WO2019204510A1

WO2019204510A1 - Programmable microchannel systems for analyte detection

Info

Publication number: WO2019204510A1
Application number: PCT/US2019/027976
Authority: WO
Inventors: Krisna Bhargava; Bryant Thompson; Noah Malmstadt
Original assignee: Reolab
Priority date: 2018-04-17
Filing date: 2019-04-17
Publication date: 2019-10-24

Abstract

Disclosed herein are systems and methods for performing enzymatic immunoassays. Systems disclosed herein are three-dimensional and modular. System hardware is reusable and microscaled, thereby minimizing reagent use, cost and waste. Systems disclosed herein generally are capable of concentrating a reaction signal. Thus, systems are capable of accurately detecting small quantities of analyte. Systems disclosed herein may also provide continuous feedback to the system or a user to manage system contaminants, thereby reducing errors in assay results. Systems and methods disclosed herein provide for detection of an analyte in minutes, thereby making them useful for point of care.

Description

PROGRAMMABLE MICROCHANNEL SYSTEMS FOR ANALYTE DETECTION

CROSS-REFERENCE

[001] This application claims the benefit of US Provisional Application Serial Number

62/659,003 filed April 17, 2018, and is incorporated herein by reference in its entirety.

BACKGROUND

[002] Enzyme immunoassays (EIA), such as an enzyme-linked immunosorbent assay (ELISA), are a gold standard technique in medical diagnostics for detecting and quantifying the presence of biomolecules. In general, an EIA experiment includes processing a biological sample for analysis, followed by optical detection of an analyte of interest. Traditionally, a microtiter plate is sequentially exposed to biochemical reagents, washing solutions and passivating blocking solutions. This process is typically performed by hand with pipettes, a process apparatus such as a plate shaker or centrifuge, and a detection apparatus such as a plate reader or fluorimeter. In some cases, the process is automated.

[003] Traditional EIA is performed on microtiter plates, by hand, robotically, or a combination thereof. For indirect capture style EIA, the wells in the plate may come pre-coated with a capture ligand solution such as an antibody. Alternatively, the EIA is provided in a kit so that the user can bind capture ligand to the well surfaces themselves. For direct capture style EIA, the wells may not come coated. The user then employs a pipette to dispense sample solution, wash solutions, other binding ligand solutions, indicator substrate solution, and substrate stop solution in a sequence in the wells. In between each dispense step, fluids are aspirated or drained from wells. After each dispense step, the plate may be incubated, centrifuged, agitated, or all of the above. Eventually, the plate is put in a plate reader apparatus to generate assay data.

[004] There are various formats for EIA, including a direct format, an indirect format, and a capture format. Typically, the direct format comprises detecting an analyte adsorbed on the surface of a microtiter plate with an antibody conjugate. In some instances, the analyte sticks non-specifically to the plate. The affinity reagent may be covalently bound or nonspecifically bound to the plate. The antibody conjugate comprises an antibody which binds the analyte and an enzyme that can process a substrate to create an optically detectable signal. If the analyte is absent from the sample, and thereby absent from the plate, there is an absence of a detectable signal. The indirect format is similar to the direct format. However, a primary antibody binds the analyte and a secondary antibody conjugated to the enzyme binds the primary antibody to process the substrate. In some instances, the analyte binds to an immobilized affinity reagent coating on the plate, referred to as a capture assay or capture format. The capture format, also referred to as the“sandwich format,” comprises adding an analyte to the microtiter plate, wherein the microtiter plate is pre-coated with a first capture antibody to the analyte. The analyte is then subsequently contacted with a second detection antibody conjugated to the enzyme. Optionally, the analyte is contacted with a second primary antibody and a secondary antibody conjugated to the enzyme.

[005] An additional format for EIA is competitive EIA. Competitive EIA generally involves mixing a primary affinity reagent ( e.g ., an antibody to an analyte of interest) with a sample potentially containing the analyte. The resulting antibody-analyte complex is then introduced to in a solution to a plate coated with purified, immobilized analyte. After incubation of the complex on the plate, the solution is washed away. An enzyme-conjugated secondary affinity reagent that binds the primary affinity reagent is then introduced to the plate. An indicator substrate is added to produce a detectable signal. If there is a low amount of analyte in the sample, there is an abundance of free primary affinity reagent in the solution that binds the purified, immobilized analyte, resulting in greater binding of the enzyme-conjugated secondary affinity reagent and a greater detectable signal, as compared to if there had been a high amount of analyte.

SUMMARY

[006] Clinicians often depend on high-resolution quantitative biomarker data to effectively manage many chronic and acute conditions. These data are typically generated in hospitals or central diagnostic laboratories from blood, sputum, urine, or other fluid biological specimens, which can take days to weeks. The biomarkers themselves can be proteins, DNA, RNA, or other biomolecules. This long period of time can cause clinicians to over- or undermanage patients by depending on empiric treatment models or by responding to nonclinical pressures to treat.

Quantitative enzyme immunoassays (EIA, ELISA) make up a significant portion of the tests executed in hospital and central labs. Traditional ELISA is highly sensitive, but complex. It is too slow for point-of-care use, requires many reagents, is sensitive to operational errors and is difficult to optimize. Methods, systems and kits disclosed herein are designed to make a quantitative EIA available at the point-of-care by returning data within minutes affordably and without need of a specialized laboratory environment.

[007] Scientists often rely on quantitative EIA data for fundamental molecular biology research, biopharmaceuticals development, clinical trials, epidemiological research, environmental screening, biodefense screening, food safety, and other non-clinical applications. Traditionally, scientists or technicians must execute these tests by hand or with robotic tools in a laboratory environment. Scientists therefore face a variety of challenges, including long R&D cycles, significant room for process errors, and a need to customize many assay parameters. Methods, systems and kits disclosed herein are designed to automate execution of any quantitative EIA quickly and in a closed system, reducing unexpected imprecisions in resulting data, and enabling faster R&D progress.

[008] In general, methods and systems disclosed herein utilize a continuous flow process. EIA is executed on immobilized beads, but process solutions may be cycled over these beads in flow. This may be referred to as active mixing or active bead homogenization, which is described in detail herein and throughout. This has the effect of eliminating the need for extensive incubation times by removing substantial mixing-related limitations from EIA reaction kinetics. In a microfluidic system, such as those disclosed herein, this may be referred to as“micromixing.” Micromixing advantageously uses substantially less fluid ( e.g ., process solutions), reagents, and overall biomaterial than traditional EIA, thereby reducing cost and waste.

[009] Methods and systems disclosed herein utilize a closed environment. Traditional EIA is executed such that reaction media (e.g., process solutions) are open to the environment, and therefore at risk of contamination. In addition, traditional EIA relies on the use of many different apparatus, such as centrifuges, agitators, pipettes, plate washers, magnetic bead separation racks, and plate readers. Such contamination can reduce or completely corrupt data quality. In contrast, methods and systems disclosed herein eliminate these risks by handling fluids in closed chambers and microchannels. Systems disclosed herein completely integrate all fluid sensing and actuation, and control functions needed to execute EIA from sample to answer in one system.

[010] Solid components of systems disclosed herein are reusable. Traditional microtiter plate- based processing depends on specialized pre-coated disposable plastic-wear, and a substantial number of disposable plastics, such as microtiter plates, pipette tips, and vials. Systems and methods disclosed herein eliminate this waste by utilizing microbeads that can be cleaned out of the system, leaving behind a system that can be re-used. In some instances, systems and methods disclosed herein require no disposables other than vials containing fluids. In addition, systems disclosed herein may be described as self-cleaning. Systems disclosed herein comprise a network of chambers and channels instead of a microtiter plate. This network may be sterilized after an assay and thus, the network may be used repeatedly for multiple assays.

[Oil] Methods and systems disclosed herein eliminate errors generated in traditional EIA. Traditional EIA depends on pipette- or syringe-based dispensing and aspiration of fluids. These techniques are intrinsically prone to significant volumetric error, especially when dealing with volumes in the tens to hundreds of microliters. In contrast, systems and methods disclosed herein utilize controlled pumps to cycle reagents over reaction surfaces (i.e. immobilized microbeads).

[012] Traditional microtiter plate-based processing is prone to errors due to inhomogeneous or poorly controlled adsorption or binding of capture ligands to well surfaces. This so called“coffee ring effect” results in unintended binding mechanics and large errors in resulting assay data. Systems and methods disclosed herein eliminate these errors by utilizing microbeads that may be pre-coated in dispersion, guaranteeing better homogeneity of capture ligand on binding surfaces. Systems and methods disclosed herein further eliminate error by tightly controlling the number of microbeads immobilized in the reactor microchannel from run to run.

[013] Unlike traditional EIA, systems and methods disclosed herein may utilize multiplexing to simultaneously measure multiple biomarkers. Systems disclosed herein may be configured to measure multiple biomarkers by running key microfluidic reactor components in parallel.

Consider the system intended to measure a single biomarker in FIG. 1. It may consist of fluids stored in vials or similar containers, a fluid logic unit, a microfluidic reactor, a detector, a waste management valve system, a pump, and a digital control unit. Multiple biomarkers may be measured individually or in parallel by adding new microfluidic reactors between the fluid logic unit and the pump, as seen in FIG. 2.

[014] Traditional EIA depends on the use of a so-called stop solution that stops the continued enzymatic processing of an indicator substrate. Often, stop solutions comprise an acid.

Inaccuracies in timing in the introduction of the stop solution can contribute to errors in assay data. In contrast to traditional EIA, systems and methods disclosed herein generally do not use a stop solution. Instead, systems and methods disclosed herein control the extent of indication via flowrate of an indicator substrate.

