cartridge. For example, in some embodiments, the complementary instrument includes an adjustable heating and detection assembly having a plurality of heat transfer devices and a fluorometer. In some embodiments, the complementary instrument includes a magnetic transfer element, for example, for moving magnetic material within a cartridge associated with the magnetic transfer element. In some embodiments, the complementary instrument comprises a pressure element, for example, drawing fluid into and/or out of the cartridge (or a chamber thereof) and dropping fluid into and/or withdrawing fluid from the cartridge. In some embodiments, the cartridge is disposable (e.g., single use). In other embodiments, the cartridge is a multi-use device, but must be cleaned and/or reloaded with reagents between uses. The cartridge is used with a multi-purpose instrument. In some embodiments, the instrument is used with a plurality of different cartridges for performing different sample treatments and/or detection assays/protocols. In some embodiments, the instrument is dedicated to a single cartridge configuration.

In some embodiments, the cartridge device includes two or more chambers for holding a volume of liquid (e.g., 0.1ml, 0.2ml, 0.5ml, 1.0ml, 1.5ml, 2.0ml, or more). In some embodiments, one or more of the chambers includes an access channel extending upward from a bottom of the chamber (e.g., a side of the bottom of the chamber) toward a top of the device, terminating at an access port. The inlet channel allows liquid in the chamber to be extracted through the inlet channel by applying a negative pressure at the inlet port. Since the inlet of the inlet channel of the chamber is at the bottom of the chamber (e.g., one side of the bottom), liquid added to the chamber will immediately collect at the bottom of the chamber and about 100% (e.g., 99.9%, 99.5%, 99%, 98.5%, 98%, 95%, or ranges therebetween) of liquid within the chamber can be removed via the inlet channel. In some embodiments, one or more (e.g., all) of the access chambers include a vent channel. The vent passage allows the chamber to remain at ambient atmospheric pressure when pressure is applied to the inlet passage, allowing liquid to be moved into/out of the inlet passage.

In some embodiments, the cartridge device comprises a transfer balloon. In some embodiments, the transfer balloon tube includes an open top at one end, an open tip, and an open lumen extending therebetween. In some embodiments, the transfer bladder tube is similar to a pipette tip. In some embodiments, the transfer balloon tube is stored within a cavity in the device (e.g., a cavity located adjacent to and aligned with a chamber of the device (e.g., within a storage section of the device)). In some embodiments, the open top of the transfer bladder tube is configured to engage with a nozzle on a pressure element of a complementary instrument (e.g., in the same manner as a pipette tip engages with a pipette). In some embodiments, the pressure element is engaged with the transfer balloon tube, capable of lifting the transfer balloon tube up and out of the storage cavity, and capable of moving the transfer balloon tube laterally along the storage and processing sections of the device. In some embodiments, the tip of the transfer balloon tube is configured to engage (e.g., be positioned within, form a seal with, etc.) an access port connected to the chamber. In some embodiments, the pressure source applies a negative pressure to draw liquid from the chamber upward, through the access channel, and into the transfer bladder tube via the access port. In some embodiments, the pressure source applies positive pressure to expel liquid from the transfer bladder tube, through the inlet port and the inlet channel, and into the chamber. In some embodiments, repeated cycles of positive and negative pressure from a pressure source are used to mix the sample within the chamber (e.g., draw liquid into and out of the chamber and transfer bladder tube). In some embodiments, a pressure source and transfer bladder are used to move liquid between the chambers. Since the transfer bladder tube can be moved into alignment with the inlet ports of the multiple chambers, the system can be used to move liquid between any of the several chambers. This is in contrast to other devices that connect chambers through channels and are therefore limited by the connectivity of the chambers or require valves to open/close fluid communication between the various chambers.

In some embodiments, the detection or reaction chamber is accessed by a microfluidic, although the processing chamber and the storage chamber are accessed via a transfer bellows. In some embodiments, the last chamber of the processing section may be accessed through a transfer bladder (via an access channel and an access port) and through a microfluidic channel that extends from the processing chamber to the detection or reaction chamber. In some embodiments, the fluid added to the last processing chamber will flow through the microfluidics to the detection or reaction chamber. In some embodiments, the pellet-like PMPs in the final processing chamber may be transferred through the microfluidics and into the detection or reaction chamber using a magnetic transfer element of a complementary instrument. Described herein are exemplary configurations for connecting a processing section and a microfluidic section of a device herein.

In some embodiments, to accurately perform a reaction (e.g., PCR) and/or detection (e.g., fluorescence detection), the detection or reaction chamber is sealed to prevent material introduction or exudation during the reaction or detection. In some embodiments, the devices herein allow for sealing of the reaction/detection chamber without a valve or the like. In some embodiments, a wax seal resides adjacent to the inlet channel of the reaction/detection chamber. In its first position, the first wax seal does not block fluid from entering the reaction/detection chamber via the inlet channel. Similarly, in some embodiments, a second wax seal resides adjacent to the vent channel of the reaction/detection chamber. In its first position, the second wax seal does not block the flow of gas from the reaction/detection chamber. However, if sufficient heat is applied to the wax seal (e.g., via a heater in a complementary instrument located adjacent and in close proximity to the reaction/detection section of the cartridge device), the wax seal melts and flows into the inlet channel and vent channel. If allowed to cool in the inlet and vent channels, the wax will form a seal in the respective channels, preventing liquid or gas from flowing into/out of the reaction/detection chamber.

In some embodiments, for example when two or more channels are in fluid communication with the chamber, the direction of flow of melted wax from the chamber may be affected by selectively placing the heater in a direction opposite the desired flow path. In such embodiments, the heater will melt the wax moving opposite the desired flow path, but allow the wax moving in the desired direction to solidify more easily in the desired channel, thereby allowing the flow direction of the melted wax to be affected.

In some embodiments, the sample preparation step and the analysis step are performed simultaneously and/or sequentially. For example, in qPCR, nucleic acid amplification and quantification steps are repeated successively.

An exemplary cartridge device containing the elements described herein and capable of performing the various functions described herein is depicted, for example, in fig. 1-16. Other cartridges comprising different combinations and configurations of the elements shown in fig. 1-16 are also within the scope of this document.

Referring to fig. 1, a cartridge 10 for detecting the level of an analyte is shown. Cartridge 10 includes a storage section 14, a processing section 18, and a microfluidic section 22. In the illustrated embodiment, the processing section 18 is positioned between the storage section 14 and the microfluidic section 22. Cartridge 10 contains all the components and chambers necessary for processing and detecting a target analyte. In some embodiments, cartridge 10 is acted upon by a processing device that includes a complementary mechanism, for example, to perform operations including, but not limited to, heat transfer, liquid transfer, magnetic transfer, and optical detection.

In general, the storage section 14 contains the sample to be analyzed, the buffer and transfer vesicles 26 used in the process; the processing section 18 is where the target is extracted, purified and bound to magnetic beads; and the microfluidic section 22 is where one or more targets are detected. In the illustrated embodiment of fig. 2, the connector 30 (or docking section) is coupled to the storage section 14 and the processing section 18. The connector 30 (or docking section) provides an interface for loading the sample and transferring liquid between the chambers. In other words, the connector 30 (or docking section) provides access to the fluid contained within the storage section 14 and the processing section 18, among other things.

With continued reference to fig. 1, the cartridge 10 includes: a first seal 34 coupled to a portion of the connector 30 (or docking section) corresponding to the storage section 14; and a second seal 38 coupled to a portion of the connector 30 (or docking section) corresponding to the processing section 18. In some embodiments, the first seal 34 and the second seal 38 are heat seal foil lidstock. The transfer bladder tube 26 is shown in fig. 1 as being positioned outside of the storage section 14. However, in some embodiments, the transfer bladder tube 26 is positioned within a cavity 42 (see fig. 2) formed in the storage section 14.

Referring to fig. 2, the cassette 10 is shown with the first seal 34 and the second seal 38 removed for clarity and the transfer bladder tube 26 positioned within the cavity 42. In the illustrated embodiment, the storage section 14 includes a first chamber 46 (e.g., for containing a test sample) and a second chamber 50 (e.g., for containing a buffer). In other embodiments, the storage section 14 includes more or less than two chambers. For example, in some embodiments, the storage section 14 includes a single chamber. In other embodiments, the storage section 14 includes at least three chambers (e.g., 3, 4, 5, 6, or more). In the illustrated embodiment, the storage section 14 defines a width 54 greater than 3mm (the width 156 in section 18 is 3 mm). In some embodiments, the width 54 is greater than 3mm (e.g., 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, or greater, or ranges therebetween). In the illustrated embodiment, the first chamber 46 and the second chamber 50 each have a volume of greater than about 1ml, although other sizes of storage chambers are also contemplated (e.g., 0.5ml, 0.75ml, 1.25ml, 1.5ml, 1.75ml, 2.0ml, or greater, or ranges therebetween). In some embodiments, the first chamber 46 is formed with sloped walls 58 (fig. 4) shaped such that all volumes except a few microliters can be transferred to the processing section 18. In other words, the sloped wall 58 at least partially forms the first chamber 46.

Referring to fig. 2 and 3, the portion of the connector 30 (or docking section) coupled to the storage section 14 includes an inlet 62 (e.g., a sample inlet), a first inlet port 66 fluidly connected to the first chamber 46, a first vent 70 fluidly connected to the second chamber 50, a second inlet port 74 fluidly connected to the second chamber 50, and an opening 78 to the cavity 42. In the illustrated embodiment, the filter 72 is positioned within the first vent 70. The first passage 82 fluidly connects the first chamber 46 with the first inlet port 66. Likewise, a second passage 86 fluidly connects the second chamber 50 with the second inlet port 74. As explained in greater detail herein, the first and second access ports 66, 74 are configured to engage with the transfer balloon tube 26.

In some implementations, additional chambers in the storage section (e.g., third storage chamber, fourth storage chamber, etc.) may each include one or more of a liquid inlet, an access port (e.g., an access port engaged with a transfer bladder tube), a vent, etc. (e.g., within the storage section and/or an associated connector).

Referring to fig. 6, the first passage 82 includes a first portion 90 coupled to the first access port 66, a second portion 94 in fluid communication with the first portion 90 and extending generally perpendicular to the first portion 90. The first passage 82 also includes a third portion 98 in fluid communication with the second portion 94 and extending generally perpendicular to the second portion 94 and generally parallel to the first portion 90. In addition, the first channel 82 includes an arcuate portion 102 positioned between the third portion 98 and the first chamber 46. The first chamber 46 has a first end 46A and a second end 46B, wherein the first end 46A is positioned closer to the connector 30 (or mating section) than the second end 46B. In the illustrated embodiment, the arcuate portion 102 is coupled to the second end 46B of the first chamber 46. In other words, the first passage 82 fluidly connects the first inlet port 66 to the end 46B of the first chamber 46 that is positioned opposite the inlet 62.

With continued reference to fig. 6, the second channel 86 is similar to the first channel 82 and also includes a first portion 106, a second portion 110, a third portion 114, and an arcuate portion 118 coupled to an end 50B of the second chamber 50 that is positioned opposite the connector 30 (or docking section).

In some embodiments, the connector 30 (or docking section) includes a cap 120 that is correspondingly received within the inlet 62 once the sample has been positioned within the first chamber 46. In the illustrated embodiment, the cap 120 is movable between an open position (fig. 2) in which the cap 120 is removed from the inlet 62 and a closed position in which the cap 120 is positioned within the inlet 62. In some embodiments, a filter similar to filter 72 is positioned in cap 120 to permit venting of first chamber 46 via first inlet port 66 during fluid aspirating/dispensing.

Referring to fig. 3, the cartridge 10 includes a body 124 (fig. 5) in which the first chamber 46, the second chamber 50, and the cavity 42 are at least partially formed. In some embodiments, the body 124 is integrally formed as a single piece.

In some embodiments, the cartridge 10 further includes a first cover 128 coupled to the body 124, for example, with a first adhesive layer 132. In the illustrated embodiment, the first cover 128 is a film cover and the first adhesive layer 132 is a Pressure Sensitive Adhesive (PSA). In the illustrated embodiment, the first cover 128 and the first adhesive layer 132 correspond to the storage section 14 of the cartridge 10. The first adhesive layer 132 includes cutouts 136A, 136B, 136C corresponding to the first chamber 46, the second chamber 50, and the cavity 42, respectively. In other embodiments, the cartridge 10 further includes a first cover 128 coupled to the body 124, such as by heat sealing.

In the illustrated embodiment, the first cover 128 is a heat transfer surface of the first chamber 46 and the second chamber 50. In other words, the first cover 128 is advantageously a film to improve heat transfer from outside the cartridge 10 to the first chamber 46 and the second chamber 50. In contrast to the first cover 128, the first chamber 46, the second chamber 50, and the cavity 42 are defined by the rigid body 124 (fig. 4). In other words, in the illustrated embodiment, the first cover 128 and the body 124 at least partially define the first chamber 46, the second chamber 50, and the cavity 42.

In some embodiments, the second chamber 50 is configured to hold a volume of buffer. In some embodiments, the device is configured such that transfer bladder tube 26 may draw buffer from second chamber 50 and move the buffer to one or more other chambers within the storage section and/or the processing section via second channel 86. In some embodiments, the first chamber 46 is configured to receive a sample or specimen. The sample or specimen can be a liquid (e.g., body fluid (e.g., blood, saliva), buffer-solubilized sample, etc.) or a solid (e.g., swab tip). In some embodiments, the device is configured such that the transfer bladder tube 26 can draw a sample from the first chamber 46 (e.g., in combination with a buffer) and move the sample to one or more other chambers within the storage section and/or the processing section via the first channel 82.

Referring to fig. 2 and 3, the processing section 18 includes a third chamber 140, a fourth chamber 144, a fifth chamber 148, and a sixth chamber 152. In other embodiments, the processing section 18 includes more than four chambers or less than four chambers. For example, in some embodiments, the processing section 18 includes less than four chambers. In other embodiments, the processing section 18 includes at least five chambers. In some embodiments, the treatment section may contain fewer (e.g., 1, 2, or 3) chambers or more (e.g., 5, 6, 7, 8, or more) chambers than shown in the figures herein. Depending on the number of chambers in the storage section and the processing section, the chambers may be referred to with appropriate numbers. In the illustrated embodiment, the width 156 of the treatment section 18 is in the range of about 1mm to about 3mm (e.g., 1mm, 1.25mm, 1.5mm, 1.75mm, 2.0mm, 2.25mm, 2.5mm, 2.75mm, 3.0mm, and ranges therebetween), with both wider and narrower treatment sections being within the scope herein. As explained in more detail herein, in the third chamber 140, fourth chamber 144, fifth chamber 148, and sixth chamber 152, the target is extracted, purified, and/or bound to magnetic beads.

With continued reference to fig. 3, the portion of the connector 30 (or docking section) coupled to the processing section 18 includes a third inlet port 160 fluidly connected to the third chamber 140, a fourth inlet port 164 fluidly connected to the fourth chamber 144, a fifth inlet port 168 fluidly connected to the fifth chamber 148, and a sixth inlet port 172 fluidly connected to the sixth chamber 156. The connector 30 (or docking section) also includes a second vent 176 fluidly connecting each of the third chamber 140, the fourth chamber 144, the fifth chamber 148, and the sixth chamber 152 to the atmosphere. In the illustrated embodiment, the filter 180 is positioned within the second vent 176. In some embodiments, any vent herein may include a filter to prevent debris from entering an associated chamber and/or to prevent liquid from seeping from the chamber (e.g., as a aspirate).

Referring to fig. 7, a third passage 184 fluidly connects third chamber 140 with third inlet port 160, and a fourth passage 188 fluidly connects fourth chamber 144 with fourth inlet port 164. Likewise, fifth passage 192 fluidly connects fifth chamber 148 with fifth inlet port 168, and sixth passage 196 fluidly connects sixth chamber 152 with sixth inlet port 172. In the illustrated embodiment, each of the channels 184, 188, 192, 196 is fluidly connected to the corresponding chamber 140, 144, 148, 152 at an end 200 positioned opposite the connector 30 (or docking section).

Referring to fig. 7 and 8, the third channel 184 includes a cross-channel portion 204 positioned adjacent to the third access port 160. In some embodiments, the lyophilization reagents 208 (e.g., in the shape of spheres) are positioned in the cross channel portion 204 and are re-hydrated as the liquid flows through the third channel 184. In other embodiments, the lyophilization reagents are added into any of the channels 184, 188, 192, 196. In some embodiments, one or more of the channels 184, 188, 192, 196 contain pockets or other openings that contain reagents (solid or liquid). In some embodiments, one or more of the channels 184, 188, 192, 196 are free of pockets or other openings that contain reagents (solid or liquid). Storing the lyophilized balls 208 within the channel 184 leading to the chamber 140 helps to physically contain the lyophilized balls 208 and is further improved to be able to effectively and uniformly re-dissolve the dried reagents (i.e., storing the lyophilized balls in the chamber sometimes results in their floating up with the fluid and not effectively mixing).

With continued reference to fig. 8, the transfer bladder tube 26 is shown fluidly coupled to a third inlet port 160. In the illustrated embodiment, a seal 212 (e.g., an O-ring) is positioned within the third access port 160 and improves the seal between the transfer bladder tube 26 and the body 124. In some embodiments, any access port in the device may include a seal (e.g., an O-ring) or may be free of a seal. The transfer pocket tube 26 includes a suction head 216 that may be configured to extend through the third access port 160 and be at least partially received within the third channel 184 (fig. 8). The transfer bladder tube 26 also includes a filter 220 and a transfer chamber 222 positioned between the filter 220 and the suction head 216. In the illustrated embodiment, the transfer pocket tube 26 includes an open end 224 positioned opposite the suction head 216. As explained in more detail herein, fluid may be drawn from a chamber (e.g., third chamber 140) into transfer chamber 222 and transferred to a different location in cartridge 10.

The transfer bladder tube 26 provides random access to the liquid in the cartridge 10 and also provides the ability to mix solids and fluids. The suspension of solid phase particles and liquid flowing through the small diameter apertures 218 in the tip 216 of the transfer pocket tube 26 is subjected to high shear forces which can fracture the aggregated particles and assist in desorbing the interfering substances absorbed on its surface. Another advantage of the transfer bladder 26 is that multiple aliquots can be removed from a common wash buffer without cross-contamination, as the transfer bladder 26 never comes into contact with the bulk solution.

In some embodiments, transfer bladder tube 26 resides within storage section 14 and/or processing section 18 of cartridge device 10. In some embodiments, the transfer bladder tube is similar to a pipette tip. In some embodiments, the tip 216 of the transfer bladder tube 26 is configured to engage with an access port connected to each chamber of the device 10. The open end 224 of the transfer balloon tube 26 is accessible from outside the device 10. In some embodiments, a pressure source from outside the device (e.g., a component of an instrument into which the device is inserted) is engaged with the open end 224 of the transfer balloon tube 26. An external pressure source applies a negative pressure differential (e.g., a reduced pressure relative to the pressure of the transfer bladder tube) in the transfer bladder tube 26 to draw fluid from the chamber through the inlet port. An external pressure source applies a positive pressure differential (e.g., an increased pressure relative to the pressure of the transfer bladder tube) in the transfer bladder tube 26 to expel fluid from the transfer bladder tube 26 through the inlet port and into the chamber. In some embodiments, the device includes a single transfer balloon tube for moving liquid (e.g., sample, buffer, etc.) between chambers. In some embodiments, the device comprises a plurality (e.g., 2, 3, 4, etc.) of transfer vesicles. In some embodiments, an external pressure source is engaged with the transfer balloon catheter, the transfer balloon catheter 26 is withdrawn from the device, the transfer balloon catheter 26 is moved to a desired position, and then the transfer balloon catheter 26 is engaged with a desired access port. The transfer bladder tube 26 is contained within the cartridge device. The external pressure source and other components of the instrument into which the device is inserted do not contact the liquid reagent within the device. In some embodiments, the transfer bladder is used to transport a plurality of different liquids between a plurality of different chambers. In some embodiments, the external pressure source and the transfer balloon tube may be disengaged and re-engaged multiple times during analysis. The use of a transfer bladder tube and an external pressure source allows for inter-chamber liquid transfer within the storage section and the processing section, as well as mixing of liquid in the chambers, without valves and without regard to the relative positions of the chambers in the device (e.g., 'skip' the chambers).

Referring to fig. 3, the body 124 (fig. 5) at least partially forms a third chamber 140, a fourth chamber 144, a fifth chamber 148, and a sixth chamber 152. Also included on the first side 10A of the cartridge 10 is a cover 228 that is coupled to the body 124 with an adhesive layer 232. In the illustrated embodiment, the cover 228 is a film cover and the adhesive layer 232 is a Pressure Sensitive Adhesive (PSA). In some embodiments, the lid 228 is thin to allow for rapid heat transfer, but is capable of withstanding temperatures up to 100 ℃. In some embodiments, the lid 228 is chemically compatible (e.g., non-reactive, chemically inert, etc.). In the illustrated embodiment, the lid 228 and the adhesive layer 232 correspond to the processing section 18 of the cartridge 10. The adhesive layer 232 includes cutouts 236A, 236B, 236C, 236D corresponding to the third chamber 140, the fourth chamber 144, the fifth chamber 148, and the sixth chamber 152, respectively.

Referring to fig. 4, on a second side 10B (opposite the first side 10A) of the cartridge 10, the processing section 18 includes a first laminate layer 240, a second laminate layer 244, a cover 248, and three adhesive layers 252, 256, 260 positioned therebetween. In the embodiment shown, the first adhesive layer 252 couples the first laminate layer 240 to the body 124. Likewise, a second adhesive layer 256 couples the second laminate layer 244 to the first laminate layer 240. Finally, a third adhesive layer 260 couples the cover 248 to the second laminate layer 244. In other embodiments, the treatment section 18 includes any number of laminate layers and an adhesive layer coupled between the laminate layers. Films 240 and 248 are particularly selected for their optical clarity, fluorescent compatibility, rapid and efficient heat transfer, chemical compatibility, and ability to withstand high temperatures (about 100 ℃).

With continued reference to fig. 4, each of the first laminate layer 240, the second laminate layer 244, the adhesive layers 252, 256, and 260 includes cutouts 264A, 264B, 264C, 264D corresponding to the chambers 140, 144, 148, 152. In the illustrated embodiment, the second laminate layer 244 and the adhesive layer 252 each at least partially define a common vent channel 268. Each of the third chamber 140, the fourth chamber 144, the fifth chamber 148, and the sixth chamber 152 are fluidly connected to the second vent 176 by a common vent passage 268. Specifically, the common vent passage 268 intersects the fifth and sixth chambers 148, 152. The third and fourth chambers 140, 144 are fluidly connected to the common vent passage 268 through connection passages 272, 276 and apertures 280, 284 (fig. 7) formed in the body 124. In other words, the connection channel 272 and the aperture 280 are positioned between the third chamber 140 and the common vent channel 268. Likewise, the connecting passage 276 and the aperture 284 are positioned between the fourth chamber 144 and the common vent passage 268. In the illustrated embodiment, the common vent channel 268 is at least partially defined by the first laminate layer 240 and the adhesive layer 260.

Referring to fig. 9, the microfluidic section 22 includes a reaction chamber 304, a microfluidic vent channel 308, and a microfluidic inlet channel 312. The microfluidic vent channel 308 is fluidly connected to the reaction chamber 304. The microfluidic inlet channel 312 is fluidly connected to the sixth chamber 152 in the processing section 18 and the reaction chamber 304. In other words, the microfluidic inlet channel 312 fluidly connects the sixth chamber 152 in the processing section 18 to the microfluidic section 22. In the illustrated embodiment, the microfluidic inlet channel 312 is connected to a side 316 (fig. 4) of the sixth chamber 152, wherein the side 316 is positioned between where the sixth channel 196 is connected to the chamber 152 and the sixth inlet port 172. In other words, the microfluidic inlet channel 312 is connected to the side 316 in the middle of the sixth chamber 152. The microfluidic inlet channel 312 serves as an interface between the processing section 18 and the microfluidic section 22 and facilitates easy transfer of liquid and PMP to the microfluidic section 22 with openings that the pipettes (transfer bladder tubes) cannot directly access. The offset of chamber 196 in the vertical orientation as compared to chamber 192 also helps to achieve a high hydrostatic head, which also helps to facilitate easy transfer of liquid to section 22.

With continued reference to fig. 9, the reaction chamber 304 is positioned between the microfluidic inlet channel 312 and the microfluidic vent channel 308. In some embodiments, the lyophilized master mix 320 is positioned within the reaction chamber 304, in which, for example, PCR is performed.

Referring to fig. 3 and 4, the microfluidic section 22 of the illustrated embodiment is at least partially defined by a cover 324 and an adhesive layer 328 (fig. 3). Further, the microfluidic section 22 is at least partially defined by a first laminate layer 240, an adhesive layer 256, a second laminate layer 244, an adhesive layer 260, and a cover 248. Thus, in the illustrated embodiment, the microfluidic section 22 includes at least seven layers (including an adhesive layer). The microfluidic section 22 defines a width 334. In the embodiment shown, width 334 is less than width 156, and width 156 is less than width 54. The cover 324 and the adhesive layer 328 include cutouts 332 corresponding to the reaction chambers 304. In other words, the cutouts 332 in the cover 324 and the adhesive layer 328 expose the reaction chamber 304 to improve optical detection and heater engagement.

Referring to fig. 4 and 9, microfluidic vent channel 308 includes a first portion 336 formed in adhesive layer 260, a second portion 340 formed in adhesive layer 256, and a third portion 344 formed in adhesive layer 260. Thus, the second portion 340 is offset (i.e., positioned in a different plane) from the first portion 336 and the third portion 344 of the microfluidic vent channel 308. The third portion 344 of the microfluidic vent channel 308 is fluidly connected to the common vent channel 268.

Referring to fig. 4, adhesive layers (e.g., adhesive layers 256, 260, 328) in the cartridge 10 add functionality to the processing section 18 and the microfluidic section 22. Adhesives are conventionally used to bond films together to form a laminate structure. However, the adhesive layer in the cartridge 10 provides additional functionality. First, the adhesive layer may be exposed to create a location where the lyophilized reagents may be bonded. For example, in some embodiments, the PCR master mix 320 in the reaction chamber 304 adheres to the adhesive layer exposed to the reaction chamber 304. In other embodiments, the spheres of the lyophilized spheres (e.g., the reagent 208 in the third channel 184) are affixed to the exposed portion 233 (fig. 8) of the adhesive layer 252. In some embodiments, the reagent 208 is adhesively bonded to the PSA 252 on the side opposite the location where it is inserted.

A second additional function provided by the adhesive layer is to form channels in the adhesive layer to equalize the air pressure within the chamber. In the illustrated embodiment, the chambers 140, 144, 148, 152 in the processing section 18 are fluidly connected to a common vent channel 268 that is at least partially formed in the adhesive layer 256 (fig. 4). In other embodiments, the microfluidic inlet channel 312 and the microfluidic vent channel 308 are at least partially formed in the adhesive layers 256, 260. Forming the channels partially with the adhesive layer reduces the overall size of the cartridge 10.

With continued reference to fig. 9, the microfluidic section 22 includes a first wax seal 348 and a second wax seal 352. A first wax seal 348 is positioned adjacent the microfluidic vent channel 308 and a second wax seal 352 is positioned adjacent the microfluidic inlet channel 312. To maintain a stable concentration of reagents and amplicons, PCR is performed in a closed system (e.g., the reaction chamber 304 is sealed). The wax seals 348, 352 are configured to seal the reaction chamber 304 from both ends (i.e., the inlet end and the vent end). The air-vented microfluidic vent channel 308 leading from the reaction chamber 304 is used to initially buffer fluid prime (via channel 312) and air purge dry reagents. After priming, the paramagnetic particles carrying the targets are transferred into the reaction chamber 304 through the microfluidic inlet channel 312. Once the target is in the reaction chamber 304, the microfluidic vent channel 308 is closed (i.e., sealed) by melting the first wax seal 348 (by, for example, a heater). The molten wax fills the void space and nearby microfluidic vent channels 308 and then hardens, thereby sealing the microfluidic vent channels 308. After the first wax seal 348 is melted, the second wax seal 352 is then melted in a similar manner, sealing the microfluidic inlet channel 312 and closing the reaction chamber 304. Thus, in some embodiments, the first wax seal 348 in the microfluidic vent channel 308 melts and hardens before the second wax seal 352 in the microfluidic inlet channel 312. This ensures that any air trapped around the second wax seal 352 creates pressure to prevent molten wax from the second wax seal 352 from flowing into the reaction chamber 304. Any trapped air tends to flow out of the reaction chamber 304 to the sixth chamber 152 via the channels 312. The wax in the reaction chamber 304 can affect the fluorescence optical reading.

Referring to fig. 10A and 10B, the first and second wax seals 348, 352 are initially in a first solid state (fig. 10A) with the respective channels 308, 312 open, and may be changed to a second solid state (fig. 10B) with the respective channels 308, 312 sealed (i.e., closed). The wax seals 348, 352 enter a molten state between a first solid state and a second solid state. In the illustrated embodiment, the first and second wax seals 348, 352 are cylindrical in shape in the initial first solid state (fig. 10A) and do not block or seal their respective channels 308, 312. In response to the application of heat, the first and second wax seals 348, 352 melt and flow into the channels 308, 312. After the application of heat is removed, the melted wax resolidifies and enters a second solid state (fig. 10B) and forms a solid wax seal positioned within the channels 308, 312.

Referring to fig. 11 and 12, a first wax seal 348 in a first solid state is initially positioned over the microfluidic vent channel 308 within a cutout 356 formed in the laminate layers 240, 244 and the adhesive layer 256 (fig. 4). The first wax seal 348 initially rests on top of the adhesive layer (260) to improve the bond quality at the interface. In the initial solid state of the first wax seal 348, air can readily pass around and under the wax seal 348. When the wax seal 348 melts, the molten wax fills the empty surrounding space. In some embodiments, the volume of the first wax seal 348 is less than the volume of the second wax seal 352.

With continued reference to fig. 11 and 12, a second wax seal 352 positioned adjacent the microfluidic inlet channel 312 extends beyond the laminate layer, creating a tent air pocket 360 (fig. 10A) around the perimeter of the second wax seal 352. In other words, the second wax seal 352 causes the cover 324 to deform during assembly, creating a tent air pocket 360. Although the second wax seal 352 is positioned over the microfluidic inlet channel 312, fluid and target transfer through the microfluidic inlet channel 312 is still possible by utilizing the hydrostatic head at the beginning of the microfluidic inlet channel (contained within the chamber 152) and a certain amount of detergent in the fluid.

In some embodiments, the microfluidic inlet channel 312 is taller than the microfluidic vent channel 308. The microfluidic ventilation channel 312 has a thickness of about 0.051mm ² For example, 0.051mm high by 1mm wide). Likewise, the microfluidic inlet channel 312 has a thickness of about 0.54mm ² Cross-sectional area (e.g., 0.36mm high by 1.5mm wide). The microfluidic vent channel 308 has a smaller cross-sectional area than the microfluidic inlet channel 312 because the microfluidic vent channel 308 directs only the airflow, whereas the microfluidic inlet channel 312 must allow the passage of liquid buffer and solid particles containing genetic targets. Thus, the amount of wax required in the wax seal 352 for the microfluidic inlet channel 312 is greater than the amount of wax in the wax seal 348 for the microfluidic vent channel 308.

In some embodiments, the tent air pocket 360 is about 0.38mm higher than the surrounding laminate. When the wax seal 352 is melted by the pinch heater, the tent air pocket 360 collapses. Air trapped within the hardened wax seal creates a potential for fluid leakage. It is therefore important that during the wax melting process, air has a path out of the system so as not to compromise seal integrity.

In the illustrated embodiment, a spacing of at least about 2mm is provided between any laser cut features and the edges of the laminate to provide sufficient surface area to form a strong adhesive bond. In other words, the narrow adhesive contact areas are prone to leakage and failure. Specifically, the perimeter 364 of the tent air pocket 360 is positioned at least about 2mm away from the reaction chamber 304. In the embodiment shown, there is a spacing of at least about 2mm from the tent air pocket to any exposed laminate edge.

Wax seals 348 and 352 provide several advantages. The hardened wax seals 348, 352 are advantageously configured to withstand the pressures experienced in the reaction chamber 304 during thermal cycling involving alternating clamping of the reaction chamber with heaters having temperatures in the range of about 50 ℃ and about 95 ℃. In other words, the combination of high temperature and fluid displacement from the mechanical clamping places stresses on the wax seals 348, 352, which are subjected to the stresses. Although the heater may not directly contact the wax seal during thermal cycling, in some embodiments a wax having a high melting temperature (e.g., paraffin wax having a melting temperature of at least about 85 ℃) is selected to ensure that the wax seal is not inadvertently remelted by the heater associated with the reaction chamber 304.

In the illustrated embodiment, the wax seal is initially cylindrical in shape (i.e., coin-shaped) (e.g., a diameter of about 4.5mm times a thickness of about 0.43 mm). The rotational symmetry of the circular geometry of the wax seal reduces the risk of misalignment during manufacture of the cartridge 10. In addition, having the same wax seal design simplifies production. In other embodiments, the wax seal is initially oval in shape (fig. 13).

The wax seals 348, 352 may melt in a range of about 86 ℃ (i.e., the wax melting point) and about 95 ℃ (i.e., the default temperature setting of the PCR heater). The duration of the melting, or the duration of the clamping of the PCR heater to the wax seal, may be adjusted in synchronization with the melting temperature to ensure a good seal. For example, if the wax seal melts at too high a temperature for too long, the melted wax may spread further away from the seal site, thereby reducing the material density and mechanical integrity of the seal. In some embodiments, the melting procedure melts the wax seals 348, 352 by applying a relatively hot heater for a duration in the range of about 4 to about 5 seconds. The wax seals 348, 352 are then clamped with a cooler heater having a set point below the wax melting temperature for a duration of about 1.5 seconds. To reduce overall processing time, the wax seals 348, 352 are melted as the thermal PCR heater cools from about 95 ℃ to about 86 ℃ (rather than at a fixed temperature).

In some embodiments, the wax seals 348, 352 have a density of about 0.9g/mL and a density slightly less than the fluid around them at 1 g/mL. In the embodiment shown, gravity acts downwardly during melting and subsequent hardening of the wax seal. Thus, the orientation of gravity affects the movement of the molten wax. For example, molten wax may flow upward when gravity acts downward. The direction of flow of the molten wax is also affected by the surface area and location of the heater used to melt the wax seal. For example, when clamped with a heater, if not concentric but offset in one direction, the molten wax will tend to flow in the offset direction. Also, the heater clamping force is adjusted according to the relative positions of the front and rear sides of the heater, which may be mounted on a low spring constant spring, for example. In some embodiments, the plastic laminate layer is less rigid and is capable of deforming in response to the heater clamping force, thereby extruding and pushing the wax seal out of the boundary of the heater surface area.

In some embodiments, the molten wax seal is cooled by clamping the molten wax seal with a cooler heater (i.e., a heater having a temperature below the melting temperature of the wax). In other embodiments, the molten wax seal is cooled in ambient air to harden. Clamping the molten wax seal with a cooler heater cools the wax so that the wax hardens more quickly. The time of cooling in ambient air is about 6 to about 8 seconds, while the time of cooling by clamping is about 2 seconds.

In some embodiments, the adhesion of the wax seals 348, 352 is improved by exposing the wax to a layer of acrylic tape (rather than other plastic films such as polyester or polycarbonate). The improved quality of the bond between the melted wax and the channel wall may reduce the likelihood of fluid leakage through the hardened seal.

Overall, the cartridge 10 provides several advantages. The cartridge 10 is self-contained with a cartridge carrier liquid and freeze-dried reagents that do not require refrigeration. The cartridge 10 allows for processing of a variety of input sample types, including input sample types from pipettes, nasal swabs, and the like. The transfer of liquid between the chambers of cartridge 10 achieves random access and rapid mixing without the need for any valves. As used herein, random access means that liquid on one chamber can be transferred to any other chamber regardless of its position in the cartridge 10. Since the cartridge 10 achieves random access of liquid, the cartridge 10 can accommodate different treatment protocols. Furthermore, the random access in the cartridge 10 enables multiple washing of the paramagnetic particles (PMP) by disposing of dirty wash in an empty processing chamber. For example, the dirty wash in the fifth chamber 148 is disposed of in the third chamber 140 where the lysis is completed. In some embodiments, PMP is used to mix fluids inside the microfluidic section 22 via magnetic coupling. Furthermore, random access enables the cartridge 10 to store PMP in the fourth chamber 144 and add PMP to the third chamber 140 after the probe has had time to bind to the target at high temperature, thereby eliminating the need for an additional heater.

Another advantage of the cartridge 10 is that the dry reagents in the wet storage section 14, as well as the processing section 18 and the microfluidic section 22, are separated by the cavity 42 that receives the transfer bladder tube 26. In other words, the cavity 42 increases the distance between the dry reagent and the liquid buffer in the cartridge 10 to increase shelf life. The two separate heat-sealed foil covers 34, 38 also provide further separation between the storage section 14 and the processing section 18.

An additional advantage of cartridge 10 is that liquid can flow into the chamber at high speed (i.e., laboratory bench-top pipette mixing), which provides good mixing of the reactants. Speed, volume and time delay allow flexibility. The cross-sectional geometry of the channel (e.g., third channel 184) facilitates fluid mixing. Additionally, air may be injected into the chamber, creating bubbles that pass from the bottom of the chamber to the vent to mix the fluid via chaotic eddies and eddies. In some embodiments, air may be vented through a channel to a single common filter (e.g., filter 180 in vent 176).

Another advantage of the cartridge 10 is that it minimizes the chance of sample or processed sample leaking out of the cartridge. When the cartridge 10 is upright (i.e., as viewed in fig. 1), the liquid is away from the outlet channel and the hydrostatic pressure pulls the fluid away from the outlet. When the cartridge 10 is inverted, the liquid is away from the inlet of the channel and the hydrostatic pressure pulls the liquid away from the inlet. When the cartridge 10 is placed side by side, gravity passes through the chamber, so there is no hydrostatic head that causes liquid to flow to the outlet. The size and shape of the channels is detrimental to any capillary driven flow.

Referring to fig. 13, a microfluidic segment 400 (similar to microfluidic segment 22) is shown according to another embodiment. The microfluidic section 400 includes an oval inlet wax seal 404 and a circular vent wax seal 408. In some embodiments, the volume of the oval inlet wax seal 404 is at least about 2 times the volume of the vented wax seal 404. The vent wax seal 404 is positioned directly above the vent passage 412. The inlet wax seal 404 is positioned offset from, but in the same plane as, the microfluidic inlet channel 416 (as viewed in fig. 13). In the illustrated embodiment, the inlet wax seal 404 has a corresponding wax vent passage 420 that merges with a vent opening 424 positioned downstream of the reaction chamber 248. The wax vent channel 420 permits air to escape through vent 424 and escape the microfluidic inlet channel 416 during the melting and hardening process.

Referring to fig. 14, a microfluidic segment 500 according to another embodiment is shown. The microfluidic section 500 includes three identical wax seals 504A, 504B, 504C in an initial cylindrical shape. In the illustrated embodiment, each of the wax seals 504A, 504B, 504C in the initial state is about 4.0mm in diameter and about 0.51mm in thickness. The same wax seal reduces manufacturing complexity by using uniform dimensions. In the embodiment shown, the wax seals 504A and 504B are positioned offset from, but in the same plane as, the microfluidic inlet channel 508 (as viewed in fig. 14). In other words, the wax seals 504A, 504B in the initial state are coplanar with the microfluidic inlet channel 508. Each of the inlet wax seals 504A, 504B includes a corresponding wax vent passage 512A, 512B that merges at a common vent 516.

Referring to fig. 15A and 15B, each access port is depicted as having an overmolded seal (blue) in the access port of the respective chamber. In some embodiments, the overmolded seal comprises a thermoplastic elastomer that is molded onto the already formed cartridge during a subsequent molding process. In some embodiments, a strong bond is created between the overmolded seal and the cartridge material. In some embodiments, over molding eliminates the need to install an O-ring on the access port, which may be more difficult to hold in place during use. As with other access port seals (e.g., O-rings) that may be used with embodiments herein, the overmolded seals create an airtight fluid path between the cassette and the suction head of the transfer bladder tube. In some embodiments, all or a portion (e.g., 1, 2, 3, 4, 5, 6, 7, or more) of the access ports on the cassette include an overmolded seal.

In some embodiments, a film or lid (e.g., first lid 128 in fig. 3) is heat sealed to the cartridge. Referring to fig. 16, in some embodiments, one surface that is heat sealed to another surface includes an energy director (e.g., green line in fig. 16). The energy director is a raised surface where melting may be initiated to ensure a firm and complete seal is formed around critical areas (e.g., chambers). In some embodiments, the energy director is a linear convex surface formed from the bulk plastic of the molded body. The membrane is attached by hot pressing on the outside of the membrane. After the application of heat, the energy director melts and as the melted plastic cools and solidifies, it will adhere the film to the cartridge body.

The cartridge arrangements and components shown in fig. 1-30 are exemplary and illustrate preferred embodiments of the present invention. However, other cartridges that include different combinations and configurations of the elements shown in fig. 1-30 and/or described herein are also within the scope of the invention. Various embodiments and aspects of the present technology are described in patents and publications and are understood by those skilled in the art. Exemplary patents and publications include U.S. patent publication No. 2020/0190506; PCT publication No. WO2018226891; PCT publication No. WO2018218053; each of the publications is incorporated by reference in its entirety.

III. apparatus

As described throughout, the cartridge devices herein may be used with instruments that provide the components and functions that are lacking in the devices. In some embodiments, the cartridge and complementary instrument (e.g., heating and optical assembly 10) form a system capable of sample processing and analyte detection/quantification. In some embodiments, such systems are provided herein.

In some embodiments, the apparatus for embodiments herein comprises one or more heaters. In some embodiments, the heater is capable of regulating the temperature of the liquid in the chamber. In some embodiments, heaters reside on both sides of the chamber within the instrument to efficiently transfer heat to the chamber. In some embodiments, one or two heaters residing within the instrument adjacent to the chamber bring the liquid within the chamber to a desired temperature (e.g., 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, or ranges therebetween) within 30 seconds (e.g., 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s, 26s, 27s, 28s, 29s, 30s, or ranges therebetween), depending on the number of heaters, the proximity of the heaters to the chamber, and the volume of liquid.

Referring to fig. 17, a heating and optical assembly 10 is shown. In some embodiments, the heating and optical assembly 10 is part of a complementary instrument for processing and analyzing samples and/or cartridges. The assembly 10 includes a first support 14 and a second support 18 that is movable relative to the first support 14. The assembly 10 also includes a plurality of heat transfer devices 22A-22E and a fluorometer 26.

In the illustrated embodiment, a first heat transfer device 22A is coupled to the first support 14 and a second heat transfer device 22B is coupled to the second support 18 and positioned opposite the first heat transfer device 22A. The first heat transfer device 22A includes a first planar surface 30 and the second heat transfer device 22B includes a second planar surface 34 positioned opposite the first planar surface 30. In some embodiments, the first heat transfer device 22A and the second heat transfer device 22B are maintained at a first temperature. In some embodiments, the first temperature is in the range of about 90 ℃ to about 100 ℃. In some embodiments, the first temperature is about 95 ℃.