[015] Disclosed herein, in some aspects, are methods of performing an assay for a target analyte in a sample, comprising: flowing a first solution of particles into a microfluidic system, wherein the particles are magnetic particles; contacting the microfluidic system at a first external surface of the microfluidic system with at least one magnet to exert a magnetic force on the magnetic particles, thereby drawing the magnetic particles to a first internal surface of the microfluidic system; flowing a second solution comprising at least one of a reagent and a first target analyte into the microfluidic system; withdrawing the magnetic force; and contacting the microfluidic system at a second external surface of the microfluidic system with the at least one magnet to exert the magnetic force on the magnetic particles, thereby drawing the magnetic particles to a second internal surface of the microfluidic system, wherein the magnetic particles are dispersed in the second solution during movement from the first internal surface to the second internal surface, thereby exposing the magnetic particles surfaces to at least one of the reagent and the first target analyte. In some instances, methods comprise repeating contacting the microfluidic system at a first external surface of the microfluidic system with at least one magnet to exert a magnetic force on the magnetic particles; and contacting the microfluidic system at a second external surface of the microfluidic system with the at least one magnet to exert the magnetic force on the magnetic particles, thereby repeatedly exposing magnetic particle surfaces to at least one of the reagent and the first target analyte. In some instances, the second solution comprises the first target analyte. In some instances, methods comprise detecting the first target analyte. In some instances, methods comprise washing the microfluidic system to remove essentially all of the first target analyte. In some instances, methods comprise contacting the microfluidic system with a second target analyte and detecting the second target analyte. In some instances, the reagent is an antibody that binds the first target analyte. In some instances, the magnetic particles are coated with an antibody that binds the first target analyte. In some instances, methods comprise contacting the microfluidic system with a first magnet at a first external surface proximal the first internal surface and contacting the microfluidic system with a second magnet at a second external surface proximal the second internal surface. In some instances, methods comprise physically moving the at least one magnet near and away from the microfluidic system to exert temporary magnetic forces on the magnetic particles. In some instances, the at least one magnet is an electromagnet, comprising powering the electromagnet to exert a magnetic force on the magnetic particles. In some instances, methods comprise modulating at least one of (a) the magnetic force of the at least one magnet and (b) flow rate of the second solution, thereby controlling the amount of magnetic beads that are drawn to at least one of the first internal surface or second internal surface.

[016] Further disclosed herein, in some aspects, are methods of performing an assay for a target analyte in a sample, comprising: flowing a first solution of particles into a microfluidic system, wherein the particles are dielectric particles; contacting the microfluidic system with at least one electrode to draw the dielectric particles to a first internal surface of the microfluidic system; flowing a second solution comprising at least one of a reagent and a first target analyte into the microfluidic system; and contacting the microfluidic system with the at least one electrode to draw the dielectric particles to a second internal surface of the microfluidic system, wherein the dielectric particles are dispersed in the second solution thereby exposing dielectric particles surfaces to at least one of the reagent and the first target analyte.

[017] Disclosed herein, in some aspects, are methods of performing an assay for a target analyte in a sample, comprising flowing a solution of particles into a microfluidic system in a first direction, wherein the particles are captured by a first frit; introducing at least one of a reagent and a target analyte to the microfluidic system; flowing the solution of particles in a second direction away from the first frit and towards a second frit, thereby exposing the particles to at least one of the reagent and the target analyte; and detecting the target analyte. In some instances, methods comprise capturing the particles on the second frit before detecting. In some instances, methods comprise repeating flowing a solution of particles into a microfluidic system in a first direction, wherein the particles are captured by a first frit; and flowing the solution of particles in a second direction away from the first frit and towards a second frit, thereby repeatedly exposing particle surfaces to at least one of the reagent and the target analyte. In some instances, methods comprise monitoring for an assay contaminant during flowing or contacting.

[018] Further disclosed herein, in some aspects, are methods of performing an assay for a target analyte in a sample, comprising: contacting a microfluidic system with a first sample solution containing a first target analyte, wherein the first target analyte binds a first set of particles in the microfluidic system; detecting the first target analyte; washing the microfluidic system to remove essentially all of the first target analyte, and optionally all of the particles; contacting the microfluidic system with a second sample solution containing a second target analyte, wherein the first target analyte binds a second set of particles in the microfluidic system; and detecting the second target analyte. In some instances, methods comprise flowing particles into the

microfluidic system thereby exposing particles surfaces to the target analyte, wherein the particles are magnetic particles; contacting the microfluidic system with at least one magnet to draw the magnetic particles to a first internal surface of the microfluidic system; and contacting the microfluidic system with the at least one magnet to draw the magnetic particles to a second internal surface of the microfluidic system. In some instances, methods comprise repeating contacting the microfluidic system with at least one magnet to draw the magnetic particles to the first internal surface of the microfluidic system; and contacting the microfluidic system with the at least one magnet, thereby repeatedly exposing magnetic particle surfaces to the first target analyte. In some instances, the assay is an enzyme-linked immunosorbent assay. In some instances, methods comprise coating the particles with a primary antibody that binds the (first or second) target analyte. In some instances, methods comprise coating the particles with a purified form of the (first or second) target analyte. In some instances, methods comprise binding the (first or second) target analyte to the particles. In some instances, methods comprise binding the (first or second) target analyte with a primary antibody, wherein the primary antibody is conjugated to an enzyme. In some instances, methods comprise binding the target analyte with a primary antibody and a secondary antibody, wherein the secondary antibody is conjugated to an enzyme. In some instances, methods comprise binding the target analyte with a primary antibody, wherein the primary antibody is conjugated to a detectable signal. In some instances, methods comprise binding the target analyte with a primary antibody and a secondary antibody, wherein the secondary antibody is conjugated to a detectable signal. In some instances, methods comprise binding the target analyte with a binding moiety comprising a target analyte substrate, peptide, or small molecule, or combination thereof, wherein the binding moiety comprises a detectable signal. In some instances, the detectable signal is fluorescent, luminescent, chemiluminescent, or colorimetric. In some instances, methods comprise operating the microfluidic system to detect a plurality of target analytes simultaneously. In some instances, the method is performed in less than about 5min. In some instances, the method is performed in about 1 min to about 60 min. In some instances, methods comprise detecting a concentration of target analyte as low as 1 pg/ml of target analyte. In some instances, methods comprise flowing a blocking buffer through the system before flowing a solution comprising a target analyte, a particle, or a reagent through the system.

[019] In some instances, methods disclosed herein comprise introducing a first set of particles into the microfluidic system; contacting the microfluidic system at the first external surface of the microfluidic system with the at least one particle manipulator to exert a force on the first set of particles, thereby drawing the first set of particles to the first internal surface of the microfluidic system; flowing a first control solution comprising purified target analyte at a first concentration into the microfluidic system; withdrawing the force; and contacting the microfluidic system at the second external surface of the microfluidic system with the at least one particle manipulator to exert the force on the first set of particles, thereby drawing the first set of particles to a second internal surface of the microfluidic system, wherein the first set of particles are dispersed in the first control solution during movement from the first internal surface to the second internal surface. In some instances, methods comprise repeating contacting the microfluidic system at the first external surface of the microfluidic system with the at least one particle manipulator, withdrawing the force, and contacting the microfluidic system at the second external surface of the microfluidic system with the at least one particle manipulator at least once, thereby exposing the first set of particles to the first control solution repeatedly. In some instances, methods comprise introducing a second set of particles into the microfluidic system; contacting the microfluidic system at the first external surface of the microfluidic system with the at least one particle manipulator to exert a force on the second set of particles, thereby drawing the second set of particles to the first internal surface of the microfluidic system; flowing a second control solution comprising purified target analyte at a second concentration into the microfluidic system; withdrawing the force; and contacting the microfluidic system at the second external surface of the microfluidic system with the at least one particle manipulator to exert the force on the second set of particles, thereby drawing the second set of particles to a second internal surface of the microfluidic system, wherein the second set of particles are dispersed in the second control solution during movement from the first internal surface to the second internal surface. In some instances, methods comprise repeating contacting the microfluidic system at the first external surface of the microfluidic system with the at least one particle manipulator,

withdrawing the force, and contacting the microfluidic system at the second external surface of the microfluidic system with the at least one particle manipulator at least once, thereby exposing the second set of particles to the second control solution repeatedly. In some instances, the first concentration and the second concentration are different. In some instances, methods comprise detecting the purified analyte via a detectable signal, and preparing a binding curve of the detectable signal versus the first and second concentrations. In some instances, methods comprise detecting the purified analyte via the detectable signal comprises binding the purified analyte with a binding moiety that is conjugated to an indicator substrate or enzyme that processes an indicator substrate.

[020] Disclosed herein, in some aspects, are microfluidic systems for performing an assay for a target analyte in a sample, the microfluidic system comprising: a reactor for binding a target analyte to particles, the reactor comprising at least one trap or mixer for homogenizing the surface of particles with the target analyte; at least one particle manipulator configured to move the particles from a first internal surface to a second internal surface of the reactor in a solution comprising the target analyte, thereby homogenizing the surface of particles with the target analyte; a flow regulator that draws a solution containing the target analyte through the system; and at least one of a computer, microcontroller, and detector for detecting the target analyte. In some instances, the at least one particle manipulator is a magnet and the particles are magnetic.

In some instances, the at least one particle manipulator is an electrode and the particles are dielectric or partially dielectric. In some instances, the at least one particle manipulator comprises the first pump, first valve, first pressure regulator, a second pump, second valve, second pressure regulator or combination thereof, and the reactor comprises the at least one trap, wherein the at least one trap comprises a frit. In some instances, at least one component of the system is 3D printed. In some instances, the at least one component is the at least one trap. In some instances, the entire system is 3D printed as one unit. In some instances, the at least one trap or mixer comprises an internal surface, wherein the internal surface comprises a material that is at least one of a plastic, a metal, and a ceramic. In some instances, the internal surface comprises a material comprises polynitrile butadiene styrene, polycarbonate, polystyrene, polyethylene, metal, glass, or silica. In some instances, the system is not produced with soft lithography. In some instances, the microfluidic system comprises at least one of the computer and

microcontroller, and the microfluidic system function is automated by at least one of the computer and microcontroller. In some instances, the flow regulator is selected from a pump, a valve, and a constant pressure regulator. In some instances, the microfluidic system comprises a fluid logic that draws a solution from a reservoir and delivers it to at least one of the flow regulator and reactor. In some instances, all components of the microfluidic system are integrated into a single device. In some instances, the single device is encompassed by a single housing unit. In some instances, the system self-sterilizes after performing the assay. In some instances, the system comprises a detector, wherein the detector comprises an optical fiber. In some instances, the microfluidic system is compatible with an optical fiber based detector. In some instances, the microfluidic system is compatible with a mass spectroscopy system, a thermal sensor, or a non-fiber based optical system. In some instances, the system comprises the particles. In some instances, the microfluidic system comprises a binding moiety that binds the target analyte, wherein the binding moiety comprise a primary antibody, a target analyte substrate, a small molecule that binds to the target analyte, a peptide that binds to the target analyte, or a combination thereof. In some instances, the system comprises the particles, wherein the particles are pre-coated with the binding moiety. In some instances, the binding moiety is provided in a solution. In some instances, the particles or binding moiety are provided in a kit. In some instances, the kit comprises a first solution in a first container and a second solution in a second container. In some instances, the microfluidic system comprises a fluid logic, wherein the fluid logic is compatible with the kit such that the fluid logic is capable of drawing up the first solution and the second solution and delivering them to the reactor. In some instances, the microfluidic system comprises the computer or microcontroller, wherein the computer or microcontroller controls the fluid logic in an automated manner. In some instances, the computer or

microcontroller controls the fluid logic in order to deliver the first solution to the reactor at a first time and the second solution to the reactor at a second time.