Likewise, a third heat transfer device 22C is coupled to the first support 14, and a fourth heat transfer device 22D is coupled to the second support 18 and positioned opposite the third heat transfer device 22C. The third heat transfer device 22C includes a third planar surface 38 and the fourth heat transfer device 22D includes a fourth planar surface 42 positioned opposite the third planar surface 38. In some embodiments, the third heat transfer device 22C and the fourth heat transfer device 22D are maintained at a second temperature. In some embodiments, the second temperature (of the third heat transfer device 22C and the fourth heat transfer device 22D) is different than the first temperature (of the first heat transfer device 22A and the second heat transfer device 22B). In some embodiments, the second temperature is in the range of about 60 ℃ to about 70 ℃. In some embodiments, the second temperature is about 65 ℃.

In the illustrated embodiment, the fluorometer 26 is coupled to the first support 14 and positioned between the first heat transfer device 22A and the third heat transfer device 22C. In the illustrated embodiment, the assembly 10 further includes a fifth heat transfer device 22E coupled to the second support 18 and positioned opposite the fluorometer 25. In particular, the fifth heat transfer device 22E includes a fifth planar surface 46 positioned opposite the fluorometer 26. The fifth heat transfer device 22E is positioned between the second heat transfer device 22B and the fourth heat transfer device 22D. In some embodiments, the second heat transfer device 22B, the fourth heat transfer device 22D, and the fifth heat transfer device 22E are each biased by a biasing member 48 (e.g., a compression spring) to move relative to the second support 18 toward the first support 14.

Referring to fig. 18, a cartridge 50 for detecting the level of an analyte is shown. Cartridge 50 includes a storage section 54, a processing section 58, and a microfluidic section 62. In the illustrated embodiment, the processing section 58 is positioned between the storage section 54 and the microfluidic section 62. Cartridge 50 contains all the components and chambers necessary for processing and detecting the target analyte. In some embodiments, cartridge 50 is acted upon by a processing device that includes a complementary mechanism, for example, to perform operations including, but not limited to, heat transfer, liquid transfer, magnetic transfer, and optical detection. In general, the storage section 54 contains the sample to be analyzed, the buffers used in the processing and the transfer vesicles; the processing section 58 is where the target is extracted, purified and bound to magnetic beads; and the microfluidic section 62 is where one or more targets are detected.

Referring to fig. 26, the microfluidic section 62 of the cartridge 50 includes a reaction chamber 66, a microfluidic vent channel 70, and a microfluidic inlet channel 74. The microfluidic vent channel 70 is fluidly connected to the reaction chamber 66. The microfluidic inlet channel 74 serves as an interface between the processing section 58 and the microfluidic section 62 and facilitates easy transfer of liquid to the microfluidic section 62 with openings that the pipettes cannot directly access. The reaction chamber 66 is positioned between the microfluidic inlet channel 74 and the microfluidic vent channel 70. In some embodiments, the lyophilized master mix 78 is positioned within the reaction chamber 66 and, for example, PCR is performed therein. In the illustrated embodiment, the reaction chamber 66 is a PCR chamber.

Referring to fig. 2, as explained in greater detail herein, the heating and optical assembly 10 is configured to receive a reaction chamber (e.g., reaction chamber 66) between the first planar surface 30 of the first heat transfer device 22A and the second planar surface 34 of the second heat transfer device 22B to bring the reaction chamber to a first temperature. Likewise, the assembly 10 is configured to receive the reaction chamber between the third planar surface 38 and the fourth planar surface 42 of the heat transfer devices 22C, 22D to bring the reaction chamber to the second temperature.

With continued reference to fig. 17 and 18, the assembly 10 further includes a first actuator 82 and a second actuator 86 configured to move portions of the assembly 10 relative to the cassette 50. The first actuator 82 is coupled to the second support 18 and is configured to move the second support 18 along the clamp axis 90 between a first position and a second position. Although the clamp axis 90 is shown as a linear axis, in some embodiments the clamp axis is a curve or circular arc. In the first position, the first planar surface 30 and the second planar surface 34 are spaced apart a first distance; and in the second position, the first planar surface 30 and the second planar surface 34 are spaced apart a second distance that is less than the first distance. In some embodiments, the second distance is in the range of about 400 microns to about 600 microns. In other words, the first actuator 82 moves the second support 18 along the clamping axis 90 to clamp the cassette 50 between the supports 14, 18. In the illustrated embodiment, the cassette 50 is clamped between the first heat transfer device 22A and the second heat transfer device 22B; between the third heat transfer device 22C and the fourth heat transfer device 22D; and between the fluorometer 26 and the fifth heat transfer device 22E.

In the illustrated embodiment, the second actuator 86 is coupled to the first support 14 and the second support 18, and the second actuator 86 is configured to move the first support 14 and the second support 18 together along the translation axis 94. Although translation axis 94 is shown as a linear axis, in some embodiments, the translation axis is a curve or circular arc. In some embodiments, the translation axis 94 is perpendicular to the clamp axis 90. In some embodiments, the translation axis 94 is vertical and the clamp axis 90 is horizontal. In other words, the second actuator 86 moves the supports 14, 18 along the translation axis 94 relative to the cassette 50. Movement along translation axis 94 aligns reaction chamber 66 of cassette 50 with different heat transfer devices 22A-22E and/or fluorometer 26. In some embodiments, the cassette 50 remains stationary as the supports 14, 18 move relative to the cassette 50 along the translation axis 94 and/or the clamp axis 90. In other embodiments, the support members 14, 18 remain stationary as the cassette 50 moves relative to the support members 14, 18.

Referring to FIG. 21, a heat transfer device 98 for heating a reaction chamber (e.g., PCR reaction chamber 66) is shown. In some implementations, the heat transfer device 98 may be any of the heat transfer devices 22A-22E in the heating and optical assembly 10. The heat transfer device 98 includes a heat reservoir 102 having a base 106, a first aperture 110, a second aperture 114, and a heat exchanger 118 extending from the base 106. The heat exchanger 118 includes a planar surface 122 (e.g., planar surfaces 30, 34, 38, 42, 46) configured to surround the reaction chamber 66. In the illustrated embodiment, the heat exchanger 118 is cylindrical and the base 106 is rectangular. In some embodiments, the heat reservoir is a highly thermally conductive material (e.g., aluminum). In the illustrated embodiment, the first and second apertures 110, 114 are formed in the base 106 and extend perpendicular to a longitudinal axis 124 of the heat exchanger 118. In the illustrated embodiment, the first aperture 110 and the second aperture 114 are formed in the same surface of the base 106 and extend parallel to one another. The heat transfer device 98 also includes a heater 126 positioned within the first aperture 110 and a temperature sensor 130 positioned within the second aperture 114. In some embodiments, the heater 126 is a resistive heater.

Referring to fig. 22 and 23, a heat transfer device 134 for heating and/or cooling a reaction chamber (e.g., reaction chamber 66) is shown. In some implementations, the heat transfer device 134 may be any of the heat transfer devices 22A-22E in the heating and optical assembly 10. The heat transfer device 134 includes a heat reservoir 138 having a base 142, a first aperture 146, a second aperture 150, and a heat exchanger 154 extending from the base 142. The heat exchanger 154 includes a planar surface 158 (e.g., planar surfaces 30, 34, 38, 42, 46) configured to abut the reaction chamber 66. In the illustrated embodiment, the heat exchanger 154 is cylindrical, the base 142 is cylindrical, and the flange 160 separates the two portions 142, 154. In some embodiments, a biasing member (e.g., spring 48) abuts flange 160 to bias heat transfer device 134 in a direction (e.g., toward another heat transfer device mounted oppositely). In the illustrated embodiment, the first and second apertures 146, 150 are formed in the base 142 and extend parallel to the longitudinal axis 162 of the heat exchanger 154. In the illustrated embodiment, the first and second apertures 146, 150 are formed in the same surface of the base 142 and extend parallel to one another. The heat transfer device 134 also includes a heater 166 positioned within the first aperture 146 and a temperature sensor 170 positioned within the second aperture 150. In some embodiments, the heater 166 is a resistive heater.

In some embodiments, the heat transfer devices 22A-22E and the assembly 10 include a processor 174 and a non-transitory memory 178. In some embodiments, the memory 178 includes instructions that when executed by the processor 174 perform closed loop temperature control of the heat reservoirs of the heat transfer devices 22A-22E. Closed loop temperature control of the heat reservoir is achieved by using temperature sensors (e.g., sensors 130, 170) as feedback and heaters (e.g., 126, 166) as controlled steering inputs. In some embodiments, the closed loop control is a proportional integral derivative ("PID") or proportional integral ("PI") controller.

In some embodiments, an apparatus for use in embodiments herein includes one or more magnetic transfer elements. In some embodiments, the magnetic transfer element comprises magnets positionable at different locations along the cartridge and at different distances from the chamber/channel of the device so as to vary the magnetic force at each location within the device. In some embodiments, the magnetic transfer element comprises a robotic arm having a magnet at its distal end. The distance between the magnet and the sides of one or more of the device chambers (e.g., C5, C6, reaction chamber) may be varied (e.g., 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, 18mm, 19mm, 20mm, or more, or ranges therebetween) to apply various magnetic forces to the contents of the chamber (e.g., PMP). In some embodiments, the robotic arm allows the magnet to move vertically from top to bottom (and from bottom to top) of the chamber, and to move laterally (e.g., associated with a different chamber, dragging the pellet through a transfer channel, etc.).

In some embodiments, the instrument comprises a fluorometer, camera, or other optical reader capable of detecting light emitted from, for example, a sample within a detection/reaction chamber of the device. In some embodiments, a fluorometer is provided that is capable of exciting a fluorophore within a detection/reaction chamber of the device and detecting the wavelength of the emitted light.

Numerous techniques may be used to detect the presence and/or concentration of an analyte in a sample within a detection/reaction chamber. For example, fluorescent labeling of the analyte may be used. Fluorescent labels (or fluorescent probes) are typically substances (i.e., which fluoresce) that absorb radiation and emit a signal (typically radiation distinguishable from stimulating radiation, e.g., by wavelength) when stimulated by an appropriate electromagnetic signal or radiation, which persists as the stimulating radiation persists. Fluorometry involves exposing a sample containing a fluorescent label or probe to stimulating (also known as excitation) radiation, such as a light source of appropriate wavelength, to excite the probe and initiate fluorescence. The emitted radiation is detected using a suitable detector, such as a photodiode, photomultiplier, charge Coupled Device (CCD), or the like. In some embodiments, the complementary instrument for the cartridge device includes an appropriate detector (e.g., photodiode, photomultiplier, charge Coupled Device (CCD), fluorometer, luminometer, etc.).

Fluorometers for use with fluorescently labeled samples are known in the art. One type of fluorometer is an optical reader, such as described by Andrews et al in U.S. patent No. 6,043,880; said patent is incorporated by reference in its entirety. The optical reader may be integrated within the reaction chamber (e.g., a thermal cycler) such that the sample may be analyzed without removing the sample from the reaction chamber (e.g., without interrupting PCR). Examples of such integrated devices are described in U.S. patent No. 5,928,907, U.S. patent No. 6,015,674, U.S. patent No. 6,043,880, U.S. patent No. 6,144,448, U.S. patent No. 6,337,435, and U.S. patent No. 6,369,863; said patent is incorporated by reference in its entirety. Such a combination device may be used in a variety of applications, as described, for example, in U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,994,056, U.S. Pat. No. 6,140,054, and U.S. Pat. No. 6,174,670; said patent is incorporated by reference in its entirety.

In some embodiments, the provided systems, devices, and components thereof include a fluorometer. In some embodiments, the complementary instrument for use with the cartridge device (e.g., as described herein) comprises a fluorometer. In some embodiments, for example with reference to fig. 28-30, fluorometer 26 is integrated with heat transfer devices 22A-22E in a single assembly (e.g., heating and optical assembly 10). Quantitative PCR performs multiple fluorescence measurements after multiple thermal cycles, as discussed herein. Thus, the time to perform the PCR is reduced by the heating and optical assembly 10, which is compact and effectively moves the heater and fluorometer 26 relative to the cassette 50.

With continued reference to fig. 28-30, the fluorometer 26 is coupled to the first support 14. In the illustrated embodiment, the fluorometer 26 includes a housing 210 defining a measurement aperture 214. As detailed herein, the measurement aperture 214 is aligned with the PCR chamber 66 to perform fluorescence measurements on the sample. In some embodiments, the measurement aperture 214 is configured to align with a planar surface (e.g., front surface) of the PCR chamber. The measurement aperture 214 defines a normal axis 218 perpendicular to the measurement aperture 214. Thus, normal axis 218 is perpendicular to PCR chamber 66 during fluorescence measurement.

Conventional fluorescence measurements are made at right angles (e.g., at right angles to the edges) to minimize excitation light collected by the emission optics. However, in some embodiments, the thickness of the PCR chamber is small (e.g., less than 500 microns) and right angle fluorescence readings from the edge are not feasible. In the embodiment shown, excitation light enters and fluorescence is detected through the front surface of the PCR chamber 66. This allows for a large part of the fluorescence in the collection chamber. Typically, this type of measurement requires the use of dichroic mirrors and/or beam splitters that reflect the excitation light and pass the emitted light, and results in complex optical systems.

Fluorometer 26 includes a plurality of light sources 222A-222D and a plurality of light detectors 226A-226D. In the illustrated embodiment, the fluorometer 26 includes four light sources 222A, 222B, 222C, and 222D. The first light source 222A is coupled to the housing 210 along a first light source axis 230A, the second light source 222B is coupled to the housing 210 along a second light source axis 230B, the third light source 222C is coupled to the housing 210 along a third light source axis 230C, and the fourth light source 222D is coupled to the housing 210 along a fourth light source axis 230D. Each of the light source axes 230A-230D intersects the measurement aperture 214. In other embodiments, the fluorometer includes 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) light sources and 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) light source axes that are not coaxial and intersect the measurement aperture.

In the illustrated embodiment, fluorometer 26 includes four light detectors 226A-226D, one of which corresponds to each light source. The first light detector 226A is coupled to the housing 210 along a first detector axis 234A, the second light detector 226B is coupled to the housing 210 along a second detector axis 234B, the third light detector 226C is coupled to the housing 210 along a third detector axis 234C, and the fourth light detector 226D is coupled to the housing 210 along a fourth detector axis 234D. Each of the detector axes 234A-234D intersects the measurement aperture 214.

In the illustrated embodiment, both the source axes 230A-230D and the detector axes 234A-234D intersect the measurement aperture 214. Light source axes 230A-230D, detector axes 234A-234D, and normal axis 218 are all not coaxial. In other words, the light source axes 230A-230D and the detector axes 234A-234D intersect each other at the measurement aperture 214, but do not otherwise overlap each other. In the illustrated embodiment, the light source axes 230A-230D and the detector axes 234A-234D are positioned circumferentially about the normal axis 218 (FIG. 28). In some embodiments, the light sources 222A-222D and the detectors 226A-226D alternate in a circumferential direction. In some embodiments, the detector axis is adjacent to the LED axis. For example, in the illustrated embodiment, the first light source 222A is positioned circumferentially between the first detector 226A and the fourth detector 226D. Likewise, the first detector 222A is positioned circumferentially between the first light source 222A and the second light source 222B.

The first light source 222A emits first excitation light along a first light source axis 230A, and the first excitation light is reflected away from a first detector axis 234A at the measurement aperture 214. In other words, the excitation beam enters the PCR chamber 66 along one optical axis (e.g., the first light source axis 230A) and the emitted light is collected along a different optical axis (e.g., the first detector axis 234A). The angle between the axes 230A, 234A and the surface of the PCR chamber 66 is selected such that excitation light is reflected away from the emission optical axis. This allows multiple pairs of excitation and emission optics to measure fluorescence in the same chamber without the use of optical fibers, dichroic mirrors, or moving filter modules. In the illustrated embodiment, the fluorometer 26 advantageously does not include a dichroic mirror or beam splitter. The fluorometer 26 is small, lightweight, and compact such that the fluorometer 26 is integrated with the heat transfer device 22A-22E for target amplification.

The four light sources 222A-222D and the four light detectors 226A-226D together create four fluorescence detection channels. In some embodiments, fluorometer 26 includes at least four fluorescence detection channels. For example, the first excitation light from the first light source 222A has a first spectrum (e.g., a first spectral power distribution), and the first light detector 226A measures a first fluorescence of the sample (e.g., a first channel) in response to the first excitation light. Likewise, a second excitation light having a second spectrum (e.g., a second spectral power distribution) is emitted from the second light source 222B, and the second light detector 226B measures fluorescence of the sample in response to the second excitation light. Advantageously, by reducing the diameter of the lens while maintaining approximately the same numerical aperture, more light source/detector pairs (e.g., channels) can be arranged about the central axis (e.g., normal axis 218) without significantly reducing signal strength.

Referring to fig. 19, a first light source 222A (which represents the structure of each light source) includes a light emitter 238, a lens 242, and a wavelength selective filter 246. In some embodiments, the light emitters 238 are Light Emitting Diodes (LEDs). For some fluorophores and LEDs, the wavelength selective filter may be omitted. The first excitation light illuminates the reaction chamber 66 and is reflected away from the first detector 226A. In other words, stray light from first light source 222A is reflected away from first detector 226A because the angle of incidence of first light source axis 230A is greater than the angle of incidence of first detector axis 234A. Fluorescence generated in response to the first excitation light is detected by the first photodetector 226A. In some embodiments, the intensity of the first excitation light is multiple orders of magnitude greater than the emitted fluorescence light.

Longer wavelength fluorescence may be emitted from the reaction chamber 66. The first light detector 226A (which represents the structure of each light detector) includes a first lens 150, a filter 254 (e.g., a wavelength selective filter), a second lens 258, and a solid state detector 262. For some fluorophores, the second lens may be omitted because the light is already sufficiently collimated by the first lens. Light incident on the solid state detector 262 is converted to electrical signal measurements that are detected and stored by the processor 174. In some implementations, the non-transitory memory 178 includes instructions that when executed by the processor 174 store four hundred (400) analog-to-digital readings obtained by the first detector 226A over a 100 millisecond period. To minimize ambient noise, radiated noise, and conducted AC noise due to visible light, four hundred modulus readings were taken over a fixed 100 millisecond period. The length of this period is exactly six cycles at 60Hz and five cycles at 50Hz, so that the signal variations due to AC power in different countries are averaged out.

Methods, kits and systems

In some embodiments, provided herein are methods of sample processing and analyte detection/quantification using the devices and systems described herein. In some embodiments, provided herein are systems, kits, and methods for preparing a target nucleic acid in a biological sample for subsequent analysis.

The devices herein can be used to detect various analytes (e.g., nucleic acids, small molecules, peptides, proteins, etc.) via various detection reagents (e.g., primers, probes, antibodies, etc.) and by various detection techniques (e.g., PCR, fluorescence, immunoassays), to treat various sample types (e.g., biological samples (e.g., tissue, blood products, saliva, etc.), environmental samples (e.g., soil or water samples), research samples (e.g., cell cultures, in vitro samples), etc.).

In some embodiments, reagents used in the methods/kits/systems herein are provided in dry (e.g., lyophilization trays, pellets, etc.) or concentrated (e.g., liquid, gel, etc.) form. In some embodiments, reagents used in the methods/kits/systems herein include components for cell lysis (e.g., detergents (e.g., SDS), etc.), components for protein digestion (e.g., proteinase K, etc.), nucleic acid capture probes (e.g., hybridization sequences linked to capture moieties (e.g., biotin, etc.), capture agent-coated magnetic beads (e.g., streptavidin-coated beads), amplification reagents (e.g., primers, nucleotides, magnesium, etc.), detection reagents (e.g., fluorescent labels), and the like.

In some embodiments, the method utilizes three dried or concentrated reagent compositions (e.g., lyophilization trays, pellets, etc.; concentrated liquids, gels, etc.) for target nucleic acid capture/sorting/purification, and optionally one additional dried or concentrated reagent composition for target nucleic acid amplification/detection. In some embodiments, the three dried or concentrated reagent compositions for target nucleic acid capture/sorting/purification are a lysis reagent (and/or protein digestion reagent), a capture reagent, and a capture reagent coated magnetic bead. In some embodiments, the method comprises one or more (e.g., all) of the following steps: (a) Combining a biological sample with a lysis reagent capable of digesting a cell membrane and degrading a protein, and allowing the lysis reagent to digest the cell membrane and degrade the protein to produce a lysate, wherein the biological sample comprises a nucleic acid; (b) Combining the lysate with a capture reagent, wherein the capture reagent comprises a nucleic acid probe tethered to a capture moiety; (c) Allowing the nucleic acid probes to hybridize with nucleic acids of the biological sample to produce a probe-bound nucleic acid solution; (d) Combining the probe-bound nucleic acid with a capture agent-coated magnetic bead; (e) Allowing the capture agent to bind to the capture moiety to produce a bead-captured nucleic acid suspension; (f) Sorting bead-captured nucleic acids within the bead-captured nucleic acid suspension by exposing a portion of the bead-captured nucleic acid suspension to a magnetic field; and (g) separating the sorted bead-captured nucleic acids from the liquid portion of the bead-captured nucleic acid suspension.