[021] Further disclosed, in some aspects, are reusable microfluidic systems for performing multiple assays for a target analyte, comprising: a reactor for binding a target analyte to particles, the reactor comprising at least one trap or mixer for homogenizing the surface of particles with the target analyte; at least one particle manipulator for moving the particles from a first location to a second location in the reactor in a solution comprising the target analyte, thereby

homogenizing the surface of particles with the target analyte; at least one flow regulator that draws a solution containing the target analyte through the system; and a wash solution for flushing the target analyte out of the system, wherein at least the trap or mixer does not comprise an internal porous surface. In some instances, the at least the trap or mixer is 3D printed. In some instances, the trap or mixer has an internal surface comprising polynitrile butadiene styrene, polycarbonate, polystyrene, polyethylene, metal, glass, or silica. In some instances, the particle manipulator is sufficiently distal from the microfluidic system during flushing the target analyte out of the system, so that the particle manipulator does not exert a force on the particles during flushing. In some instances, reusable microfluidic systems comprise a blocking buffer, wherein the blocking buffer contains molecules that passivate an interior surface of the system. In some instances, the reactor comprises a channel in connection with a detection region, and wherein the channel supports a first direction of flow that is not parallel to a second direction of flow through the detection region. In some instances, reusable microfluidic systems comprise a plurality of reactors, each reactor having a channel. In some instances, reusable microfluidic systems comprise a single detector for detecting a signal in each reactor. In some instances, two or more reactors of the plurality of reactors are characterized by a different volume. In some instances, a first reactor comprises a first trap and a second reactor comprises a second trap, and wherein the volume of the first trap and the volume of the second trap are different. In some instances, a first reactor comprises a first channel and a second reactor comprises a second channel, and wherein the volume of the first channel and the volume of the second channel are different. In some instances, reusable microfluidic systems comprise a single fixture of at least one magnet, wherein the single fixture is capable of manipulating magnetic particles in the plurality of reactors simultaneously, thereby providing consistent active bead homogenization between channels. In some instances, the assay for a target analyte is an immunosorbent assay and the particles are a substrate for antibody binding of the target analyte. In some instances, the system self-monitors for a contaminant of the system.

BRIEF DESCRIPTION OF THE DRAWINGS

[022] FIG. 1 shows architecture of an exemplary rapid ELISA reactor network.

[023] FIG. 2 shows exemplary parallelization of an ELISA reactor network.

[024] FIG. 3 shows an exemplary valve array for an ELISA reactor network.

[025] FIG. 4 shows an exemplary ELISA reactor network with a constant pressure source.

[026] FIG. 5 shows a flow diagram that exemplifies real-time feedback process control.

[027] FIG. 6 shows an exemplary reactor with one trap (left-top view cross section, middle-side view cross section, right-top view). [028] FIG. 7 shows an exemplary reactor with three traps (left-front hidden line view, middle- side hidden line view, right-front cross section).

[029] FIG. 8 shows an exemplary reactor with eight traps (left-front hidden line view, middle- side hidden line view, right-front cross section).

[030] FIG. 9 shows several views of an exemplary monopole trap in a reactor.

[031] FIG. 10 shows several views of an exemplary dipole trap in a reactor.

[032] FIG. 11 shows several views of an exemplary series dipole trap in a reactor.

[033] FIG. 12 shows an example of trapping magnetic beads in a trap of a reactor.

[034] FIG. 13 shows exemplary formation of a binding layer of magnetic beads in a trap of a reactor.

[035] FIG. 14 shows a trap of an exemplary reactor before layer formation, as exemplified in

FIG. 13

[036] FIG. 15 shows a frit capture in a reactor of an exemplary system.

[037] FIG. 16 shows a frit capture in a reactor of an exemplary system.

[038] FIG. 17 shows exemplary frits that can be used in systems disclosed herein.

[039] FIG. 18 shows an exemplary dual frit trap.

[040] FIG. 19 shows an example of active bead homogenization.

[041] FIG. 20 shows exemplary active bead homogenization.

[042] FIG. 21 shows an exemplary reactor with a helical mixer for chaotic advection micromixing.

[043] FIG. 22 shows an exemplary system comprising ports for optical fibers.

[044] FIG. 23 shows an exemplary method comprising active bead homogenization. FIG. 23A shows microbeads are immobilized on one side of a trap during reagent solution exchange. FIG. 23B shows microbeads initially engaged during reagent solution exchange are now disengaged. FIG. 23C shows the disengaged beads may now disperse in reagent solution while moving towards another magnet that is engaging the beads. FIG. 23D shows that beads have moved to another side of the trap where they are engaged by a different magnet than the magnet that engaged in FIG. 23A. FIG. 23E shows disengagement of the engaging magnet in FIG. 23D and re-engagement of the initial engaging magnet. FIG. 23F shows the disengaged beads may now disperse in reagent solution while moving towards the initially engaging magnet. FIG. 23G shows the return of beads after dispersion in the reagent solution where all surfaces of the beads were exposed to reagent. FIG. 23H shows a 90 degree rotated view of FIG. 23A along the axis of flow. FIG. 231 shows a reactor with a detection region having an axis oriented in a different direction than that of initial flow through the reactor. FIG. 23J shows a 90 degree rotated view of FIG. 231 along the axis of initial flow. FIG. 23K shows optical fiber detection of an assay signal that has been concentrated by a magnet-based trap.

[045] FIG. 24 shows an exemplary system disclosed herein with electromagnets and detection of signal in a multi-channel system.

[046] FIG. 25 shows exemplary systems with multiple reactors (microchannels), each reactor comprising a trap of various volume. FIG. 25A shows the indicator substrate before it has passed through the detection region. FIG. 25B shows the indicator substrate after it has passed through the detection region.

Certain Terminologies

[0002] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following examples are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms“a,” “an” and“the” include plural referents unless the context clearly dictates otherwise. In this application, the use of“or” means“and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as“include”,“includes,” and“included,” is not limiting.

[0003] As used herein, ranges and amounts can be expressed as“about” a particular value or range. About also includes the exact amount. For example,“about 5 pL” means“about 5 pL” and also“5 pL.” Generally, the term“about” includes an amount that would be expected to be within experimental error. The term“about” includes values that are within 10% less to 10% greater of the value provided. For example,“about 50%” means“between 45% and 55%.” Also, by way of example,“about 30” means“between 27 and 33.”

[0004] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

DETAILED DESCRIPTION OF THE INVENTION

[047] Disclosed herein, in some aspects, are systems and methods for performing an assay to detect an analyte in a sample. The assay may be an enzyme immunoassay (EIA), e.g., an ELISA. In contrast to traditional EIA, systems disclosed herein comprise three dimensional microfluidic networks. Often these networks, or components thereof, are 3D printed. A network disclosed herein may comprise a fluid reservoir, a fluid logic, a pump, a valve, a channel, a trap, a mixer, a detector, a computer/microcontroller, or a combination thereof, for carrying out various steps of EIA, e.g., immobilizing microbeads and mixing solutions. Often the system comprises an optics component for measuring an optically detectable molecule suitable for an EIA.

[048] By way of non-limiting example, consider a system intended to measure a single biomarker in FIG. 1. It comprises fluids stored in vials or similar containers, a fluid logic unit, a microfluidic reactor, a detector, a waste management valve system, a pump, and a digital control unit. In some instances, the system consists essentially of these components. Multiple biomarkers may be measured individually or in parallel by adding new microfluidic reactors between the fluid logic unit and the pump, as seen in FIG. 2. Fluids are sequentially drawn through the reactor by means of the pump. The fluid logic unit selects a fluid to flow through the reactor at any given time. Typically, the first fluid to be selected is a blocking buffer, which contains molecules that passivate the interior surfaces of the fluidic channels. This passivation coating reduces or eliminates unintended nonspecific binding of ligands that may be flowed later in the process. Next, a solution of microbeads is flowed into the reactor and immobilized in the trap subcomponent. The resulting immobilized layer acts as a solid surface on which EIA chemistries can occur. Subsequent fluids may comprise a washing buffer, a binding ligand, or an indicator substrate. In this manner, any known format of EIA can be executed in the reactor based on the selection of inlet fluids and procedural operation of the fluid logic unit. Optical measurements may be taken throughout these flows, enabling the constant monitoring of the process. Data from these optical measurements may be collected or reported in real-time or after the procedure is complete. Data may be sent directly to a database, local area network or cloud. At the end of the EIA procedure, a trap may be disengaged, re-mobilizing the microbeads. All interior surfaces of the fluidic circuit may be washed by a variety of fluids. Wash fluids may also selected by the fluid logic unit.

I. Methods

[049] Disclosed herein, in some aspects, are methods of performing an assay for a target analyte in a sample. The assay may be an immunosorbent assay, involving an antibody to the target analyte, e.g., an ELISA assay. Methods may comprise flowing a first solution of particles into a microfluidic system; contacting the microfluidic system at a first external surface of the microfluidic system with at least one particle manipulator to exert a force on the particles, thereby drawing the particles to a first internal surface of the microfluidic system; flowing a second solution comprising at least one of a reagent and a first target analyte into the microfluidic system; withdrawing the force; and contacting the microfluidic system at a second external surface of the microfluidic system with the at least one particle manipulator to exert the force on the particles, thereby drawing the particles to a second internal surface of the microfluidic system, wherein the particles are dispersed in the second solution during movement from the first internal surface to the second internal surface, thereby exposing the particles surfaces to at least one of the reagent and the first target analyte.

[050] Methods may comprise providing input to a processor ( e.g ., computer, microcontroller) used to actuate and control a system disclosed herein. Non-limiting examples of input are a solution volume, sample volume, a particle type, a particle manipulator characteristic, a particle amount, a solution flow rate, and an assay type (e.g., direct ELISA).

[051] Methods may comprise flowing a solution of particles into a microfluidic system.

Methods may comprise drawing particles to an internal surface to create a layer of particles captured against the internal surface with a particle manipulator (e.g., magnet, electrode, pressure regulator). The layer may have a high surface area-to-volume ratio. In some instances, the surface area-to-volume ratio is about 10^L2 to 10^L9. In some instances, the surface area-to-volume ratio is about 10^L3 to 10^L9. In some instances, the surface area-to-volume ratio is about 10^L4 to 10^L9. In some instances, the surface area-to-volume ratio is about 10^L2 to 10^L12. In some instances, the surface area-to-volume ratio is about 10^L2 to 10^L15.