In some embodiments, the method utilizes two dried or concentrated reagent compositions (e.g., lyophilization trays, pellets, etc.; concentrated liquids, gels, etc.) for target nucleic acid capture/sorting/purification, and optionally one additional dried or concentrated reagent composition for target nucleic acid amplification/detection. In some embodiments, the two dried or concentrated reagent compositions for target nucleic acid capture/sorting/purification are a lysis/capture reagent and a capture reagent coated magnetic bead. In some embodiments, the method comprises one or more (e.g., all) of the following steps: (a) Combining a biological sample with a lysis reagent and a capture reagent, wherein the lysis reagent comprises a component capable of digesting cell membranes and degrading cellular proteins, wherein the capture reagent comprises a nucleic acid probe tethered to a capture moiety, and wherein the biological sample comprises nucleic acids; (b) Allowing the lysis reagent to digest the cell membrane and degrade the protein to produce a lysate; (c) Allowing the nucleic acid probes to hybridize with nucleic acids of the biological sample to produce a probe-bound nucleic acid solution; (d) Combining the probe-bound nucleic acid with a capture agent-coated magnetic bead; (e) Allowing the capture agent to bind to the capture moiety to produce a bead-captured nucleic acid suspension; (f) Sorting bead-captured nucleic acids within the bead-captured nucleic acid suspension by exposing a portion of the bead-captured nucleic acid suspension to a magnetic field; and (g) separating the sorted bead-captured nucleic acids from the liquid portion of the bead-captured nucleic acid suspension.

Certain embodiments herein do not include a centrifugation step, do not include a filtration step, and/or do not include nucleic acid precipitation. In some embodiments, nucleic acids (including target nucleic acids) for a biological sample are not separated from contaminants or excess reagents (e.g., unbound probes) of the biological sample during the steps of lysing, digesting, probe hybridization, and/or capturing (e.g., bead-bound capture reagent binds to probe-bound capture moiety).

Embodiments herein require a sample comprising (or suspected of containing) nucleic acids. Suitable samples contain nucleic acids and are therefore referred to herein as biological samples. The biological sample may be of any origin or source, and may be obtained from nature or a sample produced in a laboratory. Biological samples are available from animals (including humans) and encompass fluids, solids, tissues, and gases. Some biological samples include blood products such as plasma, serum, stool, urine, and the like. The sample may be an environmental source and may include environmental materials such as surface materials, soil, mud, sludge, biological films, water, and industrial samples. In some preferred embodiments, the sample comprises nucleic acid from a pathogen (e.g., virus, bacteria, fungus, protozoa, helminth, etc.). In other embodiments, the sample comprises nucleic acid from a subject (e.g., human, non-human primate, livestock, wild animal, etc.). Any sample containing any type of nucleic acid may be used in embodiments herein. The exemplary samples provided herein should not be construed as limiting the types of samples suitable for use in the present invention.

In some embodiments, the sample is added to the chambers of the devices herein, e.g., using the open top of the chamber, and the chamber is sealed (e.g., with a cap). In the case of a liquid sample, the sample may be injected into a chamber (e.g., an empty chamber, a chamber including an appropriate buffer, etc.). In the case of a solid sample or a sample on a solid medium (e.g., a wipe, swab tip, etc.), the solid may be inserted into the chamber and the sample dissolved into the liquid in the chamber.

In some embodiments, the sample is digested in a chamber of a device herein. In some embodiments, depending on the type of sample and target analyte, an appropriate treatment reagent is used. For example, the reagents may include protein precipitation reagents (e.g., acetonitrile, methanol, or perchloric acid), cell lysis reagents (e.g., zinc sulfate, strong acids, enzymes digesters with lysozyme, cellulases, proteases, detergents (including but not limited to nonionic, zwitterionic, anionic, and cationic detergents), protein digestion reagents (e.g., serine proteases (such as trypsin, threonine, cysteine, lysine, arginine, or aspartic proteases), metalloproteases, chymotrypsin, glutamate proteases, lys-c, glu-c, and chemical trypsin (chemotopsin)), internal standards (e.g., stable isotopically labeled analytes, heavy isotopically labeled peptides, non-natural peptides or analytes, structurally similar analogs, chemically similar analogs), antibiotics (for microbial antibiotic sensitivity testing, or "AST"), protein stabilizers (including buffers, chaotropic or denaturing agents), calibration standards, and control reagents according to various embodiments, one or more of the pre-mix reagents to form a specific combination for a specific assay or set of assays, in embodiments, including in a sample (e.g., a sample) may be subjected to conditions such as to degradation in a sample, and the like.

In some embodiments, a biological sample is combined with reagents described herein (e.g., cleavage and/or digestion reagents) to initiate steps of the methods herein. In other embodiments, a liquid buffer solution is added to the biological sample (or the sample is added to a liquid buffer solution) in order to dilute the sample, to bring the sample to a volume sufficient for processing and/or to extract the sample from a substrate (e.g., swab, collection bottle, etc.). In some embodiments, the matrix comprising the biological sample or the biological sample itself is added to a liquid buffer (e.g., in a tube, well, chamber, etc.) to begin the methods herein.

In some embodiments, a biological sample alone (comprising nucleic acids) or in an appropriate buffer is treated with a lysis and/or digestion reagent. In some embodiments, the cleavage and/or digestion reagent is a complex reagent (i.e., comprising two or more separate component reagents). In some embodiments, the lysis and/or digestion reagent comprises a component reagent for degrading a cell membrane (e.g., of a bacterial or eukaryotic cell). In some embodiments, the lysis and/or digestion reagent comprises a component reagent for digesting proteins (e.g., cellular proteins, viral proteins, etc.) within the sample. In some embodiments, the lysis and/or digestion reagent comprises a component reagent suitable for releasing nucleic acid from cells and/or viruses. The lysis reagent and/or component reagents in the reagent may comprise one or more enzymes (e.g., proteases), proteases (e.g., pronases), trypsin, proteinase K, phage lytic enzymes (e.g., plyGBS), lysozyme (e.g., modified lysozyme such as ReadyLyse), cell-specific enzymes (e.g., mutanolysin for lysing group B streptococcus)) configured to reduce proteins (e.g., denature proteins). Other enzymes present in the cleavage and/or digestion reagent may include lysostaphin, a digestive enzyme, a cellulase, a mutanolysin, a xylanase, and the like. In some embodiments, the component reagents in the lysis and/or digestion reagent comprise one or more chemical cell lysis reagents, such as Triton-X, guanidinium, or SDS. In some embodiments, the cleavage and/or digestion reagent may comprise various salts (e.g., naCl, mgCl ₂ Etc.), buffers (e.g., tris, MOPS, MES, etc.), or other components. In certain embodiments, the cleavage reagent comprises proteinase K. In other embodiments, cleavageThe reagent comprises proteinase K and SDS. In some embodiments, the cleavage reagent comprises proteinase K (e.g., 1U, 2U, 5U, 10U, 15U, 20U, 25U, 30U, 40U, 50U, or more, or ranges therebetween), caCl ₂ (e.g., 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 30mM or more, or ranges therebetween) and/or HEPES (e.g., 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 15mM, 20mM, 30mM or more, or ranges therebetween). In some embodiments, the lysis reagent comprises 15U proteinase K, 5mM CaCl ₂ And 5mM HEPES.

In some embodiments, the lysing and/or digesting agent is a dry agent (e.g., a lyophilization tray or pellet), and the dry lysing agent is resuspended upon combination with the biological sample and/or buffer solution. In other embodiments, the lysis and/or digestion reagent is a concentrated liquid (or gel) and is diluted in the biological sample and/or buffer solution. Concentrated lysis reagents may be present at a concentration of 10X, 20X, 50X, 100C, 200X, 500X or more compared to the 1X working concentration after dilution into biological sample and/or buffer solution.

In some embodiments, sample processing to produce a lysate may include physical processes in addition to the chemical reagents and enzymes described above or understood in the art. For example, in some embodiments, the sample is heated to assist in lysis (e.g., >60 ℃, >65 ℃, >70 ℃, >75 ℃, >80 ℃, >85 ℃, >90 ℃, >95 ℃, etc.). In some embodiments, the sample is heated (e.g., 90 ℃ -100 ℃) after lysis to inactivate one or more of the enzymes used for lysis. In some embodiments, freeze/thaw is employed to assist in lysis. In some embodiments, the lysis is performed using mechanical means such as French press, grinding, sonication, and the like. In some embodiments, the methods herein do not employ mechanical means to lyse cells or viruses.

In some embodiments, the lysate is combined with a capture reagent upon release of the nucleic acid from the cell, virus, or the like. In some embodiments, the capture reagent comprises a nucleic acid probe comprising a hybridization sequence and a capture moiety.

In some embodiments, a capture agent (e.g., a sequence-specific capture probe) is used to bind (capture) the target analyte after the sample is appropriately processed to release the target analyte (e.g., cell lysis, contaminant component digestion, etc.). In some embodiments, the capture agent comprises a target binding moiety and a handle (handle) or affinity moiety. The target binding moiety can be any molecular entity (e.g., a nucleic acid probe, an antibody or antibody fragment, a target-specific ligand, etc.) capable of stably binding to a target analyte. The binding moiety can be, for example, a nucleic acid probe sequence that is effective to hybridize to a target nucleic acid sequence, or an antibody or functional fragment thereof that is effective to bind a target protein or other analyte. Any binding moiety having any desired specificity may be used. The handle or affinity moiety is an element having an affinity pair that can be used to capture an analyte when the analyte binds to a capture agent. Immunoreactive specific binding members include antigens or antigen fragments and antibodies or functional antibody fragments. Other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. In some embodiments, the binding member is attached to a solid support, such as a plurality of paramagnetic particles, to extract the analyte from a sample containing non-target components. In a preferred embodiment, a sequence-specific capture probe comprising a biotin moiety or another affinity handle hybridizes to a target analyte nucleic acid. Subsequently, the capture probe-bound nucleic acid is captured onto paramagnetic particles (PMPs) comprising streptavidin or another affinity agent capable of binding to a handle. In some embodiments, washing of the pellet PMP provides for removal of contaminants from the captured target analyte. In the case of non-nucleic acid targets, other suitable capture means should be understood.

In some embodiments, the hybridization sequence is a polynucleotide sequence that is complementary to all or a portion of a target sequence in a target nucleic acid. In some embodiments, the hybridization sequence is sufficiently complementary to the target sequence to allow hybridization of the probe to the target nucleic acid under the conditions of the methods herein. In some embodiments, the hybridization sequence is at least 70% complementary (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%) to the target sequence.

In some embodiments, the capture moiety is a chemical group that is capable of being stably bound by the capture agent under the conditions of the methods herein. In some embodiments, the capture moiety is biotin (and the capture agent is streptavidin). In other embodiments, the capture moiety is a haloalkane (and the capture agent isPromega), alkynes (and the capture agent is azide), and the like.

In some embodiments, the capture reagent is a dry reagent (e.g., a lyophilization tray or pellet), and the dry capture reagent is resuspended when combined with the lysate and/or buffer solution. In other embodiments, the capture reagent is a concentrated liquid (or gel) and is diluted in a lysate and/or buffer solution. Concentrated capture reagents may be present at a concentration of 10X, 20X, 50X, 100C, 200X, 500X or greater than the 1X working concentration after dilution into lysate and/or buffer solution.

In some embodiments, the lysate is added to a concentrated or dried capture reagent. In some embodiments, the lysate and capture reagents are mixed (e.g., stirred, aspirated, etc.). In some embodiments, sufficient time and conditions are provided to allow hybridization of the probe to the target nucleic acid (e.g., 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, or longer, or a range therebetween). In some embodiments, the capture reagent and lysate are incubated at a temperature sufficient to promote specific hybridization of the hybridization sequence to the target sequence (e.g., 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, or ranges therebetween). In some embodiments, the sequence of the hybridization sequence, the degree of complementarity to the target sequence, and the conditions and temperatures used prevent non-specific hybridization of the probe.

The nucleic acid probes may also comprise other nucleic acid elements useful in the methods herein. For example, the probe can comprise a primer binding site (for subsequent target nucleic acid amplification), a linker region (for attachment of a capture moiety), and the like.

In some embodiments, a combined lysis/capture reagent is employed in the methods herein. Such reagents comprise a cleavage component and a capture component. In some embodiments, the cleavage component is identical to the cleavage reagents described above (e.g., contains SDS and proteinase K). In some embodiments, the capture component is identical to the capture reagent described above (e.g., contains a nucleic acid probe tethered to the capture moiety). In such embodiments, the biological sample (alone or in a buffer) is combined with a lysis/capture reagent and exposed to conditions suitable for lysis/digestion followed by conditions suitable for probe hybridization. In some embodiments, changing from cleavage conditions to hybridization conditions includes inactivating the digestion/cleavage enzyme (e.g., exposing to elevated temperatures) and exposing to hybridization temperatures. In some embodiments, no cleavage species is removed prior to hybridization, whether using cleavage and capture reagents alone or in combination.

In some embodiments, after hybridization of the capture probes to the target nucleic acids, the probe-bound nucleic acids are combined with capture agent-coated magnetic beads. In some embodiments, the lysate and probe-containing mixture is not removed from contaminating materials or other components prior to the addition of the beads. In some embodiments, the probe-bound nucleic acid and the capture agent coated magnetic beads are mixed in suspension. In some embodiments, high temperatures (e.g., 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, or ranges therebetween) are used to facilitate the re-suspension and mixing of the beads.

In some embodiments, the capture agent coated magnetic beads are dried reagents (e.g., lyophilized trays or pellets), and the dried capture agent coated magnetic beads are resuspended upon combination with the probe-bound nucleic acid mixture and/or buffer solution. In other embodiments, the capture agent coated magnetic beads are concentrated liquids (or gels) and diluted in a probe-bound nucleic acid mixture and/or buffer solution. Concentrated capture agent coated magnetic beads may be present at a concentration of 10X, 20X, 50X, 100C, 200X, 500X or greater than the 1X working concentration after dilution into the probe-bound nucleic acid mixture and/or buffer solution.

When the capture agent coated magnetic beads and probe binding nucleic acid combination, at the capture reagent binding to capture part at a temperature (for example, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, or the range therebetween) under incubation suspension.

In some embodiments, once the cell or virus is lysed, the hybridized sequence of the probe binds to the target nucleic acid and the capture agent binds to the capture moiety, thereby forming a target nucleic acid/capture probe/magnetic bead complex; contaminants, unused reagents and/or optional components are removed for the first time in the process herein. In some embodiments, a magnetic field is applied to the magnetic beads and the beads and all components bound thereto (capture probes and target nucleic acids) are separated from unbound components, reagents and contaminants of the liquid portion of the suspension. There are a variety of techniques available for separating the beads from the liquid and unbound components of the suspension. In some embodiments, the magnetic field is head stable and liquid is drawn from a vessel containing the suspension. The liquid may be removed by any suitable means, including pipetting, inverting the container, passing through a microfluidic, and the like. In other embodiments, the magnetic field is moved to drag the beads through or cause the beads to flow across the liquid/air interface. "dragging" the magnetic beads through the liquid/air interface includes positioning a magnetic field to create a pellet of magnetic beads. The magnetic field is then moved through the liquid such that the pellets move with the magnetic field. The magnetic field moves across the liquid/air interface, thereby removing the beads from the liquid. During the "dragging" process, the magnets are positioned continuously over the magnetically induced pellets as the pellets move across the liquid/air interface. The "flowing" of the magnetic beads through the liquid/air interface similarly includes positioning a magnetic field to create a pellet of magnetic beads. The pellets are moved into close proximity to the liquid/air interface. The magnetic field is then temporarily reduced or eliminated and then reestablished on the opposite side of the liquid/air interface. The magnetic field pulls the beads across the interface. Flowing the PMP through the liquid/air interface rather than dragging (e.g., where the magnets are positioned continuously over the PMP's magnetically induced pellets) reduces elongation of the liquid/air interface and reduces the volume of undesirable liquid carried by the PMP into the air gap. In some embodiments, streaming is achieved by: (i) generating a magnetic field to pull the beads into a pellet (e.g., on the surface of a vessel containing the suspension), (ii) moving the magnetic field to bring the pellet near or adjacent to the air/liquid interface, (iii) reducing or eliminating the magnetic field experienced by the beads (e.g., by lifting the magnet off the vessel), (iv) reestablishing the magnetic field on the opposite side (air side) of the liquid/air interface, and (v) allowing the beads agglomerated in the liquid to flow out of the liquid into the air. In some embodiments, by flowing the beads through the interface, rather than pulling the entire pellet across the interface, the beads carry less contaminated liquid. However, any method of separating beads from liquids and unbound contaminants may be used in embodiments herein.

In some embodiments, after initially sorting the bead captured target nucleic acids from a majority of unbound lysate components, capture reagents, buffers, etc., the sorted beads and bead captured target nucleic acids are subjected to one or more washing steps. Typical washing steps include combining a washing buffer with the sorted beads and target nucleic acids captured by the beads, mixing the beads and buffer to allow washing away residual contaminants and unbound reagents from the beads, target nucleic acids, probes, etc., and then repeating the process of sorting the beads from the liquid (e.g., with the method steps described above). In some embodiments, one to five washing steps (e.g., 1, 2, 3, 4, 5) are performed. In some embodiments, the washing beads comprise the steps of: combining the nucleic acid captured by the sorted beads with a wash buffer; resuspending the bead captured nucleic acid in a wash buffer; sorting the bead-captured nucleic acids in the wash buffer by exposing a portion of the bead-captured nucleic acids to a magnetic field; and separating the nucleic acid captured by the sorted beads from the wash buffer.

In addition to preparing a biological sample for analysis by the methods herein, the buffer solution may also be In the methods herein are used to wash bead-bound nucleic acids, resuspend reagents, transfer components of the steps herein, perform amplification reactions, and the like. The buffer solution used in certain embodiments herein may comprise one or more of the following: naCl, mgCl ₂ EDTA, sucrose, tergitol, BME, bis Tris buffer, sorbitol, dextran, polyvinylsulfonic acid, lithium dodecyl sulfate, bovine serum albumin, triton X-100, citric acid, DTT, CHAPS, naOH, liCl, MES buffer, phosphate buffer, etc. The buffer solution may contain one or more salts, surfactants, detergents, defoamers, and the like. Suitable combinations of buffer solutions for processing, lysing and/or digesting biological samples, and/or for nucleic acid hybridization, capture (e.g., biotin/streptavidin binding), washing, re-suspending, nucleic acid amplification, and/or fluorescence detection, as well as other components, will be understood in the art and may be used in embodiments herein. In particular embodiments, the buffer solution comprises lithium dodecyl sulfate (e.g., 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% or more, or ranges therebetween), EDTA (e.g., 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 50mM, 60mM or more, or ranges therebetween), liCl ₂ (e.g., 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 500mM, or more, or ranges therebetween), an antifoaming agent (e.g., HYDROTECH, bio-rad) (e.g., 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% or more, or ranges therebetween), SDS (e.g., 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5% or more, or ranges therebetween), and/or Tris (pH 7.5-8.5 (e.g., pH 8.0)), e.g., 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 50mM, 60mM, or more, or ranges therebetween. In a particular embodiment, the buffer solution comprises 1% lithium dodecyl sulfate, 30mM EDTA, 300mM LiCl ₂ 1% (v/v) HYDROTECH (Bio-rad), 1% SDS and 30mM Tris (pH 8.0). In some embodiments, the buffer comprises 1% sds and 30mM Tris (pH 8.0).

In some embodiments, after washing the beads thoroughlyAfter the beads and target nucleic acids, the nucleic acids captured by the sorted beads are resuspended in a resuspension buffer. In some embodiments, the same buffer is used for washing and re-suspending. In some embodiments, the wash/resuspension buffer comprises glycerol (e.g., 1%, 2%, 5%, 10%, 15%, 20% or more, or a range therebetween), tris (pH 7.5-8.5) (10 mM, 20mM, 50mM, 100mM, 150mM, 200mM or more, or a range therebetween), diglycine (pH 7.5-8.5) (10 mM, 20mM, 50mM, 100mM, 150mM, 200mM or more, or a range therebetween), potassium glutamate (10 mM, 20mM, 50mM, 100mM, 150mM, 200mM or more, or a range therebetween), mnCl ₂ Or MgCl ₂ (e.g., 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 10mM, or more, or ranges therebetween), tween 20 (e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or more, or ranges therebetween), and/or a HYDROTECH defoamer (BioRad) (e.g., 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or more, or ranges therebetween). In some embodiments, for RNA applications, the buffer comprises 10% (v/v) glycerol, 100mM Tris (pH 8.0), 62.4mM diglycine (pH 8.0), 65mM potassium glutamate, 3mM MnCl ₂ 0.04% Tween 20 and 0.2% HYDROTECH defoamer (BioRad). In some embodiments, for RNA applications, the buffer comprises 10% (v/v) glycerol, 100mM Tris (pH 8.0), 62.4mM diglycine (pH 8.0), 65mM potassium glutamate, 3mM MgCl ₂ 0.04% Tween 20 and 0.2% HYDROTECH defoamer (BioRad).

In some embodiments, the re-suspending comprises: combining the nucleic acid captured by the sorted beads with a resuspension buffer; and resuspending the bead-captured nucleic acid in a resuspension buffer to produce a bead-captured nucleic acid resuspension.

In some embodiments, the sorted bead-captured nucleic acids or bead-captured nucleic acid resuspension are under conditions suitable for amplification, analysis, and/or detection of the target nucleic acid. In some embodiments, the methods herein comprise combining a nucleic acid resuspension of the sorted bead capture with an analytical reagent. In some embodiments, the analytical reagent includes reagents for amplification of target nucleic acids, reagents for detection or quantification of target nucleic acids, reagents for sequencing target nucleic acids, and the like.

In some embodiments, detection of the analyte is performed with the target analyte bound to the capture probe. In some embodiments, detection of the analyte is performed with the analyte present on a solid substrate (e.g., PMP). Detection and/or quantification of analytes can be performed by a variety of methods.

The presence or amount of a nucleic acid analyte can be determined by several methods well known in the art. In some embodiments, the quantification is absolute (i.e., related to a specific amount of target analyte), or relative (i.e., measured in arbitrary normalized units). Methods that allow absolute or relative quantification are well known in the art, for example, quantitative PCR methods are methods for relative quantification; if a calibration curve is incorporated into such an assay, then relative quantification can be used to obtain absolute quantification. Other known methods are, for example, nucleic Acid Sequence Based Amplification (NASBA) or branched DNA signal amplification assays. The amount of nucleic acid analyte can be determined by sequencing or PCR techniques (e.g., fluorescence-based real-time PCR), many examples of which are understood in the art.