[052] Once particles have been introduced to the system and drawn to an internal surface to create a layer of particles captured against the first internal surface, methods may comprise flowing a second solution into the microfluidic system. The second solution may comprise a target analyte. The second solution may comprise a binding moiety that binds the target analyte. Non-limiting examples of binding moieties are small molecules, antibodies, substrates, nucleic acids, and binding peptides/proteins. The second solution may comprise a primary antibody. The second solution may comprise a secondary antibody. In some instances, methods do not comprise flowing a second solution into the system. In some instances, the reagent and/or analyte are present in the system before introducing the particles. In some instances, particles are present in the system before introducing the reagent and/or analyte. Particles may be drawn to internal surfaces or otherwise captured within the system as needed to introduce additional solutions.

[053] In some instances, the particles are magnetic particles. Methods may comprise contacting the microfluidic system at a first external surface of the microfluidic system with at least one magnet to exert a magnetic force on the magnetic particles, thereby drawing the magnetic particles to a first internal surface of the microfluidic system. In general, methods comprise maintaining magnet position during flow and moving magnets when flow is stopped. In this way, magnetic particles may be exposed to reagent while being maintained in a given location.

Another way to put this is that magnetic particles are not flowed to downstream regions (such as the detector) until they are sufficiently coated with reagents and/or analytes as desired. Once magnetic particles have been introduced to the system and drawn to a first internal surface to create a layer of magnetic particles captured against the first internal surface, methods may comprise flowing a second solution into the microfluidic system. Similar to the description above, the second solution may comprise a target analyte, a binding moiety that binds the target analyte, or a combination thereof. In some instances, methods do not comprise flowing a second solution into the system. In some instances, the reagent and/or analyte are present in the system before introducing the magnetic particles. In some instances, the magnetic particles are present in the system before introducing the reagent and/or analyte. Once magnetic particles are present within the system with a reagent and/or analyte, magnetic forces may be withdrawn from and applied to the external surfaces of the system in order to move the magnetic particles around in a solution of the reagent and/or analyte, thereby exposing the magnetic particles surfaces to at least one of the reagent and the first target analyte.

[054] In some instances, the particles are dielectric particles. Methods may comprise contacting the microfluidic system with at least one electrode to draw the dielectric particles to a first internal surface of the microfluidic system. Methods may comprise flowing a second solution comprising at least one of a reagent and a target analyte into the microfluidic system. Methods may comprise contacting the microfluidic system with the at least one electrode to draw the dielectric particles to a second internal surface of the microfluidic system, wherein the dielectric particles are dispersed in the second solution thereby exposing dielectric particles surfaces to at least one of the reagent and the target analyte.

[055] In some instances, the particles are inert. In some instances, the particles are not magnetic. In some instances, the particles are not dielectric. In some instances, methods comprise capturing the particles on a textured surface. The textured surface may be sufficiently porous to allow a solution or fluid to pass through, but not sufficiently porous to allow a particle used with the system to pass through it. By way of non-limiting example, the textured surface may be a filter or frit. Methods may comprise flowing a solution of particles into a microfluidic system in a first direction, wherein the particles are captured by a first filter or frit. Methods may comprise flowing the solution of particles in a second direction away from the first frit or filter and towards a second frit or filter, thereby exposing the particles to at least one of a reagent and target analyte in a solution present in the microfluidics system. Various forces may be applied in these methods to flow particles from one frit or filter to another as many times as desired, including pumps, valves, and other pressure regulators.

[056] As described in the immediately foregoing paragraphs, methods disclosed herein generally comprise applying and withdrawing forces to microfluidic systems to move particles around within the systems. Methods may comprise applying and withdrawing a force at least once. Methods may comprise applying and withdrawing a force at least twice. Methods may comprise applying and withdrawing a force at least three times. Methods may comprise applying and withdrawing a force at least about 10 times. Methods may comprise applying and withdrawing a force at least about 20 times. Applying and withdrawing forces to beads is generally referred to herein as particle homogenization, also referred to as bead homogenization or active bead homogenization. As described, forces may be provided by magnets, electrodes, and flow/pressure-regulating devices, ( e.g ., pumps, valves, pressure regulators). By way of non limiting example, homogenization may be described as tumbling the particles in solution, mixing the particles in solution, or moving the particles in solution.

[057] Particle homogenization may also be described as moving particles around in solution containing a target analyte or reagent in an effort to coat the particles to a similar extent with target analyte and/or reagent. Homogenization is a particularly advantageous aspect to methods and systems disclosed herein because it allows for maximizing particle surface area and consistency between reactions, which is especially useful for detecting/quantifying target analytes present in low amounts/concentrations in test samples.

[058] Methods may comprise applying and withdrawing a force repeatedly, thereby repeatedly exposing particle surfaces to at least one of the reagent and the first target analyte. In some instances, methods comprise exposing at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of total particle surfaces to the reagent and/or target analyte. In some instances, methods comprise exposing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the total surface area of at least about 50% of the particles to the reagent and/or target analyte. In some instances, methods comprise exposing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the total surface area of at least about 70% of the particles to the reagent and/or target analyte. In some instances, methods comprise exposing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the total surface area of at least about 90% of the particles to the reagent and/or target analyte. In some instances, methods comprise exposing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% of the total surface area of each particle to the reagent and/or target analyte. [059] Due to homogenization provided by methods and systems disclosed herein, binding of target analytes and reagents to particles and to each other may also reach equilibrium faster than that of previously known systems. In some instances, binding of target analytes and reagents to particles and to each other may reach near equilibrium to provide a useful result. This means that systems and methods disclosed herein may require less reagent/sample and provide a faster time to test results than previously known methods and devices. For instance, methods and systems disclosed herein may provide for detection of a target analyte in about 1 minute to about 60 minutes from application of the target analyte to the system. Methods and systems disclosed herein may provide for detection of a target analyte in about 1 minute to about 40 minutes from application of the target analyte to the system. Methods and systems disclosed herein may provide for detection of a target analyte in about 1 minute to about 20 minutes from application of the target analyte to the system. Methods and systems disclosed herein may provide for detection of a target analyte in less than about 1 minute, less than about 5 minutes, less than about 10 minutes, less than about 20 minutes, or less than about 30 minutes from application of the target analyte to the system. Methods and systems disclosed herein may provide for detection of a target analyte in less than 5 minutes from application of the target analyte to the system. These short time frames make systems and methods disclosed herein particularly useful for point of care and patients at high risk (infants, elderly).

[060] Methods may comprise homogenizing, (moving, tumbling, or mixing) particles in a microfluidic system in the presence of a solution described herein. In some instances, methods comprise homogenizing particles in the presence of a sample solution, comprising a target analyte. In some instances, methods comprise homogenizing particles in the presence of a reagent or reagent solution. The reagent may comprise a primary antibody, secondary antibody, indicator substrate, or a combination thereof. In some instances, methods comprise homogenizing particles in the presence of a target analyte, washing the particles and homogenizing particles in the presence of a binding moiety that binds to the target analyte. The binding moiety may comprise a detectable signal. In some instances, methods comprise homogenizing particles in the presence of a target analyte wherein the target analyte coats the particles, washing the particles, and homogenizing particles in the presence of an antibody to the target analyte. In some instances, methods comprise homogenizing particles in the presence of an antibody to a target analyte wherein the antibody coats the particles, washing the particles, and homogenizing particles in the presence of the target analyte. In some instances, methods comprise

homogenizing the particles in a wash solution. In some instances, methods comprise

homogenizing the particles in a sterilization solution. For instance, methods may comprise reusing a system disclosed herein, and previously used particles must be cleared from the system. In this case, it may be useful to use forces to remove particles from an internal surface ( e.g ., a trap) as a sterilization solution is being flowed through the system. Removing essentially all particles, with any accompanying analytes and reagents, from the system before re-use is key to a reliable reusable system where such contaminants from previous tests would interfere with future tests if not sufficiently cleared. Reusable systems are described in greater detail herein.

[061] Methods disclosed herein may comprise applying and withdrawing a force to obtain desirable homogenization of beads. In some instances, the force is a magnetophoretic force. A magnetophoretic force may be applied by physically moving a magnet towards the system.

Alternatively, or additionally, the magnetophoretic force may be applied by providing power to an electromagnet, and withdrawing the magnetophoretic force by turning the electromagnet off.

In some instances, methods comprise inverting the magnetophoretic force and repelling particles. In some instances, the force is a di electrophoretic force. In some instances, methods comprise switching the dielectrophoretic force polarity, thereby repelling particles when they are initially attracted to an electrode, and vice versa. This process may be automated. For instance, a force may be applied and withdrawn at a constant rate. A magnetic force may be applied at specified locations and distances relative to the system in an automated manner. Automation generally allows for experimental control, thereby providing precise and reliable results. For example, methods may comprise withdrawing a magnetic force from the first external surface and contacting the microfluidic system at a second external surface of the microfluidic system with the at least one magnet to exert the magnetic force on the magnetic particles, thereby drawing the magnetic particles to a second internal surface of the microfluidic system, wherein the magnetic particles are dispersed in the second solution during movement from the first internal surface to the second internal surface, thereby exposing the magnetic particles surfaces to at least one of the reagent and the first target analyte. This could alternatively be performed in a similar manner with dielectric particles and an electrode, or inert particles, a frit, and a syringe pump.

[062] Methods disclosed herein may comprise applying and withdrawing a force repeatedly at a given frequency. In some instances, the force is applied at a frequency of less than 1 Hz. In some instances, the force is applied at about 1 Hz to about 100 Hz. In some instances, the force is applied at about 1 Hz to about 500 Hz. In some instances, the force is applied at about 500 Hz to about 1 kHz. In some instances, the force is applied at about 1 Hz to about 1 kHz. In some instances the force is applied at greater than about 1 kHz. In some instances, the force is applied at about 1 kHz to about 100 kHz. In some instances, the force is applied at about 100 kHz to about 500 kHz. In some instances, the force is applied at about 500 kHz to about 1000 kHz (1 MHz). In some instances, the force is applied at greater than about 1 MHz. In some instances, the force is applied at about 1 MHz to about 100 MHz. In some instances, the force is applied at about 100 MHz to about 500 MHz.