The presence or amount of a peptide or polypeptide analyte can be determined by various methods well known in the art. Direct measurement involves measuring the amount of peptide or polypeptide based on a signal obtained from the peptide or polypeptide itself and whose intensity is directly related to the number of molecules of the peptide present in the sample. Such a signal (sometimes referred to as an intensity signal) may be obtained, for example, by measuring the intensity value of a particular physical or chemical property of the peptide or polypeptide. Indirect measurement involves measuring a signal obtained from a secondary component (i.e., a component other than the peptide or polypeptide itself) or a biological readout system (e.g., a measurable cellular reaction, ligand, label, or enzymatic reaction product). Determining the amount of peptide or polypeptide may be accomplished by any known means for determining the amount of peptide in a sample. Such means include immunoassays and/or immunohistochemical methods which can utilize a labeling molecule (e.g., antibodies and antibody fragments) in a variety of sandwich, competitive, or other assay formats. The assay will produce a signal indicative of the presence or absence of the peptide or polypeptide.

In some embodiments, the assay reagent is a dry reagent (e.g., a lyophilization tray or pellet), and the dry assay reagent is resuspended when combined with the bead-captured nucleic acid resuspension and/or buffer solution. In other embodiments, the assay reagent is a concentrated liquid (or gel) and is diluted in a bead-captured nucleic acid resuspension and/or buffer solution. Concentrated assay reagents may be present at a concentration of 10X, 20X, 50X, 100C, 200X, 500X or greater than the 1X working concentration after dilution into the bead-captured nucleic acid resuspension and/or buffer solution.

In some embodiments, the methods herein utilize any suitable technique for analyzing, detecting, quantifying, sequencing, etc., a target nucleic acid. Thus, the methods herein can be utilized and/or the systems/kits herein can include any components/reagents necessary for detecting, quantifying, sequencing, etc., a target nucleic acid by techniques understood in the art. For example, the assay reagents can comprise primers (e.g., fluorescently labeled primers), probes, nucleotides, salts, and any other reagents understood in the art that can be used in known nucleic acid analysis techniques.

In some embodiments, the analysis of the target nucleic acid comprises amplification of the target sequence. Known nucleic acid amplification methods can be used in the methods herein, and known reagents for performing such amplification techniques can be used in the kits/systems herein. For example, the methods herein may utilize amplification techniques such as Polymerase Chain Reaction (PCR), real-time PCR, probe hydrolysis PCR, digital PCR, reverse transcription PCR, isothermal amplification, nucleic Acid Sequence Based Amplification (NASBA), ligase chain reaction, transcription mediated amplification, and the like.

Embodiments herein include various reagents for performing amplification reactions, including but not limited to PCR reactions. Such PCR reactions and other amplification/detection/quantification techniques can be performed as described herein with any suitable analytical reagent. DNA polymerases that can be used according to these embodiments include, but are not limited to, any polymerase capable of replicating a DNA molecule. In some embodiments, the DNA polymerase is a thermostable polymerase particularly useful in PCR applications. Thermostable polymerases are selected from a wide variety of thermophilic bacteria, such as Thermus aquaticus (Taq), thermus brucei (Tbr), thermus flavus (Tfl), thermus red (Tru), thermus thermophilus (Tth), thermus thermophilus (Tli), and other species of Thermococcus; thermophilic species (Thermoplasma acidophilum) (Tac), thermotoga new Apollo (Tne), thermotoga maritima (Tma) and other species of the genus Thermoplasma; other species of the genus pyrococcus intensive (Pfu), pyrococcus vortioides (Pyrococcus woesei) (Pwo) and pyrococcus; bacillus stearothermophilus (Bacillus sterothermophilus) (Bst), sulfolobus acidophilus (Sac), sulfolobus solfataricus (Sso), thermomyces crypticus (Poc), thermomyces profundus (Pyrodictium abyssi) (Pab) and Methanobacterium thermophilum (Methanobacterium thermoautotrophicum) (Mth), and mutants, variants or derivatives thereof.

According to embodiments provided herein, various other PCR reagents may include amplification reagents (which may comprise at least one primer or at least one pair of primers for nucleic acid target amplification), at least one probe and/or dye capable of detecting amplification, a ligase, a detergent (e.g., a non-ionic detergent), nucleotides (dntps and/or NTPs), divalent magnesium ions, or any combination thereof, as well as other reagents that will be recognized by one of ordinary skill based on the present disclosure. In some embodiments, the amplification reagents and/or nucleic acid targets can each be present in an effective amount, such as an amount sufficient to effect amplification of the desired nucleic acid target in the presence of other necessary reagents.

According to embodiments provided herein, the assay reagents may comprise one or more primers, or any nucleic acid capable of and/or used to prime replication of a nucleic acid template. The primer may be DNA, RNA, an analog thereof (e.g., an artificial nucleic acid), or any combination thereof. The primer may have any suitable length, such as at least about 10, 15, 20, or 30 nucleotides. Exemplary primers are chemically synthesized. The primers may be supplied as at least one pair of primers for amplifying at least one nucleic acid target. A pair of primers may be a sense primer and an antisense primer that together define opposite ends (and thus define a length) of the resulting amplicon. In some embodiments, the primers are labeled for detection of the resulting amplicon. Suitable labels include fluorescent labels that are detected by known fluorescent detection methods.

According to embodiments provided herein, the assay reagents may also comprise one or more probes, or any nucleic acid linked to at least one label (such as at least one dye). The probe may be a sequence specific binding partner for the nucleic acid target and/or amplicon. Probes can be designed to be able to detect target amplification based on fluorescence or Fluorescence Resonance Energy Transfer (FRET). The methods herein may include 5' nuclease assays, such as with TAQMAN probes. The assay reagents may comprise one or more labels or reporter molecules. Exemplary reporters include at least one dye, such as a fluorescent dye or energy transfer pair, and/or at least one oligonucleotide. Exemplary reporters for use in nucleic acid amplification assays may include probes and/or intercalating dyes (e.g., SYBR Green, ethidium bromide, etc.).

In some embodiments, one or more assay reagents are combined to form a composition or kit. In some embodiments, reagents required for amplification are combined into concentrated or dried analytical reagents. In some embodiments, the composition may comprise any suitable PCR reagents required to perform an amplification reaction. For example, the analytical reagent may include one or more PCR reagents, such as one or more of the following: primers or a pair of primers for nucleic acid target amplification, probes and/or dyes capable of detecting amplification, ligases, polymerases, nucleotides (dntps and/or NTPs), divalent magnesium ions, or any combination thereof, as well as other reagents that will be recognized by one of ordinary skill in the art based on the present disclosure. According to embodiments provided herein, the concentration of the PCR reagents described above may vary, depending on the particular reaction conditions and reagents used, as well as the desired DNA target to be amplified. Those skilled in the art will readily recognize that any particular concentration or concentration range provided herein for any PCR reagent will vary depending on the particular reaction conditions and reagents used, and is not meant to be limiting.

In some embodiments, the present invention provides systems, kits, and methods for research, screening, and diagnostic applications. For example, in some embodiments, diagnostic applications provide for the detection and/or quantification of nucleic acids from pathogenic entities (e.g., viruses, bacteria, etc.) in a biological sample (e.g., from a subject). In some embodiments, the level, presence or absence of a pathogen is used to provide a diagnosis or prognosis. In some embodiments, the subject is tested. Exemplary diagnostic methods are described herein. In some embodiments, nucleic acid from a pathogen is detected in a sample from a subject. In some embodiments, nucleic acids from pathogens are identified using the methods and reagents described herein.

Some embodiments herein utilize nucleic acid sequencing to detect/quantify a target nucleic acid (e.g., from a pathogen) in a sample from a subject. As used herein, the term "sequencing" refers to a method of obtaining the identity of at least 10 consecutive nucleotides of a polynucleotide (e.g., the identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides). The term "next generation sequencing" refers to the so-called parallel sequencing-by-synthesis or sequencing-by-ligation platforms currently employed by Illumina, life Technologies and Roche, et al. The next generation sequencing methods may also include nanopore sequencing methods or methods based on electronic detection, such as Ion Torrent technology commercialized by Life Technologies. In some embodiments, nucleic acids are amplified using primers compatible with the use of a reversible terminator method such as Illumina, a pyrosequencing method of Roche (454), sequencing by ligation (SOLiD platform) of Life Technologies, or Ion Torrent platform of Life Technologies. Examples of such methods are described in the following references: margulies et al (Nature 2005 437:376-80); ronaghi et al (Analytical Biochemistry 1996 242:84-9); shendure et al (Science 2005 309:1728-32); imelfort et al (Brief Bioinfo.2009:10:609-18); fox et al (Methods Mol biol.2009; 553:79-108); appleby et al (Methods Mol biol.2009; 513:19-39) and Morozova et al (genomics.200892:255-64), which references are incorporated by reference herein for a general description of Methods and specific steps of Methods, including all starting products, reagents and end products of each step. In another embodiment, nanopore sequencing can be used to sequence a target nucleic acid (e.g., as described in Soni et al, clin Chem 2007 53:1996-2001, or as described by Oxford Nanopore Technologies). Nanopore sequencing techniques are disclosed in U.S. patent nos. 5,795,782, 6,015,714, 6,627,067, 7,238,485 and 7,258,838, and U.S. patent publication nos. 2006003171 and 20090029477. The sorted target nucleic acids can be directly sequenced, or in some embodiments, the target nucleic acids can be amplified (e.g., by PCR) to produce sequenced amplification products. In certain embodiments, the amplification product may contain sequences compatible with use of, for example, the reversible terminator method of Illumina, the pyrosequencing method of Roche (454), the ligation sequencing of Life Technologies (SOLiD platform), or the Ion Torrent platform of Life Technologies, as described above.

In some embodiments, provided herein is a method of detecting/analyzing a target nucleic acid in a biological sample for subsequent analysis, the method comprising: (a) Combining a biological sample comprising cells with a dried or concentrated lysis reagent comprising protease K, SDS and a salt, and re-suspending the dried or concentrated lysis reagent in the biological sample to produce a lysate; (b) Combining the lysate with a dried or concentrated capture reagent and resuspending the dried or concentrated capture reagent in the lysate, wherein the capture reagent comprises a nucleic acid probe tethered to the capture moiety, wherein the capture moiety is biotin, and wherein the nucleic acid probe comprises a hybridization sequence complementary to a target sequence within a nucleic acid of the biological sample; (c) Incubating the nucleic acid probes and nucleic acids of the lysate at a temperature of 63-73 ℃ to produce a probe-bound nucleic acid solution; (d) Combining the probe-bound nucleic acid with dried or concentrated capture agent coated magnetic beads and resuspending the dried or concentrated capture agent coated magnetic beads in the probe-bound nucleic acid at 70 ℃ to 80 ℃, wherein the capture agent is streptavidin; (e) Incubating the probe-bound nucleic acid and the capture agent-coated magnetic beads at 63-73 ℃ and allowing the capture agent to bind to the capture moiety to produce a bead-captured nucleic acid suspension; (f) Sorting bead-captured nucleic acids within the bead-captured nucleic acid suspension by exposing a portion of the bead-captured nucleic acid suspension to a magnetic field, and (a) holding the magnetic field in place while removing the liquid portion from the bead-captured nucleic acid suspension, or (B) moving the magnetic field to drag the bead-captured nucleic acids out of the liquid portion; and (g) separating the sorted bead-captured nucleic acids from the liquid portion of the bead-captured nucleic acid suspension; (h) Combining the nucleic acid captured by the sorted beads with a wash buffer; (i) Resuspending the bead captured nucleic acid in a wash buffer; (j) Sorting the bead-captured nucleic acids in the wash buffer by exposing a portion of the bead-captured nucleic acids to a magnetic field; and (k) separating the sorted bead captured nucleic acid from the wash buffer; (i) Combining the nucleic acid captured by the sorted beads with a resuspension buffer; (m) resuspending the bead-captured nucleic acids in a resuspension buffer to produce a bead-captured nucleic acid resuspension; (n) combining the bead-captured nucleic acid resuspension with a dried or concentrated analytical reagent and resuspending the dried or concentrated analytical reagent in the bead-captured nucleic acid resuspension, wherein the analytical reagent comprises primers and a detectable label for amplifying and detecting the target nucleic acid; and amplifying and detecting the target nucleic acid hybridized to the bead-bound capture reagent; wherein the method does not comprise a centrifugation step, a filtration step or a nucleic acid precipitation step; wherein the nucleic acid is not separated from contaminants within the biological sample in steps (a) through (e); and wherein the wash buffer and the resuspension buffer comprise the same components.

In some embodiments, provided herein are methods of preparing a target nucleic acid in a biological sample for subsequent analysis, the methods comprising: (a) Combining the biological sample with a drying or concentrating reagent comprising a lysis/digestion component and a capture component, and allowing the drying or concentrating reagent to re-suspend in the biological sample, wherein the lysis/digestion component comprises a protease K, SDS and a salt, wherein the capture component comprises a nucleic acid probe tethered to the capture component, wherein the capture component is biotin, and wherein the nucleic acid probe comprises a hybridization sequence complementary to a target sequence within a nucleic acid of the biological sample; (b) Incubating the biological sample at a temperature of 90 ℃ to 100 ℃ to allow cell membrane lysis and protein digestion to occur within the sample to produce a lysate; (c) Incubating the lysate at a temperature of 63-73 ℃ to allow the hybridization sequences of the nucleic acid probes to bind to the target sequences of the nucleic acids of the biological sample to produce a probe-bound nucleic acid solution; (d) Combining the probe-bound nucleic acid with dried or concentrated capture agent coated magnetic beads and resuspending the dried or concentrated capture agent coated magnetic beads in the probe-bound nucleic acid at 70 ℃ to 80 ℃, wherein the capture agent is streptavidin; (e) Incubating the probe-bound nucleic acid and the capture agent-coated magnetic beads at 63-73 ℃ and allowing the capture agent to bind to the capture moiety to produce a bead-captured nucleic acid suspension; (f) Sorting bead-captured nucleic acids within the bead-captured nucleic acid suspension by exposing a portion of the bead-captured nucleic acid suspension to a magnetic field, and (a) holding the magnetic field in place while removing the liquid portion from the bead-captured nucleic acid suspension, or (B) moving the magnetic field to drag the bead-captured nucleic acids out of the liquid portion; and (g) separating the sorted bead-captured nucleic acids from the liquid portion of the bead-captured nucleic acid suspension; (h) Combining the nucleic acid captured by the sorted beads with a wash buffer; (i) Resuspending the bead captured nucleic acid in a wash buffer; (j) Sorting the bead-captured nucleic acids in the wash buffer by exposing a portion of the bead-captured nucleic acids to a magnetic field; (k) Separating the nucleic acid captured by the sorted beads from the wash buffer; (i) Combining the nucleic acid captured by the sorted beads with a resuspension buffer; and (m) resuspending the bead-captured nucleic acid in a resuspension buffer to produce a bead-captured nucleic acid resuspension; (n) combining the bead-captured nucleic acid resuspension with a dried or concentrated analytical reagent and resuspending the dried or concentrated analytical reagent in the bead-captured nucleic acid resuspension, wherein the analytical reagent comprises primers and a detectable label for amplifying and detecting the target nucleic acid; and (o) amplifying and detecting target nucleic acids hybridized to the bead-bound capture reagent; wherein the method does not comprise a centrifugation step, a filtration step or a nucleic acid precipitation step; wherein the nucleic acid is not separated from contaminants within the biological sample in steps (a) through (e); and wherein the wash buffer and the resuspension buffer comprise the same components.

In some embodiments, the methods herein are performed using any suitable reaction vessel (e.g., tube, well, chamber within a device, etc.), manual laboratory instrument (e.g., manual pipette, heating block, vortex shaker, etc.), or automated instrument (e.g., robot, microfluidic, liquid handler, self-contained cartridge, fluorometer, etc.). In some embodiments, the method is performed manually (e.g., under control of a human operator). In some embodiments, the moving, combining, and/or mixing of the liquid and the reagent is performed by manual pipetting. In some embodiments, the performance of one or more (e.g., all) of the method steps is automated. In some embodiments, one or more (e.g., all) of the method steps are performed within a single use cartridge. In some embodiments, the single-use cartridge contains all dry and liquid reagents and buffers for performing the method steps. In some embodiments, the single-use cartridge is engaged with an instrument that includes components for combining and mixing reagents, a heating element, a magnet, and a fluorescence detector. Suitable systems including single use cartridges and complementary instruments are described in U.S. provisional application 63/180,270; said application is incorporated by reference in its entirety. In some embodiments, one or more (e.g., all) of the method steps are performed by an automated instrument (e.g., a high throughput robotic platform capable of transferring liquid, mixing solutions/suspensions, heating, moving magnetic fields, detecting fluorescence, etc. between wells).

In some embodiments, provided herein are systems or kits comprising: (a) A lysis reagent (dried or concentrated) capable of digesting cell membranes and degrading cellular proteins; (b) A capture reagent (dried or concentrated) comprising a nucleic acid probe tethered to a capture moiety; (c) magnetic beads coated with a capture agent (dried or concentrated); (d) amplification/detection reagents (dried or concentrated); and (e) a wash/resuspension buffer solution.

In other embodiments, provided herein are systems or kits comprising: (a) A lysis/capture reagent (dried or concentrated) comprising (1) a lysis/digestion component capable of digesting cell membranes and degrading proteins; and (2) a nucleic acid probe tethered to the capture moiety; (b) magnetic beads coated with capture agent (dried or concentrated); (c) amplification/detection reagents (dried or concentrated); and (d) a wash/resuspension buffer solution.

In other embodiments, provided herein are systems or kits comprising: (a) A lysis reagent (dried or concentrated) comprising protease K, SDS and one or more salts; (b) A capture reagent (dried or concentrated) comprising a nucleic acid probe tethered to a biotin capture moiety; (c) Magnetic beads coated with a capture agent (dried or concentrated), wherein the capture agent is streptavidin; (d) Amplification/detection reagents (dried or concentrated) comprising primers, detectable labels and nucleotides; and (e) a buffer solution.

In other embodiments, provided herein are systems or kits comprising: (a) A lysis/capture reagent (dried or concentrated) comprising (1) a lysis/digestion component comprising protease K, SDS and one or more salts; and (2) a nucleic acid probe tethered to the capture moiety; (b) Magnetic beads coated with a capture agent (dried or concentrated), wherein the capture agent is streptavidin; (c) Amplification/detection reagents (dried or concentrated) comprising primers, detectable labels and nucleotides; and (d) a buffer solution.

In some embodiments, the systems and kits further include a biological sample comprising nucleic acid (e.g., within a virus or cell (e.g., bacteria, protozoa, etc.).

In some embodiments, the systems and kits further include disposable laboratory products for manually using the systems or kits to capture, sort, amplify, and detect target nucleic acids from a biological sample comprising cells. In some embodiments, the disposable laboratory product includes a pipette tip, a reaction tube, and/or a microplate.

In some embodiments, the systems and kits further comprise a single-use cartridge comprising components of the systems/kits herein, wherein the single-use cartridge is capable of being engaged with an instrument comprising components for combining and mixing reagents, a heating element, and a magnet.

In an exemplary version, for an exemplary cartridge device (e.g., the device shown in fig. 1-9), the device is provided to contain buffer (e.g., 800 μl) in the second chamber (50) and sample (e.g., a liquid sample (e.g., about 1,000 μl)) in the first chamber (46). The device 10 is inserted into a complementary instrument that includes a pressure source for engaging the transfer bladder tube 26 of the device 10. The pressure source removes transfer bladder 26 from cavity 42 and draws buffer (e.g., about 350 μl) from the second chamber (50) via second inlet port 74 and transfers buffer to the sixth chamber (152) via inlet port 172 of the sixth chamber. A portion of the transferred buffer flows via the microfluidic to the reaction chamber 304.

The pressure source and transfer bladder 26 then draws the sample from the first chamber (46) through the inlet port 66 of the chamber and transfers the sample to the third chamber (140) through the inlet port 160 of the chamber. As the sample flows through the cross-channel portion 204 from the third inlet port 160, the lyophilized reagent 208 positioned in the cross-channel portion 204 is rehydrated as the liquid flows through the third channel 184. The lyophilization reagents 208 contain reagents for cell lysis and digestion of cellular components (e.g., SDS, proteinase K, etc.), as well as nucleic acid capture probes (e.g., sequence specific probes comprising a handle (e.g., biotin) that allows capture of nucleic acids hybridized to the probes). The orientation of the third chamber (140) within the instrument allows heating the contents of the third chamber (140), for example to about 95 ℃ to aid in cell lysis. The sample is lysed and digested in a third chamber (140). The pressure source and transfer bladder tube 26 are used to mix the sample and reagent by drawing the sample/reagent into and out of the transfer bladder tube through the third inlet port 160 and third channel 184 and by injecting bubbles into the sample/reagent.

The pressure source and transfer bladder 26 then draws sample/reagent from the third chamber (140) through its inlet port 160 and transfers the sample to the fourth chamber (144) through its inlet port 164. The sample/reagent is cooled in a fourth chamber (144) to allow the capture probes to hybridise to complementary nucleic acids in the sample.

The pressure source and transfer bladder 26 then draws sample/reagent from the fourth chamber (144) via its inlet port 164 and transfers the sample to the fifth chamber (148) via its inlet port 168. Upon entry of the sample/reagent into the fifth chamber (148), the paramagnetic particles (PMP) comprising the binding moiety (e.g., streptavidin) for binding to the handle on the capture probe are redissolved by the sample/reagent. Resolubilization is aided by the drawing and withdrawal of fluid from the transfer bladder tube into and out of the fifth chamber (148).

The pressure source and transfer bladder 26 then draws sample/reagent from the fifth chamber (148) via its inlet port and transfers the sample to the fourth chamber (144) via its inlet port 168. PMP is allowed to bind to the capture probes, thereby capturing the associated nucleic acids.

The pressure source and transfer bladder 26 then draws sample/reagent from the fourth chamber (144) via its inlet port 164 and transfers the sample to the fifth chamber (148) via its inlet port 168. When transferring from the fourth chamber to the fifth chamber, the instrument magnet is held at the tip of the transfer balloon so that PMP is collected during dispensing and aspiration. The instrument magnet is then placed adjacent to the bottom of the fifth chamber, forming PMP pellets with capture probes and bound nucleic acids attached. Liquid from the fifth chamber (148) is removed by the pressure source and transfer bladder tube 26 via inlet port 168 of that chamber and drops into the third chamber (140) via inlet port 160 of that chamber.

The pressure source and transfer bladder 26 then draws buffer from the second chamber (50) via the inlet port 74 of that chamber and transfers the buffer to the fifth chamber (148) via the inlet port 168 of that chamber. PMP in the fifth chamber (148) is resuspended in buffer by mixing with the transfer bladder tube 26 using a pressure source. The instrument magnet is then placed adjacent to the bottom of the fifth chamber, forming PMP pellets with capture probes and bound nucleic acids attached. Liquid from the fifth chamber (148) is removed by the pressure source and transfer bladder tube 26 via inlet port 168 of that chamber and drops into the third chamber (140) via inlet port 160 of that chamber.

The pressure source and transfer bladder 26 then draws buffer from the sixth chamber (152) via the inlet port 172 of that chamber and transfers the buffer to the fifth chamber (148) via the inlet port 168 of that chamber. PMP in the fifth chamber (148) is resuspended in buffer by mixing with the transfer bladder tube 26 using a pressure source.

The pressure source and transfer bladder 26 then draws buffer from the fifth chamber (148) via inlet port 168 and transfers the buffer to the sixth chamber (152) via inlet port 172. When liquid is added to the chamber, the instrument magnet is positioned adjacent the bottom of the sixth chamber (152) so as to pre-collect PMP at the bottom of the chamber.

The instrument magnet is then used to agglomerate and transfer the PMP to the reaction chamber 304 via the inlet channel 312. The instrument heater is used to melt the wax seals of the vent channel (308) and the inlet channel (312) and allow solidification in the vent and inlet channels of the reaction chamber. PCR is then performed on the PMP-bound nucleic acid using fluorescently labeled primers, and the amplified nucleic acid is detected using instrument-based fluorescent detection.

In some embodiments, a cartridge device is provided that includes two storage chambers (C1 and C2), four processing chambers (C3, C4, C5, and C6), and one reaction chamber. In some embodiments, C1 is a sample chamber. An environmental, biological, or research sample (e.g., comprising cells and/or target analytes) is placed into C1 and the chamber is sealed (e.g., capped). In some embodiments, C2 is a buffer storage chamber. Buffers for sample processing and analyte detection are contained in C2. In some embodiments, a single buffer is used. In embodiments where multiple buffers are desired, the device may include multiple buffer storage chambers (e.g., C2A, C B, etc.). In some embodiments, the storage chamber is sized to hold a sufficient volume of sample and buffer to perform various processing and detection steps (e.g., the storage chamber has a width that is greater than the processing chamber). In some embodiments, C3 is a sample digestion and/or cell lysis chamber. After the sample is added to C3, the reagent pellet within the access channel of C3 is dissolved, exposing the sample to reagents (e.g., lysis reagents, digestion reagents, capture probes, etc.) required for sample processing and/or analyte detection. In some embodiments, C3 is positioned on the cartridge in alignment with one or more heaters of a complementary instrument. In some embodiments, the sample in C3 is exposed to an appropriate temperature to facilitate appropriate sample processing steps for a particular sample type and assay protocol. In some embodiments, C4 is an analyte binding/hybridization chamber. In some embodiments, the C4 is positioned on the cartridge in alignment with one or more heaters of a complementary instrument. In some embodiments, C4 is maintained at a temperature (e.g., a temperature below C3) to allow the capture reagent to bind/hybridize to the target analyte. In some embodiments, the widths of C3 and C4 are adapted to accommodate the tight alignment of the chamber with the heater (e.g., C3 and C4 are narrower than the reservoir chamber). In some embodiments, C5 is a capture chamber. In some embodiments, after the sample is added to C5, the PMP-containing pellet within the access channel of C5 is dissolved, exposing the analyte (e.g., bound to the capture probe) to PMP capable of binding to the capture probe. In some embodiments, C5 is positioned on the cartridge in alignment with the magnetic transfer element of the instrument. The magnetic transfer element allows PMPs in C5 (e.g., capture probes bound to analyte binding) to cluster, move, and otherwise physically manipulate (e.g., smear). In some embodiments, the width of C5 is adapted to accommodate the tight alignment of the chamber with the magnetic transfer element of the complementary instrument. In some embodiments, C6 is a transfer chamber. In some embodiments, the pellet-shaped PMPs in C6 may be transferred into the reaction chamber via a transfer channel using a magnetic transfer element of a complementary instrument. In some embodiments, the width of C6 is adapted to accommodate the tight alignment of the chamber with the magnetic transfer element of the complementary instrument. In some embodiments, after the pellet/sample is added to the reaction chamber, the detection reagent pellet is dissolved, exposing the sample to reagents (e.g., primers, probes, antibodies, etc.) required for analyte detection. In some embodiments, the reaction chamber is sized and configured to allow the chamber to be closely aligned with the heater, fluorometer, and/or other components of a complementary instrument to allow analyte detection.

Alternatives for the exemplary devices, systems, and components thereof, as well as alternative devices, systems, and components having different layouts, are within the scope of the embodiments herein.

Referring to fig. 24 and 25, PCR may be performed by cycling the temperature of the reaction chamber 66 between two temperatures. For example, when the first heat transfer device 22A and the second heat transfer device 22B (high temperature pair) at a first temperature are in contact with the PCR chamber at a lower temperature, thermal energy flows from the heat transfer devices 22A, 22B into the PCR chamber. In some embodiments, the thermal energy flow rate is proportional to the temperature difference, and the thermal energy flow stops when the PCR chamber is warmed and the temperature difference approaches zero. In contrast, when the third heat transfer device 22C and the fourth heat transfer device 22D (low temperature pair) at the second temperature are in contact with the PCR chamber at the higher temperature, thermal energy flows out of the PCR chamber and into the heat transfer devices 22C, 22D. FIG. 24 is a graph of the temperature of liquid in a PCR chamber over time as the PCR chamber is cycled between low temperature heat transfer devices (e.g., 22C, 22D) and high temperature heat transfer devices (e.g., 22A, 22B). In the illustrated embodiment, about 40 thermal cycles are achieved over a 300 second time frame. Fig. 25 is an enlarged portion of the graph of fig. 24 and shows one of a plurality of thermal cycles.

Referring to fig. 26, the microfluidic section 62 of the cartridge 50 includes a first wax seal 182 and a second wax seal 186. A first wax seal 182 is positioned adjacent the microfluidic vent channel 70 and a second wax seal 186 is positioned adjacent the microfluidic inlet channel 74. To maintain stable concentrations of reagents and amplicons and to prevent contamination of the instrument, PCR is performed in a closed system (e.g., reaction chamber 66 is sealed). The wax seals 182, 186 are configured to seal the reaction chamber 66 from both ends (i.e., the inlet end and the vent end). An air-vented microfluidic vent channel 70 leading from the reaction chamber 66 is used for initial buffer fluid priming and air purging of dry reagents. After priming, the paramagnetic particles carrying the targets are transferred into the reaction chamber 66 through the microfluidic inlet channel 74. Once the target is in the reaction chamber 66, the microfluidic vent channel 70 is closed (i.e., sealed) by melting the first wax seal 182 (by, for example, a heater, a heat transfer device, etc.). The molten wax fills the empty space and nearby microfluidic vent channels 70 and then hardens, thereby sealing the microfluidic vent channels 70. After the first wax seal 182 is melted, the second wax seal 186 is then melted in a similar manner, sealing the microfluidic inlet channel 74 and closing the reaction chamber 66. Thus, in some embodiments, the first wax seal 182 in the microfluidic vent channel 70 melts and hardens before the second wax seal 186 in the microfluidic inlet channel 74. This ensures that any air trapped around the second wax seal 186 creates pressure to prevent molten wax from the second wax seal 186 from flowing into the reaction chamber 66. The wax in the reaction chamber 66 can affect the fluorescence optical reading.

Referring to fig. 27A and 27B, the first and second wax seals 182, 186 are initially in a first solid state (fig. 27A) with the respective channels 70, 74 open, and may be changed to a second solid state (fig. 27B) with the respective channels 70, 74 sealed (i.e., closed). The wax seals 182, 186 enter a molten state between a first solid state and a second solid state. In the illustrated embodiment, the first and second wax seals 182, 186 are cylindrical in shape in the initial first solid state (fig. 27A) and do not block (block or seal) their corresponding channels 70, 74. In response to the application of heat, the first and second wax seals 182, 186 melt and flow into the channels 70, 74. After the application of heat is removed, the melted wax resolidifies and enters a second solid state (fig. 27B) and forms a solid wax seal positioned within the channels 70, 74.

A first wax seal 348 in a first solid state is initially positioned over the microfluidic vent channel 308 within the cutout 190 (fig. 27A). In the initial solid state of the first wax seal 182, air can readily pass around and under the wax seal 182. When the wax seal 182 melts, the molten wax fills the empty surrounding space (fig. 27B). In some embodiments, the volume of the first wax seal 182 is less than the volume of the second wax seal 186.

The second wax seal 186 is positioned adjacent the microfluidic inlet channel 74 and extends beyond the laminate layer, creating a tent air pocket 194 around the perimeter of the second wax seal 186 (fig. 27A). In other words, the second wax seal 186 causes the lid to deform during assembly, creating a tent air pocket 194. Although the second wax seal 186 is positioned over the microfluidic inlet channel 74, fluid and target transfer through the microfluidic inlet channel 74 is still possible by utilizing the hydrostatic head at the beginning of the microfluidic inlet channel and a certain amount of detergent in the fluid.

In some embodiments, the microfluidic inlet channel 74 is taller than the microfluidic vent channel 70. The microfluidic ventilation channel 70 has a thickness of about 0.051mm ² For example, 0.051mm high by 1mm wide). Likewise, the microfluidic inlet channel 74 has a thickness of about 0.54mm ² Cross-sectional area (e.g., 0.36mm high by 1.5mm wide). The microfluidic vent channel 70 has a smaller cross-sectional area than the microfluidic inlet channel 74, because the microfluidic vent channel 70 directs only the airflow, whereas the microfluidic inlet channel 74 must allow the passage of liquid buffer and solid particles containing genetic targets. Thus, the amount of wax required in the wax seal 186 for the microfluidic inlet channel 74 is greater than the amount of wax in the wax seal 182 for the microfluidic vent channel 70.

In some embodiments, the tent air pocket 194 is about 0.38mm higher than the surrounding laminate. When the wax seal 186 is melted by the pinch heater, the tent air pocket 194 collapses. Air trapped within the hardened wax seal creates a potential for fluid leakage. It is therefore important that during the wax melting process, air has a path out of the system so as not to compromise seal integrity. In the illustrated embodiment, a spacing of at least about 2mm is provided between any laser cut features and the edges of the laminate to provide sufficient surface area to form a strong adhesive bond. In other words, the narrow adhesive contact areas are prone to leakage and failure. Specifically, the perimeter 198 of the tent air pocket 194 is positioned at least about 2mm away from the reaction chamber 66. In the illustrated embodiment, there is a spacing of at least about 2mm from the tent air pocket 194 to any exposed laminate edge.

Wax seals 182, 186 provide several advantages. The hardened wax seals 182, 186 are advantageously configured to withstand the pressures experienced in the reaction chamber 66 during thermal cycling involving alternating clamping of the reaction chamber with heaters having temperatures in the range of about 50 ℃ and about 95 ℃. In other words, the combination of high temperature and fluid displacement from the mechanical clamping places stresses on the wax seals 182, 186, which are subjected to them. Although the heater may not directly contact the wax seal during thermal cycling, in some embodiments a wax having a high melting temperature (e.g., paraffin wax having a melting temperature of at least about 85 ℃) is selected to ensure that the wax seal is not inadvertently remelted by the heater associated with the reaction chamber. In some embodiments, the wax seal undergoes more than one melting and hardening cycle (i.e., more than one thermal cycle).

In the illustrated embodiment, the wax seals 182, 186 are initially cylindrical in shape (i.e., coin-shaped) (e.g., a diameter of about 4.5mm times a thickness of about 0.43 mm). The rotational symmetry of the circular geometry of the wax seal reduces the risk of misalignment during manufacture of the cartridge 50. In addition, having the same wax seal design simplifies production. In other embodiments, the wax seal is initially oval in shape.

The wax seals 182, 186 may melt in a range of about 86 ℃ (i.e., the wax melting point) and about 95 ℃ (i.e., the default temperature setting of the PCR heater). The duration of melting, or the amount of time that the PCR heater is clamped to the wax seal, may be adjusted in synchronization with the melting temperature to ensure a good seal. For example, if the wax seal melts at too high a temperature for too long, the melted wax may spread further away from the seal site, thereby reducing the material density and mechanical integrity of the seal. In some embodiments, the melting procedure melts the wax seals 182, 186 by applying a relatively hot heater for a duration in the range of about 4 to about 5 seconds. The wax seals 182, 186 are then clamped with a cooler heater having a set point below the wax melting temperature for a duration of about 1.5 seconds. To reduce overall processing time, the wax seals 182, 186 are melted as the thermal PCR heater cools from about 95 ℃ to about 86 ℃ (rather than at a fixed temperature).

In some embodiments, the wax seals 182, 186 have a density of about 0.9g/mL and a density slightly less than the fluid around them at 1 g/mL. In the embodiment shown, gravity acts downwardly during melting and subsequent hardening of the wax seal. Thus, the orientation of gravity affects the movement of the molten wax. For example, molten wax may flow upward when gravity acts downward. The direction of flow of the molten wax is also affected by the surface area and location of the heater used to melt the wax seal. For example, when clamped with a heater, if not concentric but offset in one direction, the molten wax will tend to flow in the offset direction. Also, the heater clamping force is adjusted according to the relative positions of the front and rear sides of the heater, which may be mounted on a low spring constant spring, for example. In some embodiments, the plastic laminate layer is less rigid and is capable of deforming in response to the heater clamping force, thereby extruding and pushing the wax seal out of the boundary of the heater surface area.

In some embodiments, the adhesion of the wax seals 182, 186 is improved by exposing the wax to a layer of acrylic tape (rather than other plastic films such as polyester or polycarbonate). The improved quality of the bond between the melted wax and the channel wall may reduce the likelihood of fluid leakage through the hardened seal.

In some embodiments, the devices, systems, components, reagents, and methods described herein can be used for amplification and/or detection of nucleic acids in a sample. In some embodiments, such devices, systems, components, reagents, and methods can be used with single-use cartridges, multi-use cartridges, cartridge/instrument combinations, high-throughput multiplexing instruments, robots, separate sample preparation and amplification/detection components, combined sample preparation and amplification/detection components, and the like. In some embodiments, provided herein are methods for analyzing nucleic acid amplification reactions performed using the devices, systems, components, reagents, and methods described herein, and/or with other devices, systems, components, reagents, and methods as understood in the art.

Certain embodiments herein utilize real-time PCR or quantitative PCR (qPCR) to amplify and detect a target nucleic acid. In conventional PCR, amplified DNA products or amplicons are detected in an endpoint assay. In real-time PCR, the accumulation of amplified products is measured in real time as the reaction proceeds, while product quantification is performed after each cycle. By analyzing the accumulation of product after each cycle, the amount of target nucleic acid in the original sample can be quantified. In some embodiments, real-time detection of PCR products is achieved by including a reporter in the PCR reaction well that generates an increased signal as the amount of product DNA increases (e.g., the signal from the reporter is proportional to the amount of amplicon generated). In particular embodiments, the reporter is a fluorescent reporter (fluorophore) and the signal detected after each completed PCR cycle is a fluorescent signal. Various fluorescent chemicals may be used as reporter, including DNA binding dyes, fluorescently labeled target sequence specific probes, and/or fluorescently labeled primers.

Real-time PCR allows for accurate and highly sensitive determination of the initial copy number of a template nucleic acid (target sequence) over a wide dynamic range. Real-time PCR results can be qualitative (presence or absence of sequence) or quantitative (copy number). In some embodiments, the amount of an organism or pathogen (e.g., viral load) is determined based on the amount of target nucleic acid detected in the sample.

Amplification during qPCR was performed in two phases: an initial exponential phase followed by a non-exponential plateau phase. During the exponential phase, the amount of PCR product approximately doubles in each cycle. However, as the reaction proceeds, the reaction components are consumed and eventually one or more of the components become limited. At this point, the reaction slowed down and entered a non-exponential plateau phase. During the initial part of the exponential phase, fluorescence remains at background levels, and increases in fluorescence are not readily detected, but the reaction products accumulate exponentially. Once sufficient amplification product has accumulated, a detectable fluorescent signal can be detected through the remainder of the reaction. The cycle in which the fluorescence of the amplified product exceeds the background fluorescence has been referred to as the threshold cycle (Ct) or the quantification cycle (Cq). Since the Cq value is measured during an exponential phase where the reagents are not limited, real-time qPCR can be used to reliably and accurately calculate the initial amount of template present in the reaction based on a known exponential function describing the progress of the reaction. The Cq of the reaction is mainly determined by the amount of template present at the beginning of the amplification reaction. If a large amount of template is present at the beginning of the reaction, relatively few amplification cycles are required to accumulate enough product to give a fluorescent signal above background. Thus, the reaction will have a lower or earlier Cq. In contrast, if a small amount of template is present at the beginning of the reaction, more amplification cycles are required to raise the fluorescent signal above background. Thus, the reaction will have a higher or later Cq. This relationship forms the basis for real-time PCR quantification.

In some embodiments, provided herein are methods of performing qPCR, analyzing the data to determine Cq, and determining the copy number of the target sequence based thereon. In some embodiments, methods are provided for making Cq determinations based on qPCR results independent of absolute levels of fluorescence readings. Such methods eliminate the need to calibrate each instrument and allow the signal strength to vary with the aging of the optical components (LEDs, interference filters). In some embodiments, the methods herein allow for comparison of Cq values determined on separate instruments (or at the same instrument at different points in time) without the need for calibration between instruments or points in time.

In some embodiments, an essential step of the method for performing and analyzing qPCR is to obtain a fluorescence reading after each cycle (e.g., 40 cycles) of the qPCR protocol; reducing variability in fluorescence readings by calculating a moving average for each cycle; identifying a cycle with a maximum increase in fluorescence (norm delta); if the maximum norm delta is greater than the cutoff value, the moving average signal from the previous cycle is checked to identify the earliest cycle for which the norm delta exceeds the second cutoff value (e.g., lower cutoff); fitting a straight line to a plurality (e.g., 3, 4, 5, 6, 7, 8, etc.) of signals (moving average signals) (e.g., sequential signals, signals immediately preceding the earliest cycle) prior to the earliest cycle in which norm delta exceeds a second cutoff value (e.g., lower cutoff); fitting a quadratic curve to a plurality (e.g., 3, 4, 5, 6, 7, 8, etc.) of signals (moving average signals) after an earliest cycle (e.g., sequential signal, signal immediately after the earliest cycle) in which norm delta exceeds a second cutoff value (e.g., lower cutoff); and Cq is determined as a cycle calculated in the case that the difference between the fitting line and the fitting curve differs by a specified value.

In some embodiments, the method comprises: (a) Subjecting a sample suspected of containing a target nucleic acid to a multicycle amplification reaction in the presence of a detectable reporter to produce an amplified product; (b) Detecting a signal from the detectable reporter, the signal being related to the amount of detectable reporter incorporated into the amplification product after each cycle of the amplification reaction; (c) Identifying an earliest cycle in which the increase in signal is greater than the cutoff value; (d) Fitting a linear equation to the plurality of signals from loops earlier than the earliest loop in which the increase in signal is greater than the threshold; (e) Fitting a quadratic curve to the plurality of signals from loops later than the earliest loop in which the increase in signal is greater than the threshold; (f) Identifying a cycle (Cq) in which the difference of the signal of the linear equation and the quadratic curve is equal to a threshold; wherein Cq is inversely proportional to the amount of target nucleic acid present in the sample.

In some embodiments, the method comprises: (a) Subjecting a sample suspected of containing a target nucleic acid to a multicycle amplification reaction in the presence of a detectable reporter to produce an amplified product; (b) Detecting a signal from the detectable reporter, the signal being related to the amount of detectable reporter incorporated into the amplification product after each cycle of the amplification reaction; (c) identifying the cycle with the greatest increase in signal; (d) If the maximum increase in the signal is greater than the cutoff value, determining an earliest cycle of the signal that is greater than a second cutoff value (e.g., a lower cutoff) before the cycle having the maximum increase in the signal; (e) Fitting a linear equation to the plurality of signals from earlier cycles than the earliest cycle of the signal having an increase greater than a second cutoff value (e.g., a lower cutoff); (g) Fitting a quadratic curve to the plurality of signals from loops later than the earliest loop for which the increase in signal is greater than a second cutoff value (e.g., a lower cutoff); (h) Identifying a cycle (Cq) in which the difference of the signal of the linear equation and the quadratic curve is equal to a threshold; wherein Cq is inversely proportional to the amount of target nucleic acid present in the sample.