[063] Methods disclosed herein generally comprise contacting microfluidic systems with at least one source of force ( e.g ., magnet, electrode, pump, or combination thereof). In some instances, a microfluidic system comprises more than one reactor, wherein multiple sources are required. In some instances, methods comprise contacting the microfluidic system with at least two sources. In some instances, methods comprise contacting the microfluidic system with at least two, at least three, at least four, at least five, or at least six sources. In some instances, multiple sources are integrated. By way of non-limiting example, a system may have three reactors, wherein each reactor is subjected to a set of two magnets, resulting in a system with six magnets. The six magnets may be integrated on a single support that can be manipulated by a user or automated controller to move all six magnets in a similar direction and distance simultaneously, effectively treating all reactors and contained particles, consistently. In some instances, the magnets are integrated electromagnets and are powered on and off simultaneously to treat all reactors and contained particles, consistently.

[064] In some instances, forces may be removed from the system entirely, by either physically removing the source (e.g., taking away a magnet) or turning the source off (e.g., turning off an electromagnet, pump or electrode). Use of one or more electromagnets may eliminate the need for physical movements of a magnet. In some instances, two electromagnets are engaged simultaneously, as exemplified in FIG. 24. In some instances, the two electromagnets are on opposite sides of the channel, as shown in FIG. 24, and have opposite polarity, such that one repels and one attracts. In some instances, the two electromagnets are on opposite sides of the channel and have similar polarity, such that both magnets attract particles, creating a layer of particles on both sides of the channel, as shown in FIG. 24. Use of electromagnets may enable elimination of a lipped recess through magnetic bottling. This is particularly useful when the system needs to be washed or sterilized for re-use. In general, removing a source means that it no longer exerts any forces on particles within the system.

[065] In some instances, methods comprise modulating at least one of a force and a flow rate to draw the particles to an internal surface of a system. The force may be generated by a particle manipulator disclosed herein. In general, the force is strong enough to cause a particle to change its trajectory. The force may be in the pN to nN range. The force may be about 1 pN to about 500 nN. The force may be about 10 pN to about 100 nN. The force may be about 100 pN to about 10 nN. The force may be about 10 pN to about 1 nN. The force may be about 1 nN to about 900 nN. [066] In some instances, methods comprise modulating at least one of a force and a flow rate to control a thickness of a layer of particles on an internal surface of a system. In order for a layer to form, there must be a sufficient concentration of beads in the system. In some instance, the concentration of beads in solution in the system is greater than 10 pg/mL In some instance, the concentration of beads in solution in the system is between about 10 pg/mL and about 1 mg/mL. In some instance, the concentration of beads in solution in the system is between about 50 pg/mL and about 500 pg/mL. In some instance, the concentration of beads in solution in the system is between about 50 pg/mL and about 250 pg/mL. In some instances, enough shear force is created over the layer of particles to maintain a flat or even surface of particle layer exposed to solution. This shear force may be obtained by modulating solution flow rate and force applied to the particles to obtain a suitable combination for maintaining such a layer.

[067] In some instances, methods comprise forming a layer of beads. The layer thickness may depend on channel size and bead size. In some instances, the layer of beads has a thickness of about 1 micron to about 200 microns. In some instances, the layer of beads has a thickness of about 10 microns to about 200 microns. In some instances, the layer of beads has a thickness of about 20 microns to about 200 microns. In some instances, the layer of beads has a thickness of about 50 microns to about 200 microns. In some instances, the layer of beads has a thickness of about 1 micron to about 180 microns. In some instances, the layer of beads has a thickness of about 1 micron to about 160 microns. In some instances, the layer of beads has a thickness of about 1 micron to about 140 microns. In some instances, the layer of beads has a thickness of about 1 micron to about 120 microns. In some instances, the layer of beads has a thickness of about 1 micron to about 100 microns. In some instances, the layer has a thickness that does not exceed about 1/10 the width/diameter of the channel at a location of a trap in the reactor. In some instances, the layer has a thickness between about 1/10,000 and about 1/8 the width/diameter of the channel at a location of a trap in the reactor. In some instances, the layer has a thickness between about 1/1000 and about 1/8 the width/diameter of the channel at a location of a trap in the reactor. In some instances, the layer has a thickness between about 1/100 and about 1/8 the width/diameter of the channel at a location of a trap in the reactor.

[068] In some instance, methods comprise performing ELISA with systems disclosed herein. In some instances, the methods comprise performing a direct format ELISA. Methods comprising performing direct format ELISA may comprise contacting particles with a target analyte ( e.g ., a sample solution). The target analyte may stick non-specifically to the particles. In some instances, the target analyte binds to an immobilized affinity reagent coating on the particles. The affinity reagent may be covalently bound or nonspecifically bound to the particles. Methods may comprise homogenizing the particles with the target analyte. Homogenizing particles is described in greater detail herein and throughout. Methods may comprise contacting the particles (having the target analyte) with an antibody to the target analyte. Methods may comprise homogenizing the particles with the antibody. Often, the antibody is conjugated to an enzyme. The antibody conjugate may comprise an enzyme that can process a substrate to create a detectable signal. Methods generally comprise adding the substrate to the system as well. Methods may comprise homogenizing the particles with the substrate. If the analyte is absent from the sample, and thereby absent from the particles, there is an absence of a detectable signal.

[069] Methods of direct ELISA may comprise drawing a solution of particles into a system disclosed herein and trapping the particles, followed by introducing a solution of target analyte, optionally homogenizing the particles with the target analyte, introducing a solution of an antibody-enzyme conjugate to the target analyte, optionally homogenizing the particles with the antibody-enzyme conjugate, introducing a solution of substrate corresponding to the enzyme, and detecting the substrate. Methods may comprise washing away solution containing unbound analyte and reagents between any of the foregoing steps wherein washing comprises applying a force ( e.g ., magnet) to the particles. Introducing solutions may comprise the use of a fluid logic, a pump, valve or other pressure regulator, computer/microcontroller, or a combination thereof.

Any step may be automated. Methods may comprise sterilizing the system after performing the direct ELISA so that another test may be run with the system, wherein the test is not

contaminated by essentially any analytes, reagents or particles from the direct ELISA. In some instances, the system comprises multiple reactors, and multiple direct ELISAs may be performed simultaneously.

[070] In some instances, the methods comprise performing an indirect format ELISA with a system disclosed herein. The indirect format is similar to the direct format. However, methods comprise contacting the particles (having the target analyte) with a primary antibody, followed by a secondary antibody conjugated to the enzyme. Methods of indirect ELISA may comprise drawing a solution of particles into a system disclosed herein and trapping the particles, followed by introducing a solution of target analyte, optionally homogenizing the particles with the target analyte, introducing a solution of a primary antibody to the target analyte, optionally

homogenizing the particles with the primary antibody, introducing a solution of a secondary antibody-enzyme conjugate to the target analyte, optionally homogenizing the particles with the secondary antibody-enzyme conjugate, introducing a solution of substrate corresponding to the enzyme, and detecting the substrate. Methods may comprise trapping particles and washing away solution containing unbound analyte and reagents between any of the foregoing steps wherein washing comprises applying a force ( e.g ., magnet) to the particles. Introducing solutions may comprise the use of a fluid logic, a pump, valve or other pressure regulator,

computer/microcontroller, or a combination thereof. Any step may be automated. Methods may comprise sterilizing the system after performing the indirect ELISA so that another test may be run with the system, wherein the test is not contaminated by essentially any analytes, reagents or particles from the indirect ELISA. In some instances, the system comprises multiple reactors, and multiple indirect ELISAs may be performed simultaneously.

[071] In some instances, the methods comprise performing a capture format ELISA. The capture format, also referred to as the“sandwich format,” may comprise adding a target analyte to the particles, wherein the particles are pre-coated with a first primary antibody to the analyte. The analyte is then subsequently contacted with a second primary antibody conjugated to the enzyme. Optionally, the target analyte is contacted with a second primary antibody and a secondary antibody conjugated to the enzyme. Methods comprising performing indirect or sandwich ELISA may comprise homogenizing particles at respective steps with the target analyte, primary antibody, secondary antibody, enzyme substrate, or any combination thereof. Methods may comprise washing away solution containing unbound analyte and reagents between any of the foregoing steps wherein washing comprises applying a force (e.g., magnet) to the particles. Introducing solutions may comprise the use of a fluid logic, a pump, valve or other pressure regulator, computer/microcontroller, or a combination thereof. Any step may be automated. Methods may comprise sterilizing the system after performing the capture ELISA so that another test may be run with the system, wherein the test is not contaminated by essentially any analytes, reagents or particles from the capture ELISA. In some instances, the system comprises multiple reactors, and multiple capture ELISAs may be performed simultaneously.

[072] In some instances, the methods comprise performing competitive ELISA with a system disclosed herein. Methods may comprise mixing an enzyme-conjugated purified target analyte and a primary affinity reagent (e.g., an antibody to the analyte) with a sample potentially containing the target analyte. The enzyme-conjugated purified target analyte and the sample analyte, if present, compete to bind a secondary affinity reagent that binds the primary affinity reagent. The solution is then introduced to an immobilized primary affinity reagent coating on particles. The secondary affinity reagent binds this coating. Since there is more free enzyme- conjugated purified analyte when the target is present, there will be increased binding of enzyme- conjugated purified analyte to the particles, and therefore a greater detectable signal from the processed substrate. Methods comprising performing competitive ELISA may comprise homogenizing particles at respective steps with the purified target analyte, sample target analyte, primary antibody, secondary antibody, enzyme substrate, or any combination thereof. Methods may comprise washing away solution containing unbound analyte and reagents between any of the foregoing steps wherein washing comprises applying a force ( e.g ., magnet) to the particles. Introducing solutions may comprise the use of a fluid logic, a pump, valve or other pressure regulator, computer/microcontroller, or a combination thereof. Any step may be automated. Methods may comprise sterilizing the system after performing the competitive ELISA so that another test may be run with the system, wherein the test is not contaminated by essentially any analytes, reagents or particles from the competitive ELISA. In some instances, the system comprises multiple reactors, and multiple competitive ELISAs may be performed

simultaneously.

[073] Methods disclosed herein generally comprise detecting a target analyte through a detectable signal conjugated/bound to a target analyte binding moiety or produced by a target analyte binding moiety. Non-limiting examples of detectable signals are fluorescent molecules (including chemifluorescent molecules), luminescent molecules (including chemiluminescent molecules), and chromogenic molecules. A particularly useful chromogenic molecule is 3, 3', 5,5'- Tetramethylbenzidine (TMB). A particularly useful chemifluorescent molecule is QuantaRed™ because it provides greater sensitivity than many other detectable signals. In some instances, detecting comprises quantifying. In some instances, methods comprise contacting the system with a light source or energy source. Light sources and energy sources are described in greater detail herein and throughout.