In some embodiments, the method comprises: (a) Subjecting a sample suspected of containing a target nucleic acid to a multicycle amplification reaction in the presence of a detectable reporter to produce an amplified product; (b) Detecting a signal from the detectable reporter, the signal being related to the amount of detectable reporter incorporated into the amplification product after each cycle of the amplification reaction; (c) Calculating a moving average signal of the detected signal for each cycle of the amplification reaction; (d) Identifying an earliest cycle in which the increase in the moving average signal is greater than the cutoff value; (e) Fitting a linear equation to a plurality of moving average signals from loops earlier than the earliest loop in which the increase in moving average signal is greater than a threshold; (f) Fitting a quadratic curve to the plurality of moving average signals from loops later than the earliest loop in which the increase in moving average signal is greater than the threshold; (g) Identifying a cycle (Cq) in which the difference between the linear equation and the moving average signal of the quadratic curve is equal to a threshold value; wherein Cq is inversely proportional to the amount of target nucleic acid present in the sample.

In some embodiments, the method comprises: (a) Subjecting a sample suspected of containing a target nucleic acid to a multicycle amplification reaction in the presence of a detectable reporter to produce an amplified product; (b) Detecting a signal from the detectable reporter, the signal being related to the amount of detectable reporter incorporated into the amplification product after each cycle of the amplification reaction; (c) Calculating a moving average signal of the detected signal for each cycle of the amplification reaction; (d) Identifying a cycle having a greatest increase in the moving average signal; (e) If the maximum increase in the moving average signal is greater than the cutoff value, determining an earliest cycle in which the increase in the moving average signal is also greater than a second cutoff value (e.g., a lower cutoff) before the cycle having the maximum increase in the moving average signal; (f) Identifying an earliest cycle in which the increase in the moving average signal is greater than a second cutoff value (e.g., a lower cutoff); (g) Fitting a linear equation to a plurality of moving average signals from loops earlier than the earliest loop in which the increase in moving average signal is greater than a threshold; (h) Fitting a quadratic curve to the plurality of moving average signals from loops later than the earliest loop in which the increase in moving average signal is greater than the threshold; (i) Identifying a cycle (Cq) in which the difference between the linear equation and the moving average signal of the quadratic curve is equal to a threshold value; wherein Cq is inversely proportional to the amount of target nucleic acid present in the sample.

In some embodiments herein, cq is the number of cycles calculated to increase fluorescence to a specified percentage above baseline. In certain embodiments, such calculations are performed at breakpoint cycles, wherein fluorescence is determined to be significantly higher than baseline.

Due to the variability of the fluorescence readings (Fi), in some embodiments herein, a Moving Average (MAFi) of each cycle (i) is calculated and used for subsequent analysis steps. In some embodiments, MAF [ i ] for each cycle i is calculated using the cyclic signal F [ i ] and the immediately preceding and following cyclic signals (e.g., the immediately preceding and following 1-3 cycles), for example, according to the following equation:

MAF[i]＝(F[i-2]+F[i-1]+F[i]+F[i+1]+F[i+2])/5

in some embodiments, the rate of change of the signal for each cycle is determined by calculating the difference between the signals (moving average signals) of x cycles (e.g., 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, or more) before and after a given cycle, and dividing it by the signal (moving average signal) of that cycle. For example, if the number of cycles is i, when calculated using a series of signals of 5 cycles before and 5 cycles after i, the normalized difference in percent (norm Delta [ i ]) can be determined according to the following:

normDelta[i]＝100*(MAF[i+5]-MAF[i-5])/MAF[i]

In some implementations, if the maximum norm delta between cycle 5 and cycle 35 is less than cutoff highnorm delta, then no breakpoint exists. In some embodiments, the cutoff (cutoff highnormdelta) value depends on the loop. In some embodiments, between cycle 5 and cycle 24, cutoff highnormdelta is 8; between cycle 25 and cycle 34, cutoff highnormdelta is 4, and at cycle 35 cutoff highnormdelta is 2.5. In some embodiments, the cut-off can range between 1.5 and 15 (e.g., 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, or ranges therebetween). In some embodiments, different cutoffs may be applied to different circulation ranges (e.g., 5-10, 11-15, 16-20, 21-25, 26-30, 31-35, or any other suitable circulation range).

If the maximum norm delta is greater than the cutoff, the search begins to look at the earlier loop to find when it falls below cutoff lownormdelta. In some embodiments, cutoff lownormdelta is lower than cutoff highnormdelta. In some embodiments, the cutoff (cutoff lownormdelta) value depends on the loop. In some embodiments, cutoff lownormdelta is not cycle-based. In some embodiments, the cutoff (cutoff lownormdelta) can range between 1.5 and 6 (e.g., 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, or ranges therebetween). In some embodiments, the earliest cycle above the cutoff lownormdelta is the breakpoint cycle.

In some implementations, if a breakpoint loop is found, a linear equation is fitted to the breakpoint loop and the fluorescence signal (e.g., moving average signal) at several loops (e.g., 2-6 previous loops) before it. If j=0 is a breakpoint loop, then in some embodiments, the equation for the linear fit is:

FitL [ j ] = b0l+b1lxcj ], j= -4 to 0

Where Cj is the cycle number and FitL j is a linear estimate of fluorescence. b0L is the intercept of a straight line and b1L is the slope.

In some implementations, if a breakpoint loop is found, a curve (e.g., conic) equation is fitted to the breakpoint loop and fluorescence signals (e.g., moving average signals) at several loops (e.g., 2-6 later loops) after it. If j=0 is a breakpoint loop, then in some embodiments, the equation for the quadratic fit is:

FitR [ j ] =b0R+b1RxCi ] +b1RxCi ]. Sup.2, i=0 to 4

Where FitR [ j ] is a second estimation of fluorescence under parameters b0R, b R and b 2R. The coefficients of FitL (B0L and B1L) are estimated by minimizing the sum of squares of the difference between FitL and MAF, as are the coefficients of FitR (B0R, B1R, B R). Cq is the solution of the quadratic equation:

100*{(b0L+b1L x Cq)-(b0R+b1R x Cq+b1R x Cq^2)}/b0L＝

calcCq

wherein calccq=2

FIG. 33 depicts an example of Cy5 fluorescence readings of positive SARS-CoV-2 samples. FitL and FitR are depicted. Fig. 34 shows the area surrounding the breakpoint of fig. 33. The Cq value was calculated with a difference between FitR and FitL of 1% (shown in orange).

In some embodiments, normalizing the fluorescent signal using the methods described herein counteracts the difference in optical gain. The LED optical gain is the power of the light excited fluorescence in the PCR chamber divided by the electrical power to drive the LED. This is affected by the variability of the properties of the LEDs, bandpass filters and projection lenses, as well as the variability of the manner in which they are assembled into a fluorometer. The detector optical gain is the intensity of the fluorescent light emitted from the PCR chamber divided by the electrical signal produced by the solid state detector. This is affected by the variability of the properties of the two lenses, the bandpass filter and the solid state detector, as well as the variability of their placement during assembly. The methods described herein allow analysis and comparison of amplification results, regardless of LED optical gain or other variability of the instrument used.

The methods described herein may be used with the devices (e.g., cartridges), instruments (e.g., having complementary components for cartridge processing), and systems described herein, but may also be used with other devices and systems independently. The components described herein (e.g., wax seals, fluorometers, microfluidics, transfer vials, etc.) can be used with the devices (e.g., cartridges), instruments (e.g., having complementary components for cartridge processing) and systems herein, and with the methods described herein, but can also be used independently with other methods, devices, and systems.

Experiment

Example 1

Exemplary sample preparation and nucleic acid analysis protocol #1

350 μl of a liquid biological sample (e.g., a liquid sample, a sample suspended in a buffer, etc.) is combined with the lyophilized lysis/capture reagent, thereby allowing the lysis/probe reagent to be resuspended in the liquid biological sample. The lyophilized lysis/capture reagent contains (1) reagents for cell lysis and digestion of cellular components (e.g., SDS, proteinase K, etc.), and (2) nucleic acid hybridization probes (i.e., sequence specific probes) tethered to a biotin capture moiety that allows subsequent capture of nucleic acids hybridized to the probes. The biological sample and lysis/capture reagent are incubated together for about 60 seconds at a temperature rise to 90-100 ℃ to facilitate cell lysis. The suspension of cell lysates is then mixed and incubated at 68 ℃ to allow the probes to bind to target sequences within the sample nucleic acids. Mu.l of the probe-bound nucleic acid solution was combined with lyophilized streptavidin-coated magnetic beads. The resulting suspension was mixed at 75 ℃ to allow dissolution of the beads, and then the temperature was reduced to 68 ℃ to facilitate capture of probe-bound nucleic acids to the beads by binding of biotin on the probes to streptavidin on the beads. The resulting suspension was thoroughly mixed. A magnetic field is then applied to a single location within the suspension, thereby sorting the beads and any probes/target nucleic acids bound thereto into pellets within the suspension. The pellet and supernatant were separated from each other by either: (1) Placing the magnetic field outside the liquid, thereby causing the beads to be dragged or flow across the liquid/air interface, or (2) removing the liquid while maintaining the magnetic field such that the liquid is removed and the pellets remain in place. Mu.l of wash buffer was added to the pellet and the beads resuspended by mixing. The process of forming pellets and separating the beads from the liquid is repeated. The beads were then resuspended in 300 μl of resuspension buffer. The suspension containing the washed beads is then combined with a lyophilized amplification/detection reagent containing fluorescently labeled primers and nucleotides. Thermal cycling is then applied to the sample to amplify the target nucleic acid with an amplification reagent and the amplified target nucleic acid is detected.

Alternatives utilizing different reagents, different sequence of steps, different volumes, different temperatures, concentrated liquid reagents (rather than dry reagents), and the like are also within the scope of embodiments herein, as discussed throughout.

Example 2

Exemplary sample preparation and nucleic acid analysis protocol #2

350 μl of a liquid biological sample (e.g., a liquid sample, a sample suspended in a buffer, etc.) is combined with the lyophilized lysis/digestion reagent, thereby allowing the lysis/digestion reagent to be resuspended in the liquid biological sample. The lyophilized lysis/digestion reagents contain reagents for cell lysis and digestion of cellular components (e.g., SDS, proteinase K, etc.). The biological sample and lysis/digestion reagent are incubated together for about 60 seconds at a temperature elevated to 90 ℃ to 100 ℃ to aid in cell lysis. 300-340 μl of the resulting cell lysate is combined with a lyophilized capture reagent containing a nucleic acid hybridization probe (i.e., a sequence specific probe) tethered to a biotin capture moiety that allows for the subsequent capture of nucleic acid hybridized to the probe. The suspension of cell lysate and capture probes was mixed and incubated at 68 ℃. Mu.l of the probe-bound nucleic acid solution was combined with lyophilized streptavidin-coated magnetic beads. The resulting suspension was mixed at 75 ℃ to allow dissolution of the beads, and then the temperature was reduced to 68 ℃ to facilitate capture of probe-bound nucleic acids to the beads by binding of biotin on the probes to streptavidin on the beads. The resulting suspension was thoroughly mixed. A magnetic field is then applied to a single location within the suspension, thereby sorting the beads and any probes/target nucleic acids bound thereto into pellets within the suspension. The pellet and supernatant were separated from each other by either: (1) Placing the magnetic field outside the liquid, thereby causing the beads to be dragged or flow across the liquid/air interface, or (2) removing the liquid while maintaining the magnetic field such that the liquid is removed and the pellets remain in place. Mu.l of wash buffer was added to the pellet and the beads resuspended by mixing. The process of forming pellets and separating the beads from the liquid is repeated. The beads were then resuspended in 300 μl of resuspension buffer. The suspension containing the washed beads is then combined with a lyophilized amplification/detection reagent containing fluorescently labeled primers and nucleotides. Thermal cycling is then applied to the sample to amplify the target nucleic acid with an amplification reagent and the amplified target nucleic acid is detected.

Example 3

Exemplary Cq determination Using variable light Source

Table 1 shows the variability of LED excitation light and marker fluorescence detection. The optical gain of 10 different LEDs and detectors is ordered for each of the 4 channels, with the highest optical gain in each channel being ranked 1. For example, in channel 1, the LED with the highest optical gain has the Sequence Number (SN) 1-004, while the LED with the lowest optical gain is SN 1-006. The detector with the highest optical gain is SN 1-002 and the detector with the lowest gain is SN 1-005.

Table 1.

To determine the effect of absolute signal strength on Cq determination for both process control (FAM) and SARS-CoV-2 (Cy 5), the highest gain LED and detector pairs were paired and compared to the lowest LED-detector pair. The high FAM LED-detector pair is placed in the channel 1 position and the low pair is placed in the channel 3 position. The high Cy5 LED-detector pair is placed in the channel 2 position and the low pair is placed in the channel 4 position. The Cq determinations for a range of target concentrations (copy number per reaction) using the methods described herein are shown in table 2.

Table 2.

For 1,000 copies, the average of three FAM reactions was 27.6 and 28.0, and the average of two Cy5 reactions was 27.6. At 50 copies, the average of FAM reactions was 32.3 and the average of Cy5 reactions was 31.8. For very low concentrations (5 and 10 copies), the results were similar when testing for Cq.

Claims

1. A cartridge for analyte detection, the cartridge comprising:

a storage section comprising a storage chamber;

a processing section comprising a processing chamber;

a microfluidic section in fluid communication with the processing section; and a transfer bladder configured to transfer fluid between the storage chamber and the processing chamber.

2. The cartridge of claim 1, wherein the processing section is positioned between the storage section and the microfluidic section.

3. The cassette of claim 1 or 2, further comprising a docking section having an inlet port in fluid communication with the storage chamber and a process inlet port in fluid communication with the process chamber.

4. The cartridge of one of claims 1-3, further comprising a body forming at least a portion of the storage section, at least a portion of the processing section, and at least a portion of the docking section.

5. A cartridge as in one of claims 1-3, wherein a first channel fluidly connects the storage inlet port and the storage chamber, and a second channel fluidly connects the process inlet port and the process chamber.

6. The cartridge of one of claims 1-5 wherein said storage chamber comprises a first end and a second end opposite said first end, said first end being positioned closer to said storage access port than said second end, and wherein said first channel is connected to said storage chamber at said second end.

7. The cassette of one of claims 1-5, wherein the process chamber comprises a first end and a second end opposite the first end, the first end being positioned closer to the process access port than the second end, and wherein the second channel is connected to the process chamber at the second end.

8. The cartridge of one of claims 1-7, wherein the storage chamber is a first storage chamber and the storage section further comprises a second storage chamber.

9. The cartridge of one of claims 1-8, wherein the process chamber is a first process chamber and the process section further comprises a second process chamber.

10. The cartridge of one of claims 1-9, wherein said storage section comprises a cavity configured to receive said transfer balloon tube.

11. The cartridge of one of claims 1-10, further comprising a first vent fluidly coupled to the storage chamber and a second vent fluidly coupled to the processing chamber.

12. The cartridge of one of claims 1-11, wherein the microfluidic section comprises a reaction chamber, a microfluidic vent channel fluidly connected to the reaction chamber, and a microfluidic inlet channel fluidly connecting the processing chamber and the reaction chamber.

13. The cartridge of claim 12, wherein the microfluidic section further comprises a wax seal.

14. The cartridge of claim 13, wherein the wax seal is a first wax seal and the microfluidic section further comprises a second wax seal, wherein the first wax seal is positioned adjacent the microfluidic inlet channel and the second wax seal is positioned adjacent the microfluidic vent channel.

15. The cartridge of claim 14, wherein the second wax seal is positioned at a distance from the reaction chamber, wherein the distance is at least 2mm.

16. The cartridge of claim 12, further comprising an offset vent channel fluidly connected to the microfluidic inlet channel.

17. The cassette of claim 16, wherein the offset vent channel is a first offset vent channel and the cassette further comprises a second offset vent channel fluidly connecting the first offset vent channel with the second vent.

18. A microfluidic device, the microfluidic device comprising:

(i) The reaction chamber is provided with a plurality of reaction chambers,

(ii) An inlet channel in fluid communication with the reaction chamber;

(iii) A vent passage in fluid communication with the reaction chamber;

(iv) A first wax seal positioned adjacent to and in fluid communication with the inlet channel, wherein when the first wax seal is in a first position, the first wax seal does not block the inlet channel and allows fluid to enter the reaction chamber through the inlet channel, and wherein when the first wax seal is in a second position, the first wax seal blocks the inlet channel and prevents fluid from entering or seeping out of the reaction chamber through the inlet channel;

(v) A second wax seal positioned adjacent to and in fluid communication with the vent channel, wherein when the second wax seal is in a first position, the second wax seal does not block the vent channel and allows gas to exit the reaction chamber through the vent channel, and wherein when the second wax seal is in a second position, the second wax seal blocks the vent channel and prevents fluid from entering or exiting the reaction chamber through the vent channel;

Wherein a liquid reagent is introducible into the reaction chamber through the inlet channel; and is also provided with

Wherein heating the wax seal above a threshold temperature melts the wax seal, and subsequently cooling the wax seal below the threshold temperature solidifies the wax seal in the second position.

19. A system comprising a cartridge according to one of claims 1-17 and an instrument into which the cartridge can be inserted, wherein the instrument comprises means for imparting heating, magnetic transfer, fluid transfer and/or analyte detection functions into the cartridge.

20. Use of the system of claim 19 for sample processing and analyte detection.

21. A microfluidic system comprising an inlet channel, a vent channel, and a reaction chamber; wherein the inlet channel is in fluid communication with the reaction chamber, wherein the vent channel is in fluid communication with the reaction chamber;

the system further comprises a heating element capable of raising the temperature of the reaction chamber;

the system further comprises a first seal positioned adjacent to and in fluid communication with the inlet channel, wherein when the first seal is in a first position, the first seal does not block the inlet channel and allows fluid to enter the reaction chamber through the inlet channel, and wherein when the first seal is in a second position, the first seal blocks the inlet channel and prevents fluid from entering or seeping out of the reaction chamber through the inlet channel;

The system further comprises a second seal positioned adjacent to and in fluid communication with the vent channel, wherein when the second seal is in a first position, the second seal does not block the vent channel and allows gas to exit the reaction chamber through the vent channel, and wherein when the second seal is in a second position, the second seal blocks the vent channel and prevents fluid from entering or exiting the reaction chamber through the vent channel; and is also provided with

Wherein heating the seal above a threshold temperature melts the seal and allows the seal to flow from the first position to the second position, and wherein subsequently cooling the seal below the threshold temperature solidifies the wax seal on the second position.

22. The system of claim 21, wherein the first seal and the second seal comprise wax or a polymeric material.

23. A heat transfer device for heating and cooling a reaction chamber, the heat transfer device comprising:

a heat reservoir comprising a base, a first aperture, a second aperture, and a heat exchanger extending from the base; the heat exchanger includes a planar surface configured to surround the reaction chamber;

A heater positioned within the first aperture; and

a temperature sensor positioned within the second aperture.

24. The heat transfer device of claim 23, wherein the first and second holes are formed in the base.

25. The heat transfer device of claim 23, wherein the heat exchanger is cylindrical.

26. The heat transfer device of claim 23, wherein the heat reservoir is aluminum.

27. The heat transfer device of claim 23, wherein the heater is a resistive heater.

28. The heat transfer device of claim 23, wherein the reaction chamber is a PCR reaction chamber.

29. The heat transfer device of claim 23, further comprising a processor and a non-transitory memory comprising instructions that when executed by the processor perform closed loop temperature control of the heat reservoir.

30. An assembly, the assembly comprising:

a first support;

a second support movable relative to the first support;

a first heat transfer device having a first planar surface, the first heat transfer device coupled to the first support;

A second heat transfer device having a second planar surface positioned opposite the first planar surface, the second heat transfer device coupled to the second support; wherein the first heat transfer device and the second heat transfer device are at a first temperature;

a third heat transfer device having a third planar surface, the third heat transfer device coupled to the first support;

a fourth heat transfer device having a fourth planar surface positioned opposite the third planar surface, the fourth heat transfer device coupled to the second support; wherein the third heat transfer device and the fourth heat transfer device are at a second temperature different from the first temperature.

31. The assembly of claim 30, further comprising a fluorometer coupled to the first support and positioned between the first heat transfer device and the third heat transfer device.

32. The assembly of claim 31, further comprising a fifth heat transfer device having a fifth planar surface positioned opposite the fluorometer, the fifth heat transfer device coupled to the second support and positioned between the second heat transfer device and the fourth heat transfer device.

33. The assembly of claim 30, wherein the assembly is configured to receive a reaction chamber between the first planar surface and the second planar surface to bring the reaction chamber to the first temperature; and receiving the reaction chamber between the third planar surface and the fourth planar surface to bring the reaction chamber to the second temperature.

34. The assembly of claim 33, wherein the reaction chamber is a PCR chamber.

35. The assembly of claim 33, further comprising an actuator coupled to the second support and configured to move the second support along a clamp axis between a first position in which the first planar surface is spaced apart from the second planar surface by a first distance and a second position in which the first planar surface is spaced apart from the second planar surface by a second distance less than the first distance.

36. The assembly of claim 35, wherein the second distance is in the range of 400 microns to 600 microns.

37. The assembly of claim 33, wherein the actuator is a first actuator, and the assembly further comprises a second actuator coupled to the first support and the second support, wherein the second actuator is configured to move the first support and the second support together along a translation axis.

38. The assembly of claim 37, wherein the translation axis is perpendicular to the clamp axis.

39. The assembly of claim 30, wherein the first temperature is in the range of 80 ℃ and 100 ℃.

40. The assembly of claim 30, wherein the second temperature is in the range of 50 ℃ and 70 ℃.

41. A fluidic device, the fluidic device comprising:

a reaction chamber;

a channel in fluid communication with the reaction chamber; and

a wax seal, wherein when the wax seal is in a first position, the wax seal does not block the channel and allows fluid to enter or exit the reaction chamber through the channel, and when the wax seal is in a second position, the wax seal blocks the channel and prevents fluid from entering or exiting the reaction chamber through the channel;

42. A fluidic device according to claim 41, wherein the reaction chamber is in fluid communication with an inlet channel; and the fluidic device further comprises a vent channel in fluid communication with the reaction chamber.