[074] In some instances, methods comprise operating a microfluidic system disclosed herein to detect a plurality of target analytes simultaneously. This may be possible with systems that comprise multiple reactors or multiple channels. Non-limiting examples of such reactors are shown in FIGS. 23A-23K and 25A-25B, and also described in Example 7. In some instances, methods comprise subjecting at least two channels to a different average flow velocity, wherein the flow rate is conserved between the two channels, and the channels have different path lengths to a multi-channel, single piston syringe pump (see FIG. 25A). In some instances samples added to different channels have different concentrations of the same analyte, and methods comprise detecting the different concentrations. In some instances, methods comprise delaying transit of a solution or reagent in a channel relative to that of another channel in order to detect a signal in two or more channels individually. This eliminates the need for an optical multiplexer, free-space optics, or an optics positioner/aligner system. Delaying transit may comprise stopping flow. Delaying transit may comprise continuous flow. Delaying transit may comprise initiating flow in two or more channels at different time points. In some instances, delaying transit may comprise utilizing an independent syringe pump for more than one channel. Running multiple samples and controls simultaneously with the same parameters on the same system may provide for unprecedented accuracy in diagnostics, and particularly immunoassays.

[075] Methods generally are performed with reusable systems disclosed herein. Thus, methods disclosed herein may comprise contacting a microfluidic system with a first sample solution containing a first target analyte, wherein the first target analyte binds a first set of particles in the microfluidic system; detecting the first target analyte; washing the microfluidic system to remove essentially all of the first target analyte, and optionally all of the particles; contacting the microfluidic system with a second sample solution containing a second target analyte, wherein the first target analyte binds a second set of particles in the microfluidic system; and detecting the second target analyte. The second target analyte may be the same as the first target analyte. The second target analyte may be different from the first target analyte. Regardless, detection of the second target analyte is not compromised or interfered with by the first target analyte or any reagent from the previous reaction.

[076] In some instances, methods comprise washing the microfluidic system to remove essentially all of a target analyte. In some instances, methods comprise washing the microfluidic system to remove essentially all of a reagent. In some instances, methods comprise washing the microfluidic system to remove essentially all particles. In some instances, washing is sterilizing. In some instances, methods comprise removing at about least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or at least about 99.99% of a target analyte from the system.

In some instances, methods comprise removing all of the target analyte from the system. In some instances, washing is sterilizing. In some instances, methods comprise removing at about least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or at least about 99.99% of a reagent from the system. In some instances, methods comprise removing all of the reagent from the system. In some instances, washing is sterilizing. In some instances, methods comprise removing at about least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9%, or at least about 99.99% of particles from the system. In some instances, methods comprise removing all of the particles from the system. [077] In some instances, methods comprise washing the microfluidic system to passivate the surfaces of the channels -“blocking” them from nonspecific binding of reagents or analytes that may contribute to background signal.

II. Systems

[078] Disclosed herein, in some aspects, are microfluidic systems for performing an assay for a target analyte in a sample. In some instances, the assay is an immunosorbent assay and the particles are a substrate for antibody binding of the target analyte. In some instances, the immunosorbent assay is an ELISA. Immunosorbent assays, and their accompanying reagents, are described herein and throughout, any component of which may be incorporated in a system described herein.

[079] Systems generally comprise a reactor for binding a target analyte to particles. The reactor typically comprises at least one trap or mixer for homogenizing the surface of particles with the target analyte. Systems also often comprise at least one particle manipulator for moving the particles from a first location to a second location in the reactor in a solution comprising the target analyte, thereby homogenizing the surface of particles with the target analyte. Systems may comprise a flow regulator that draws a solution containing the target analyte through the system. In general, systems comprise a detector for detecting the target analyte. In some instances, systems comprise a computer or microcontroller. In some instances, the computer is an embedded system. By way of non-limiting example, the computer may be a BeagleBoard or BeagleBone. The computer or microcontroller may automate the system, may comprise the detector, monitor for a contaminant ( e.g ., air bubble) of the system, or any combination thereof. In some instances, the computer or microcontroller reports data produced by the system to a communication or information network.

[080] In many instances, the particle manipulator is a magnet and the particles are magnetic.

The particle manipulator may be an electromagnet. In some instances, the particle manipulator is an electrode and the particles are dielectric or partially dielectric (e.g., core-shell). In some instances, the particle manipulator is a pump, valve, flow regulator or pressure regulator that can move particles around the inside of the system. In some instances, the particle manipulator is a filter. In some instances, the particle manipulator is a membrane. By way of non-limiting example, a filter or membrane may be used for cross-flow dialysis or field flow fractionation, wherein a first solution is flowed above the filter or membrane, and a second solution is flowed below the filter or membrane in order to separate, place or move particles in a desired direction. [081] Systems disclosed herein may be produced by additive manufacturing, also referred to as 3D printing. In some instances, the entire system is 3D printed as one unit. In some instances, at least one component of the system is 3D printed. In some instances, multiple components of the system are 3D printed and assembled. In some instances, multiple components of the system are 3D printed and sealed together. In some instances, components of the system are 3D printed to be compatible with off-the-shelf components, including an optical fiber, a valve, a fluid logic, etc., all of which are described herein and throughout. In some instances, the system comprises one or more traps and at least the one or more traps is 3D printed. In some instances, the system comprises one or more mixers and at least the one or more mixers is 3D printed. In some instances, the system comprises one or more channels and at least the one or more channels is 3D printed. In some instances the system comprises optical fiber ports that are 3D manufactured.

This allows for very precise alignment of optical fibers.

[082] Generally, systems disclosed herein are made of materials such that reagent, analytes, particles and other solution components do not“stick” to the systems. When forces are applied to the system, particles along with any accompanying analytes or reagents are drawn to internal surfaces of the systems, e.g., the trap of a reactor. However, when the force is removed, particles, analytes and reagents do not remain on the internal surfaces. Instead, they move into solution. This may require forces described herein, agitation, flow, wash solutions, or a combination thereof. This feature makes systems disclosed herein reusable. That is, the system does not become contaminated by previous reaction components. Non-limiting examples of appropriate materials for internal surfaces of systems disclosed herein are polymers such as acrylonitrile butadiene styrene, polycarbonate, polystyrene, and polyethylene. In some instances, internal surfaces of systems disclosed herein comprise a metal. Non-limiting examples of appropriate metals are steel and aluminum. In some instances, the internal surface comprises glass. In some instances, the internal surface comprises fused silica. In some instances, the internal surface comprises quartz. In some instances, the internal surface comprises a photoresin. Non-limiting examples of photoresins are acrylates and epoxies.

[083] Previous microfluidic devices in the field have been produced by soft lithography using elastomeric materials such as polydimethylsiloxane (PDMS). In contrast to the systems disclosed herein, systems produced by soft lithography are porous and foul over time due to these materials. To“foul,” as used herein, is to absorb compounds permanently or semi-permanently in an unintended manner, changing the intrinsic properties of a surface so as to negatively affect the performance of the system or reusability. For example, small molecules in the blood may get into PDMS during a test, and later release back into flow in another test, causing a false result. Also, by way of non-limiting example, proteins adsorbing and absorbing to or in a material can change its optical properties, causing incorrect readouts in the detection step. PDMS is an elastomeric, breathable material, which means gas as can nucleate in channels that cause failures in performance. PDMS is also prone to operational errors due to mechanical stresses. As a result, systems with internal surfaces that foul are not suitable for re-use. In general, systems disclosed herein are not produced by soft lithography. In general, systems disclosed herein do not comprise PDMS. In general, systems disclosed herein are not tape or film based systems either.

[084] Often systems disclosed herein are integrated into a single device. In some instances, the single device is encompassed by a single housing unit. In some instances, the system self- sterilizes after performing an assay. Systems disclosed herein are generally lightweight, e.g., at least less than about fifty pounds. This makes the system amenable to point of care and use outside of labs and clinics.

[085] Systems disclosed herein are configured for performing particle homogenization which allows for maximizing particle surface area, which is especially useful for detecting/quantifying target analytes present in low amounts/concentrations in test samples. Thus, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of less than about 100 pg/ml. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of less than about 10 pg/ml. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of less than about 1 pg/ml. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 pg/L to about 999 pg/L. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 pg/L to about 900 pg/L. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 pg/L to about 500 pg/L. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 pg/L to about 100 pg/L. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 fg/mL to about 1 ug/mL. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 fg/mL to about 10 ug/mL. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 fg/mL to about 100 ug/mL. In some instances, systems disclosed herein may be capable of detecting an analyte in a sample at a concentration of about 1 fg/mL to about 1 mg/mL. [086] In general, systems disclosed herein comprise a processor, a fluid logic unit, a (microfluidic) reactor, a detector, a valve, and a pump. These components are described in greater detail herein, throughout the application, and as follows. Methods disclosed herein are generally performed using these systems. These systems may be provided as multi-part systems of separate components, as kits, and as integrated devices.

Processor

[087] Systems and methods disclosed herein may comprise a processor or a use thereof. The processor may comprise a digital processing device. The processor may comprise a computer. The processor may comprise a digital control unit. In some instances, the processor comprises a controller that controls a pump, a valve, a fluid logic, or a combination thereof. The processor may direct a pump, syringe, valve, or other flow or pressure regulator to move a solution containing a reagent or sample throughout a system disclosed herein. By way of non-limiting example, a processor may provide or receive given flow rates and/or solution volumes, and translate those values to various parts of the system ( e.g ., pumps, syringes) to perform an assay based on those parameters.

[088] The controller may control a system component digitally. The processor may collect data. Data, by way of non-limiting example, may comprise a signal from an indicator substrate described herein. The processor may process data. By way of non-limiting example, processing data may comprise quantifying a signal from an indicator substrate described herein. The processor may store data. The processor may transmit data. For example, data generated by a system disclosed herein may be transferred in real time to, or from, a Laboratory Information Management System (LIMS) or an electronic health or medical record system (EMR/EHR). In some instances, the processor displays data or a conclusion drawn from processed data.

[089] Preferably, devices, systems and kits disclosed herein comprise an information storage unit, e.g., a computer chip. In some instances, the devices, systems and kits disclosed herein comprise means to store data securely. For example, devices, systems and kits disclosed herein may comprise a data chip or a connection (wired or wireless) to a hard drive, server, database or cloud.