43. A fluid device according to claim 42, wherein the inlet channel wax seal is a first wax seal and the fluid device further comprises a second wax seal, wherein when the second wax seal is in a first position, the second wax seal does not block the vent channel and allows fluid to leave the reaction chamber through the vent channel, and when the second wax seal is in a second position, the second wax seal blocks the vent channel and prevents fluid from entering or exiting the reaction chamber through the vent channel.

44. The fluid device of claim 43, wherein said first wax seal is positioned adjacent said inlet channel and said second wax seal is positioned adjacent said vent channel.

45. The fluid device of claim 41, further comprising a first high temperature movable heater positionable within a range of the wax seal to heat the wax seal above the threshold temperature.

46. The fluid device of claim 45, further comprising a second cryogenically movable heater positionable within a range of the wax seal to cool the wax seal below the threshold temperature.

47. The fluid device of claim 46, wherein the wax seal has a diameter that is smaller than the diameters of the first and second heaters.

48. The fluidic device of claim 41, wherein the wax seal is coated in an adhesive.

49. The fluidic device of claim 48, wherein the adhesive is an acrylic adhesive.

50. The fluid device of claim 41, wherein the first heater and the second heater are positionable at selected locations relative to the wax seal.

51. A fluidic device according to claim 41, wherein the second wax seal is positioned at a distance from the reaction chamber, wherein the distance is at least 2mm.

52. A fluidic device according to claim 42, wherein a plurality of liquid reagents are introducible into the reaction chamber through the inlet channel.

53. A fluorometer, the fluorometer comprising:

a housing including a measurement aperture;

a first light source coupled to the housing along a first light source axis;

a second light source coupled to the housing along a second light source axis;

a third light source coupled to the housing along a third light source axis;

A fourth light source coupled to the housing along a fourth light source axis;

a first light detector coupled to the housing along a first detector axis;

a second light detector coupled to the housing along a second detector axis;

a third light detector coupled to the housing along a third detector axis;

a fourth light detector coupled to the housing along a fourth detector axis;

wherein the first, second, third, fourth, first, second, third, and fourth light source axes intersect the measurement aperture.

54. A fluorometer according to claim 53, wherein a circular measurement aperture defines a normal axis through its center and perpendicular to the plane of the measurement aperture.

55. The fluorometer of claim 54, wherein the normal axis, the first light source axis, the second light source axis, the third light source axis, the fourth light source axis, the first detector axis, the second detector axis, the third detector axis, and the fourth detector axis are not coaxial.

56. The fluorometer of claim 54, wherein the first light source axis, the second light source axis, the third light source axis, the fourth light source axis, the first detector axis, the second detector axis, the third detector axis, and the fourth detector axis are positioned circumferentially about the normal axis.

57. The fluorometer of claim 53, wherein the first light source is positioned circumferentially adjacent to the first detector.

58. The fluorometer of claim 53, wherein the fluorometer does not comprise a dichroic mirror or beam splitter.

59. The fluorometer of claim 53, further comprising a processor and a non-transitory memory comprising instructions that when executed by the processor store 400 analog-to-digital readings taken by the first detector over a 100 millisecond period.

60. The fluorometer of claim 53, wherein the first light source emits first excitation light along the first light source axis; and wherein the first excitation light is reflected away from the first photodetector axis at the measurement aperture.

61. The fluorometer of claim 53, wherein the measurement aperture is configured to receive a sample; and wherein the first excitation light from the first light source has a first spectrum and the first light detector measures a first fluorescence of the sample in response to the first excitation light.

62. The fluorometer of claim 61, wherein the second excitation light from the second light source has a second spectrum and the second light detector measures a second fluorescence of the sample in response to the second excitation light.

63. A fluorometer according to claim 53, wherein the measurement aperture is configured to align with a planar surface of a PCR chamber.

64. The fluorometer of claim 53, wherein the first detector comprises a first lens, a filter, a second lens, and a solid state detector.

65. A method of nucleic acid quantification, the method comprising:

(a) Subjecting a sample suspected of containing a target nucleic acid to a multicycle amplification reaction in the presence of a detectable reporter to produce an amplified product;

(b) Detecting a signal from the detectable reporter, the signal being related to the amount of detectable reporter incorporated into the amplification product after each cycle of the amplification reaction;

(c) Identifying an earliest cycle in which the normalized increase of the signal is greater than the cutoff value;

(d) Fitting a linear equation to the plurality of signals from cycles earlier than the earliest cycle of signals by more than a threshold;

(e) Fitting a curve to a plurality of signals from loops later than the earliest loop in which the normalized increase in signal is greater than the threshold;

(f) -identifying a cycle (Cq) in which the normalized difference of the signals of the linear equation and the curve is equal to a threshold;

wherein Cq is inversely proportional to the amount of target nucleic acid present in the sample.

66. The method of claim 65, wherein step (c) comprises:

(i) Identifying a cycle having a maximum normalized increase in signal;

(ii) If the maximum normalized increase of the signal is greater than the cutoff value, determining an earliest cycle of the signal before the cycle having the maximum normalized increase of the signal that is greater than a lower cutoff value.

67. The method of claim 65, further comprising the steps of: calculating a moving average of the detected signal for each cycle of the amplification reaction and using the moving average for each cycle of steps (c) - (f).

68. The method of claim 67, wherein the moving average is calculated as an average of the signal at each cycle, calculated with the signals at two immediately earlier cycles and two immediately later cycles.

69. The method of claim 65, wherein the curve is a conic.

70. The method of claim 65, wherein the multicycle amplification reaction is a 30-50 cycle amplification reaction.

71. The method of claim 70, wherein the multicycle amplification reaction is a 40 cycle amplification reaction.

72. The method of claim 65, wherein the multicycle amplification reaction is a quantitative polymerase chain reaction (qPCR).

73. The method of claim 65, wherein the detectable reporter is a fluorophore and the signal is fluorescence.

74. The method of claim 65, wherein each cycle comprises a nucleic acid denaturation step, an annealing/extension step and a detection step.

75. The method of claim 65, wherein each cycle comprises a nucleic acid denaturation step, an annealing step, an extension step and a detection step.

76. The method of claim 65, wherein the sample is a biological sample.

77. The method of claim 76, wherein the target nucleic acid is a viral nucleic acid.

78. The method of claim 77, wherein the amount of target nucleic acid present in the sample is proportional to the viral load in the sample.

79. A method of preparing a target nucleic acid in a biological sample for subsequent analysis, the method comprising:

(a) Combining the biological sample with a lysis reagent capable of digesting cell membranes and degrading proteins and allowing the lysis reagent to digest cell membranes and degrade proteins to produce a lysate, wherein the biological sample comprises nucleic acids;

(b) Combining the lysate with a capture reagent, wherein the capture reagent comprises a nucleic acid probe tethered to a capture moiety;

(c) Allowing the nucleic acid probes to hybridize with the nucleic acids of the biological sample to produce a probe-bound nucleic acid solution;

(d) Combining the probe-bound nucleic acid with a capture agent-coated magnetic bead;

(e) Allowing a capture agent to bind to the capture moiety to produce a bead-captured nucleic acid suspension;

(f) Sorting bead-captured nucleic acids within the bead-captured nucleic acid suspension by exposing a portion of the bead-captured nucleic acid suspension to a magnetic field; and

(g) Separating the sorted bead-captured nucleic acids from the liquid portion of the bead-captured nucleic acid suspension.

80. A method of preparing a target nucleic acid in a biological sample for subsequent analysis, the method comprising:

(a) Combining the biological sample with a lysis reagent and a capture reagent, wherein the lysis reagent comprises a component capable of digesting cell membranes and degrading cellular proteins, wherein the capture reagent comprises a nucleic acid probe tethered to a capture moiety, and wherein the biological sample comprises a nucleic acid;

(b) Allowing the lysing reagent to digest the cell membrane and degrade the protein to produce a lysate;

81. The method of claim 79 or 80, wherein the lysing reagent is a dry lysing reagent, and wherein the dry lysing reagent is resuspended in the biological sample.

82. The method of claim 79 or 80, wherein the lysing reagent is a concentrated liquid lysing reagent, and wherein the concentrated liquid lysing reagent is diluted in the biological sample.

83. The method of claim 79 or 80, wherein the capture reagent is a dry capture reagent, and wherein the dry capture reagent is resuspended in the lysate.

84. The method of claim 79 or 80, wherein the capture reagent is a concentrated liquid capture reagent, and wherein the concentrated liquid capture reagent is diluted in the lysate.

85. The method of claim 79 or 80, wherein the capture agent coated magnetic beads are dried and resuspended in the probe-bound nucleic acid solution.

86. The method of claim 79 or 80, wherein the capture agent coated magnetic beads are in a concentrated liquid and diluted in the probe-bound nucleic acid solution.

87. The method of claim 79 or 80, wherein the method does not comprise a centrifugation step.

88. The method of claim 79 or 80, wherein the method does not comprise a filtration step.

89. The method of claim 79 or 80, wherein the method does not comprise nucleic acid precipitation.

90. The method of claim 79 or 80, wherein the nucleic acid is not separated from contaminants within the biological sample in steps (a) through (e).

91. The method of claim 79 or 80, wherein allowing the lysing reagent to digest cell membranes and degrade proteins to produce a lysate comprises mixing the biological sample and the lysing reagent at a temperature that is increased to 90 ℃ -100 ℃.

92. The method of claim 79 or 80, wherein the lysing agent comprises a protease capable of digesting cellular proteins.

93. The method of claim 92, wherein the protease is proteinase K.

94. The method of claim 79 or 80, wherein the lysing reagent comprises a detergent in a concentration sufficient to lyse cells.

95. The method of claim 94, wherein the detergent is SDS.

96. The method of claim 79 or 80, wherein the lysing reagent comprises one or more salts.

97. The method of claim 79 or 80, wherein the capture moiety is biotin and the capture agent is streptavidin.

98. The method of claim 79 or 80, wherein the nucleic acid probe comprises a hybridization sequence complementary to a target sequence in the nucleic acid of the biological sample.

99. The method of claim 98, wherein allowing the nucleic acid probe to hybridize to the nucleic acid of the biological sample comprises mixing the lysate and the capture reagent.

100. The method of claim 98, wherein allowing hybridization of the nucleic acid probes to the nucleic acids of the biological sample comprises incubating the lysate and the capture reagents at 63-73 ℃.

101. The method of claim 100, wherein allowing hybridization of the nucleic acid probes to the nucleic acids of the biological sample comprises incubating the lysate and the capture reagents at about 68 ℃.

102. The method of claim 98, wherein the lysate and the capture reagent are mixed and/or incubated for 30 seconds to 5 minutes.

103. The method of claim 102, wherein the lysate and the capture reagent are mixed and/or incubated for 1 to 3 minutes.

104. The method of claim 103, wherein the lysate and the capture reagent are incubated for about 2 minutes.

105. The method of claim 79 or 80, wherein combining the probe-bound nucleic acid with the capture agent-coated magnetic bead comprises mixing the probe-bound nucleic acid with the capture agent-coated magnetic bead.

106. The method of claim 79 or 80, wherein combining the probe-bound nucleic acid with the capture agent-coated magnetic beads comprises incubating the probe-bound nucleic acid and the capture agent-coated magnetic beads at 70 ℃ -80 ℃.

107. The method of claim 106, wherein combining the probe-bound nucleic acid with the capture agent-coated magnetic beads comprises incubating the probe-bound nucleic acid and the capture agent-coated magnetic beads at about 75 ℃.

108. The method of claim 79 or 80, wherein allowing the capture agent to bind to the capture moiety comprises incubating the probe-bound nucleic acid and the capture agent-coated magnetic beads at 63-73 ℃.

109. The method of claim 108, wherein allowing the capture agent to bind to the capture moiety comprises incubating the probe-bound nucleic acid and the capture agent-coated magnetic beads at about 68 ℃.

110. The method of claim 79 or 80, further comprising aspirating the bead-captured nucleic acid suspension prior to exposure to the magnetic field.

111. The method of claim 79 or 80, wherein separating the sorted bead-captured nucleic acids from the liquid portion of the bead-captured nucleic acid suspension comprises holding the magnetic field in place and removing the liquid portion from the bead-captured nucleic acid suspension.

112. The method of claim 79 or 80, further comprising:

(h) Combining the nucleic acid captured by the sorted beads with a wash buffer;

(i) Resuspending the bead-captured nucleic acids in the wash buffer;

(j) Sorting the bead-captured nucleic acids within the wash buffer by exposing a portion of the bead-captured nucleic acids to a magnetic field; and

(k) Separating the nucleic acid captured by the sorted beads from the wash buffer.

113. The method of claim 112, further comprising repeating steps (h) - (k) one or more times.

114. The method of claim 79 or 80, further comprising:

(h) Combining the sorted bead captured nucleic acids with a resuspension buffer; and

(i) The bead-captured nucleic acids are resuspended in the resuspension buffer to produce a bead-captured nucleic acid resuspension.

115. The method of claim 114, the method further comprising:

(j) Combining the bead-captured nucleic acid resuspension with an analytical reagent.

116. The method of claim 112, the method further comprising:

(l) Combining the sorted bead captured nucleic acids with a resuspension buffer; and

(m) resuspending the bead-captured nucleic acid in the resuspension buffer to produce a bead-captured nucleic acid resuspension.

117. The method of claim 116, the method further comprising:

(n) combining the bead-captured nucleic acid resuspension with an analytical reagent.

118. The method of claim 115 or 117, wherein the assay reagent is a dry assay reagent and combining the bead-captured nucleic acid resuspension with the dry assay reagent comprises resuspension of the dry assay reagent in the bead-captured nucleic acid resuspension.

119. The method of claim 115 or 117, wherein the analytical reagent comprises a detection reagent.

120. The method of claim 115 or 117, wherein the assay reagents comprise amplification reagents.

121. The method of claim 115 or 117, further comprising amplifying and/or detecting the target nucleic acid hybridized to a bead-bound capture reagent.

122. The method of claim 78 or 80, wherein the wash buffer and the resuspension buffer contain the same components.

123. A method of preparing a target nucleic acid in a biological sample for subsequent analysis, the method comprising:

(a) Combining the biological sample comprising cells with a dry lysis reagent comprising a protease K, SDS and a salt; and resuspending the dried lysis reagent in the biological sample to produce a lysate;

(b) Combining the lysate with a dried capture reagent and resuspending the dried capture reagent in the lysate, wherein the capture reagent comprises a nucleic acid probe tethered to a capture moiety, wherein the capture moiety is biotin, and wherein the nucleic acid probe comprises a hybridization sequence complementary to a target sequence within the nucleic acids of the biological sample;

(c) Incubating the nucleic acid probes and the nucleic acids of the lysate at a temperature of 63 ℃ to 73 ℃ to produce a probe-bound nucleic acid solution;

(d) Combining the probe-bound nucleic acid with dried capture agent coated magnetic beads and resuspending the dried capture agent coated magnetic beads in the probe-bound nucleic acid at 70 ℃ -80 ℃, wherein the capture agent is streptavidin;

(e) Incubating the probe-bound nucleic acids and the capture agent-coated magnetic beads at 63-73 ℃ and allowing the capture agent to bind to the capture moiety to produce a bead-captured nucleic acid suspension;

(f) Sorting bead-captured nucleic acids within the bead-captured nucleic acid suspension by exposing a portion of the bead-captured nucleic acid suspension to a magnetic field, and (a) holding the magnetic field in place while removing a liquid portion from the bead-captured nucleic acid suspension, or (B) moving the magnetic field to drag the bead-captured nucleic acids out of the liquid portion; and

(g) Separating the sorted bead-captured nucleic acids from the liquid portion of the bead-captured nucleic acid suspension;

(h) Combining the nucleic acid captured by the sorted beads with a wash buffer;

(i) Resuspending the bead-captured nucleic acids in the wash buffer;

(k) Separating the nucleic acid captured by the sorted beads from the wash buffer;

(m) resuspending the bead-captured nucleic acids in the resuspension buffer to produce a bead-captured nucleic acid resuspension;

(n) combining the bead-captured nucleic acid resuspension with a dried analytical reagent and resuspending the dried analytical reagent in the bead-captured nucleic acid resuspension, wherein the analytical reagent comprises primers and a detectable label for amplifying and detecting the target nucleic acid; and

(o) amplifying and detecting the target nucleic acid hybridized to the bead-bound capture reagent;

wherein the method does not comprise a centrifugation step, a filtration step or a nucleic acid precipitation step; wherein the nucleic acid is not separated from contaminants within the biological sample in steps (a) through (e); and wherein the wash buffer and the resuspension buffer contain the same components.

124. A method of preparing a target nucleic acid in a biological sample for subsequent analysis, the method comprising:

(a) Combining the biological sample with a dried reagent comprising a lysis/digestion component and a capture component, and allowing the dried reagent to be resuspended in the biological sample, wherein the lysis/digestion component comprises a protease K, SDS and a salt, wherein the capture component comprises a nucleic acid probe tethered to a capture moiety, wherein the capture moiety is biotin, and wherein the nucleic acid probe comprises a hybridization sequence complementary to a target sequence within the nucleic acid of the biological sample;

(b) Incubating a biological sample at a temperature of 90 ℃ to 100 ℃ to allow cell membrane lysis and protein digestion to occur within the sample to produce a lysate;

(c) Incubating the lysate at a temperature of 63-73 ℃ to allow the hybridization sequences of the nucleic acid probes to bind to the target sequences of the nucleic acids of the biological sample to produce a probe-bound nucleic acid solution;

(h) Combining the nucleic acid captured by the sorted beads with a wash buffer;

(i) Resuspending the bead-captured nucleic acids in the wash buffer;

125. The method of one of claims 79-124, wherein the method is performed manually.

126. The method of claim 125, wherein the moving and combining of the liquids is performed by manual pipetting.

127. The method of one of claims 79-124, wherein the method is automated.

128. The method of claim 127, wherein the method steps are performed in a single use cartridge.

129. The method of claim 127, wherein the single-use cartridge comprises all dry and liquid reagents and buffers for performing the method steps of the method.

130. The method of claim 128, wherein the single-use cartridge is engaged with an instrument comprising components for combining and mixing reagents, a heating element, and a magnet.

131. The method of claim 127, wherein the method steps are performed by an automated instrument within one or more tubes or wells.

132. A system or kit, the system or kit comprising:

(a) A lysing agent capable of digesting the cell membrane and degrading cellular proteins;

(b) A capture reagent comprising a nucleic acid probe tethered to a capture moiety;

(c) Magnetic beads coated with capture agents;

(d) Amplification/detection reagents; and

(e) Wash/resuspend buffer solution.

133. A system or kit, the system or kit comprising:

(a) A lysis/capture reagent comprising (1) a lysis/digestion component capable of digesting cell membranes and degrading proteins, and (2) a nucleic acid probe tethered to a capture moiety;

(b) Magnetic beads coated with capture agents;

(c) Amplification/detection reagents; and

(d) Wash/resuspend buffer solution.

134. The system or kit of claim 132 or 133, wherein the reagents are in dry or concentrated liquid form.

135. The system or kit of claim 132 or 133, further comprising a biological sample comprising cells.

136. The system or kit of claim 132 or 133, wherein the lysis reagent or the lysis/digestion component comprises a protease capable of digesting cellular proteins.

137. The system or kit of claim 136, wherein the protease is proteinase K.

138. The system or kit of claim 132 or 133, wherein the lysis reagent or the lysis/digestion component comprises a detergent in an amount sufficient to lyse cells when combined with a biological sample.

139. The system or kit of claim 138, wherein the detergent is SDS.

140. The system or kit of claim 132 or 133, wherein the lysing reagent or the lysing/digestion component comprises one or more salts.

141. The system or kit of claim 132 or 133, wherein the capture moiety is biotin and the capture agent is streptavidin.

142. The system or kit of claim 132 or 133, wherein the nucleic acid probe comprises a hybridization sequence complementary to a target sequence in a target nucleic acid.

143. The system or kit of claim 132 or 133, wherein the dry amplification/detection reagents comprise primers, a detectable label, and nucleotides.

144. A system or kit, the system or kit comprising:

(a) A cleavage reagent comprising a protease K, SDS and one or more salts;

(b) A capture reagent comprising a nucleic acid probe tethered to a biotin capture moiety;

(c) A capture agent-coated magnetic bead, wherein the capture agent is streptavidin;

(d) An amplification/detection reagent comprising a primer, a detectable label, and a nucleotide; and

(e) Buffer solution.

145. A system or kit, the system or kit comprising:

(a) A lysis/capture reagent comprising (1) a lysis/digestion component comprising protease K, SDS and one or more salts, and (2) a nucleic acid probe tethered to a capture moiety;

(b) A capture agent-coated magnetic bead, wherein the capture agent is streptavidin;

(c) An amplification/detection reagent comprising a primer, a detectable label, and a nucleotide; and

(d) Buffer solution.

146. The system or kit of claim 144 or 145, wherein the reagents are in dry or concentrated liquid form.

147. The system or kit of claim 144 or 145, further comprising a biological sample comprising cells.

148. The system or kit of claim 132, 133, 144, or 145 further comprising a disposable laboratory product for manually using the system or kit to capture, sort, amplify, and detect target nucleic acids from a biological sample comprising cells.

149. The system or kit of claim 148, wherein the disposable laboratory product comprises a pipette tip, a reaction tube, and/or a microplate.

150. The system or kit of claim 149, further comprising a single-use cartridge comprising components of (a) - (e), wherein the single-use cartridge is capable of being engaged with an instrument comprising components for combining and mixing reagents, a heating element, and a magnet.