[090] In some embodiments, devices, systems, kits, and methods described herein include a digital processing device, or use of the same. In further embodiments, the digital processing device includes one or more hardware central processing units (CPUs) or general purpose graphics processing units (GPGPUs) that carry out the device’s functions. In still further embodiments, the digital processing device further comprises an operating system configured to perform executable instructions. In some embodiments, the digital processing device includes a communication interface ( e.g ., network adapter) for communicating with one or more peripheral devices, one or more distinct digital processing devices, one or more computing systems, one or more computer networks, and/or one or more communications networks.

[091] In some embodiments, the digital processing device is communicatively coupled to a computer network (“network”) with the aid of the communication interface. Suitable networks include, a personal area network (PAN), a local area networks (LAN), a wide area network (WAN), an intranet, an extranet, the Internet (providing access to the World Wide Web) and combinations thereof. The network in some cases is a telecommunication and/or data network. The network, in various cases, includes one or more computer servers, which enable distributed computing, such as cloud computing. The network, in some cases and with the aid of the device, implements a peer-to-peer network, which enables devices coupled to the device to behave as a client or a server.

[092] In accordance with the description herein, suitable digital processing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, and personal digital assistants. Those of skill in the art will recognize that many smartphones are suitable for use in the system described herein. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity are suitable for use in the system described herein. Suitable tablet computers include those with booklet, slate, and convertible configurations, known to those of skill in the art.

[093] In some embodiments, the digital processing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Those of skill in the art will recognize that suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD^®, Linux, Apple^® Mac OS X Server^®, Oracle^® Solaris^®, Windows Server^®, and Novell^® NetWare^®. Those of skill in the art will recognize that suitable personal computer operating systems include, by way of non-limiting examples, Microsoft^® Windows^®, Apple^® Mac OS X^®, UNIX^®, and UNIX- like operating systems such as GNU/Linux^®. In some embodiments, the operating system is provided by cloud computing. Those of skill in the art will also recognize that suitable mobile smart phone operating systems include, by way of non-limiting examples, Nokia^® Symbian^® OS, Apple^® iOS^®, Research In Motion^® BlackBerry OS^®, Google^® Android^®, Microsoft^® Windows Phone^® OS, Microsoft^® Windows Mobile^® OS, Linux^®, and Palm^® WebOS^®. Those of skill in the art will also recognize that suitable media streaming device operating systems include, by way of non-limiting examples, Apple TV^®, Roku^®, Boxee^®, Google TV^®, Google Chromecast^®, Amazon Fire^®, and Samsung^® HomeSync^®. In some instances, the operating system comprises an Internet of Things (IoT) device. Non -limiting examples of an IoT device include Amazon’s Alexa^®, Microsoft’s Cortana^®, Apple Home Pod^®, and Google Speaker^®. In some instances, devices, systems, and kits disclosed herein comprise a virtual reality and/or augmented reality system.

[094] In some embodiments, devices, systems, and kits disclosed herein comprise a storage and/or memory device. The storage and/or memory device is one or more physical apparatuses used to store data or programs on a temporary or permanent basis. In some embodiments, the device is volatile memory and requires power to maintain stored information. In some embodiments, the device is non-volatile memory and retains stored information when the digital processing device is not powered. In further embodiments, the non-volatile memory comprises flash memory. In some embodiments, the non-volatile memory comprises dynamic random- access memory (DRAM). In some embodiments, the non-volatile memory comprises

ferroelectric random access memory (FRAM). In some embodiments, the non-volatile memory comprises phase-change random access memory (PRAM). In other embodiments, the device is a storage device including, by way of non-limiting examples, CD-ROMs, DVDs, flash memory devices, magnetic disk drives, magnetic tapes drives, optical disk drives, and cloud computing based storage. In further embodiments, the storage and/or memory device is a combination of devices such as those disclosed herein.

[095] In some embodiments, the digital processing device includes a display to send visual information to a user. In some embodiments, the display is a liquid crystal display (LCD). In further embodiments, the display is a thin film transistor liquid crystal display (TFT-LCD). In some embodiments, the display is an organic light emitting diode (OLED) display. In various further embodiments, on OLED display is a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display. In some embodiments, the display is a plasma display. In other embodiments, the display is a video projector. In yet other embodiments, the display is a head- mounted display in communication with the digital processing device, such as a VR headset.

[096] In some embodiments, the digital processing device includes an input device to receive information from a user. In some embodiments, the input device is a keyboard. In some embodiments, the input device is a pointing device including, by way of non-limiting examples, a mouse, trackball, track pad, joystick, game controller, or stylus. In some embodiments, the input device is a touch screen or a multi-touch screen. In other embodiments, the input device is a microphone to capture voice or other sound input. In other embodiments, the input device is a video camera or other sensor to capture motion or visual input. In further embodiments, the input device is a Kinect, Leap Motion, or the like. In still further embodiments, the input device is a combination of devices such as those disclosed herein.

[097] In some instances, the devices, systems and kits disclosed herein are capable of communicating data to a communication device. In some instances, devices, systems and kits disclosed herein comprise an interface for receiving information based data obtained from a system disclosed herein. The communication device or interface may allow for sharing data obtained with others not physically present. In some instances the communication device is connected to the internet. In some instances the communication device is not connected to the internet. In some instances, devices, systems and kits disclosed herein are capable of

communicating data through the communication device to the internet. Non-limiting examples of communication devices are cell phones, electronic notepads, and computers.

[098] In some instances, devices, systems and kits disclosed herein comprise a communication connection or a communication interface. In some embodiments, the communication interface provides a wired interface. In further embodiments, the wired communications interface utilizes Universal Serial Bus (USB) (including mini-USB, micro-USB, USB Type A, USB Type B, and USB Type C), IEEE 1394 (FireWire), Thunderbolt, Ethernet, and optical interconnect.

[099] In some embodiments, the communication interface provides a wireless interface. In further embodiments, the wireless communications interface utilizes a wireless communications protocol such as infrared, near-field communications (NFC) (including RFID), Bluetooth, Bluetooth Low Energy (BLE), ZigBee, ANT, IEEE 802.11 (Wi-Fi), Wireless Local Area Network (WLAN), Wireless Personal Area Network (WPAN), Wireless Wide Area Network (WWAN), WiMAX, IEEE 802.16 (Worldwide Interoperability for Microwave Access

(WiMAX)), or 3G/4G/LTE/5G cellular communication methods.

[0100] In some embodiments, devices, systems, kits, and methods disclosed herein comprise a digital processing device, or use of the same, wherein the digital processing device is provided with executable instructions in the form of a mobile application. In some embodiments, the mobile application is provided to a mobile digital processing device at the time it is

manufactured. In other embodiments, the mobile application is provided to a mobile digital processing device via the computer network described herein. Mobile digital processing and mobile applications may be particularly useful for point-of-care. [0101] In view of the disclosure provided herein, a mobile application is created by techniques known to those of skill in the art using hardware, languages, and development environments known to the art. Those of skill in the art will recognize that mobile applications are written in several languages. Suitable programming languages include, by way of non-limiting examples,

C, C++, C#, Objective-C, Java™, Javascript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof.

[0102] Suitable mobile application development environments are available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator^®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments are available without cost including, by way of non-limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (iOS) SDK, Android™ SDK, BlackBerry^® SDK, BREW SDK, Palm^® OS SDK, Symbian SDK, webOS SDK, and Windows^® Mobile SDK.

[0103] Those of skill in the art will recognize that several commercial forums are available for distribution of mobile applications including, by way of non-limiting examples, Apple^® App Store, Google^® Play, Chrome WebStore, BlackBerry^® App World, App Store for Palm devices, App Catalog for webOS, Windows^® Marketplace for Mobile, Ovi Store for Nokia^® devices, and Samsung^® Apps.

[0104] In some embodiments, devices, systems, kits, and methods disclosed herein comprise computer encoded instructions to communicate with a device. By way of non-limiting example, Python™ software may be used to communicate with a device. In some embodiments, devices, systems, kits, and methods disclosed herein comprise computer encoded instructions to perform data analysis. By way of non-limiting example, GraphPad™ Prism, R programming, or

Python™ SciPy may be employed for data analysis.

Fluid Reservoirs

[0105] Disclosed herein are systems and methods that comprise a fluid reservoir or a use thereof. The fluid reservoir may be connected to an inlet of the reactor disclosed herein. When a system is actuated, fluid may be drawn from the fluid reservoir through the reactor. In some instances, the fluid reservoir contains a fluid comprising a chemical required for a reaction, e.g., a reaction that occurs in an EIA. The fluid comprising the chemical may also refer to herein as a process solution. Non-limiting examples of process solutions include a solution with an assay reagent (e.g, an antibody, a purified analyte, or indicator substrate), a target solution (e.g., a solution comprising an analyte), a wash buffer, and a microbead dispersion. In some instances, the fluid reservoir contains a calibration standard. In some instances, the fluid reservoir contains a control standard. In some instances, the fluid reservoir comprises a cleaning fluid. In some instances, the fluid reservoir comprises an inlet that can be moved to a cleaning fluid after performing an assay. Non-limiting examples of cleaning fluids are an acid solution, a detergent solution, a base solution, and deionized water. In some instances, the fluid reservoir comprises a solution comprising a solid support. A non-limiting example of a solid support is a bead, e.g., a microbead.

[0106] In some instances, the system comprises a kit or use thereof, wherein the kit comprises one or more fluid reservoirs. In some instances, the one or more fluid reservoirs contains all solutions, fluids, and reagents necessary to detect an analyte. Thus, the one or more fluid reservoirs may serve as a self-contained kit for detecting a chosen analyte of interest. The fluid reservoir may comprise a label for distinguishing it appropriate for a chosen analyte of interest. Non-limiting examples of the label include bar codes, RFID, and Bluetooth chips.

[0107] In some instances, the fluid reservoir comprises an inlet that can be moved to a cleaning fluid after performing an assay. In some instances, the fluid reservoir comprises an outlet that can be moved to a cleaning fluid after performing an assay. In some instances, the fluid reservoir may be cleaned or sterilized.

Pumps

[0108] Systems and methods disclosed herein generally comprise at least one pump or use thereof. Pumps cause fluids to flow through the system. Pumps may cause fluids to flow through a network of the system, the network comprising at least one of a channel, input, trap, mixer, and output. In some instances, systems disclosed herein are configured to pump a fluid in more than one direction in the network. By way of non-limiting example, a pump may cause fluid to flow into the network and then reverse the flow to mix/homogenize a solution, sample or bead in the system. The pump may provide a constant flow rate (CQ) from source (e.g., fluid reservoir) to valves and/or reactor. The pump may be used to draw a fluid containing a reagent, solution, sample, or combination thereof, from a fluid reservoir of a system disclosed herein. The pump may draw the fluid through the fluidic logic. The pump may draw the fluid through the reactor.

As exemplified in FIGS. 1 & 2, a syringe pump is used to expose fluids to the reactor(s) and move fluids towards a waste container(s).

[0109] In some instances, systems comprise multiple reactors that can be operated

simultaneously or sequentially. For parallel operation of multiple reactors, a syringe pump may comprise a single piston with multiple syringes that move in parallel. Alternatively, multiple independent syringe pumps may be utilized.

[0110] By drawing the fluid through the system and components thereof, dead volume is minimized, which would otherwise contribute to reagent waste and cross-contamination of the network. By drawing the fluid through the system and components thereof, highly accurate and precise results may be obtained relative to results obtained through classical aspiration techniques e.g ., pipetting). In some instances, reagents in the syringe are infused through a valve into a waste reservoir via a dedicated line, thereby washing the valve and/or other components of the system.

[0111] Often a pump used in a system disclosed herein is a syringe pump. A syringe pump may comprise a syringe that holds a volume of liquid ranging from about 10 microliters to about 10 ml. In some instances, the syringe pump holds a volume of about 50 microliters to about 5 milliliter. In some instances, the syringe pump holds a volume of about 100 microliters to about 1 milliliter. The syringe pump may comprise a user interface, wherein the user interface controls syringe activity. The user interface may comprise a communication port (e.g., a USB port) so that the user interface may relay information to, or receive instructions from, an external source, such as a computer. A syringe pump disclosed herein may be capable of maintaining a minimum flow rate of about 1 pl/min. In some instances, syringe pumps disclosed herein deliver a flow rate of about 100 pl/min to about 10 ml/min. In some instances, syringe pumps disclosed herein deliver a flow rate of about 1 nl/min to about 1 ml/min. In some instances, syringe pumps disclosed herein deliver a flow rate of about 1 pl/min to about 500 pl/min. In some instances, syringe pumps disclosed herein deliver a flow rate of about 1 pl/min to about 500 pl/min. Non-limiting examples of syringe pumps include KD Scientific Legato® syringe pumps, (e.g, KD Scientific Legato® 950 OEM syringe pump), and Tecan Cavro OEM pumps (e.g., Tecan Cavro® Omni Flex and Tecan Cavro® Omni Robot). Syringe pumps disclosed herein may comprise features, characteristics and capabilities of any of these non-limiting examples of syringe pumps, or any combination thereof.

[0112] In some instances, the pump is a pressure pump. The pressure pump may provide constant pressure (CP) to the network. The pressure pump may require an air pump or compressed gas. In some instances, pressure pumps at the reservoir and reactor outlet replace the syringe pump and waste system. With CP systems, CQ may be established through the characterization of hydraulic resistance of the input network. A pressure pump may eliminate the need for a valve to a waste reservoir. In this case, the outlet of the reactor may be the waste reservoir. Typically, there is no “syringe” in a system with a pressure pump. Fluids exiting the reactor directly exit into a waste vial. A pressure pump may provide faster time to results than a system without a pressure pump. See FIG. 4 for an example of a network to be used with a pressure pump. Pressure pumps disclosed herein may provide a pressure range from 0 bar to about 10 bar. Non-limiting examples of pressure pumps or microfluidic flow control systems used with pressure pumps, are

Elveflow® OB1 MK3 microfluidic flow control system, Fluigent MFCS™ systems, and

Dolomite Mitos-P-Pump. Pressure pumps disclosed herein may comprise features, characteristics and capabilities of any of these non-limiting examples of pressure pumps, or any combination thereof.

[0113] In some instances, systems disclosed herein comprise a pressure pump and a syringe pump. In these instances, reservoirs near the inlet are pressurized and syringe pumps are present at an outlet. The fluid logic unit may be open to pulling two or more fluids at once, like in a competitive assay (mixing analyte, enzyme-conjugate, analyte, etc). Without pressurization, the syringe pump would only pull fluid from one inlet due to inequalities in hydraulic resistance between the two inlet reservoirs and the node where they join after the logic unit. This may be useful in situations where there is a lack of gravity to hold liquids down in a vial.

[0114] In some instances, the pump is a peristaltic pump. The peristaltic pump may provide a constant flow rate (CQ) to the network. The peristaltic pump may be operated between a fluid reservoir and a reactor. Peristaltic pumps may eliminate the need for a valve to a waste reservoir. In this case, the out let of the reactor may be the waste reservoir. In general, systems with peristaltic pumps can be manufactured at a lower cost than systems with other types of pumps. Peristaltic pumps may comprise rollers. Peristaltic pumps may comprise channels. A peristaltic pump disclosed herein may comprise about 4 rollers to about 16 rollers. Depending on the number of rollers, the peristaltic pump may provide a flow range of about 0.0001 mL/min per channel to about 25 mL/min per channel. The peristaltic pump may have a speed range of about 0.1 to about 100 rpm. The peristaltic pump may have about 0.001 to about 0.1 rpm speed resolution. The peristaltic pump may provide a maximum of about 25 psi. Non-limiting examples of peristaltic pumps are Ismatech® REGLO Independent Channel Control (ICC) peristaltic pumps and Omegaflex® OEM Style peristaltic pumps. Peristaltic pumps disclosed herein may comprise features, characteristics and capabilities of any of these non-limiting examples of peristaltic pumps, or any combination thereof.

[0115] In some instances, the system does not comprise a pump. By way of non-limiting example, pressure may be provided by hand, by capillary action, electroosmotic pumping, and gravitometric pumping. In some instances, solutions are pushed/infused into the reactor from a reservoir that is pressurized. In some instances, a solution is withdrawn from a reservoir to a negatively pressurized outlet.

Reactor

[0116] Disclosed herein are systems and methods that comprise a reactor or a use thereof.

Reactors disclosed herein may comprise an inlet, a trap, a mixer, a detector, and an outlet, or any combination thereof. In general, the reactor comprises at least one microfluidic channel. Often the reactor comprises a trap, as described in greater detail herein. In general, reactions such as bead homogenization and micromixing occur in the reactor. Bead homogenization may comprise introducing beads to the reactor, trapping the beads, releasing the beads, homogenizing the beads into flow, washing the beads back towards a first reagent to increase exposure time of the bead to the first reagent, trapping the bead, and introducing a second reagent. See, e.g., FIG. 20. This process can be performed repetitively which new reagents being introduced. The reactor is generally responsible for executing the EIA chemistry and detection processes critical for producing data. The reactor may consist essentially of an inlet, a trap, a mixer, a detector and an outlet, as exemplified in FIG. 8.

[0117] Reactors disclosed herein are generally on the scale of an easily portable device. In some instances, the greatest dimension of the reactor is not more than about 20 cm. This is often referred to as the length of the reactor and is overall aligned with the axis of flow through the reactor. Occasionally, greatest dimension of the reactor is more than 20 cm. In some instances, the greatest dimension of the reactor is not more than about 18 cm. In some instances, the greatest dimension of the reactor is not more than about 16 cm. In some instances, the length of the reactor is about 1.5 cm to about 4 cm. In some instances, the length of the reactor is about 1.5 cm to about 6 cm. In some instances, the length of the reactor is about 1.5 cm to about 8 cm. In some instances, the length of the reactor is about 1.5 cm to about 10 cm. In some instances, the length of the reactor is about 1.5 cm to about 12 cm. In some instances, the length of the reactor is about 1.5 cm to about 14 cm. In some instances, the reactor has a width (perpendicular to the length) or diameter between about 0.5 cm and about 4 cm. In some instances, the width or diameter is about 0.5 cm to about 3 cm. In some instances, the width or diameter is about 0.5 cm to about 2 cm. In some instances, the width or diameter is about 0.5 cm to about 1 cm. In some instances, the width or diameter is about 1 cm to about 3 cm.

[0118] In general, reactors disclosed herein comprise a channel. The channel may be a microchannel. The channel may comprise various components of the reactor. The channel may connect various components of the reactor. Various components of the reactor may include an inlet, a trap, a mixer, a detector, and an outlet. In some instances, channels disclosed herein have a total volume of about 0.5 nL to about 5 mL. In some instances, channels disclosed herein have a total volume of about 1 nL to about 1 mL. In some instances, channels disclosed herein have a total volume of about 10 nL to about 100 pL. In some instances, channels disclosed herein have a total volume of about 1 pL to about 1 mL. In some instances, channels disclosed herein have a total volume of about 1 pL to about 100 mL. In some instances, channels disclosed herein have a total volume of about 1 pL to about 50 mL. In some instances, channels disclosed herein have a total volume of about 0.5 nL to about 5 mL. The total volume may comprise the volume of the channel only. The total volume may comprise the volume of all components ( e.g ., inlet, trap(s), mixer, outlet) and microchannels. By way of non-limiting example, a channel that has a total volume of about 0.5 nL may have an external dimension of about 10 pm x 10 pm x 5 mm.

[0119] Channels disclosed herein may have a square opening, and thereby characterized by width. Channels disclosed herein may have an, at least roughly, circularly opening, and thereby characterized by diameter. The width or diameter may be about 10 pm to about 5 mm. The width or diameter may be about 10 pm to about 3 mm. The width or diameter may be about 10 pm to about 1 mm. The width or diameter may be about 100 pm to about 5 mm. The width or diameter may be about 100 pm to about 3 mm. The width or diameter may be about 100 pm to about 1 mm. The length of the channel from a first opening to a second opening may be about 5 mm to about 10 cm. The length of the channel from a first opening to a second opening may be about 5 mm to about 5 cm. The length of the channel from a first opening to a second opening may be about 10 mm to about 5 cm. The length of the channel from a first opening to a second opening may be about 20 mm to about 8 cm. In some instances, the dimensions of the channel are about 500 pm x 500 pm x 25 mm. In some instances, the dimensions of the channel are about 1000 pm x 1000 pm x 50 mm.

[0120] Often, reactors disclosed herein comprise a trap. In some instances, the volume of the trap is about 10% to about 50% of the total volume of the channel. In some instances, the reactor comprises a trap, and the volume of the trap is about 15% to about 35% of the total volume of the channel. In some instances, the reactor comprises a trap, and the volume of the trap is about 20% to about 30% of the total volume of the channel. In some instances, the reactor comprises a trap, and the volume of the trap is about 25% of the total volume of the channel. In some instances, the system comprises multiple traps. In some instances, the total volume of all traps in the reactor is about 40% to about 80% of the total volume of the channel. In some instances, the total volume of all traps in the reactor is about 50% to about 90% of the total volume of the channel.

[0121] In some instances, reactors disclosed herein comprise a channel. The channel may be a microchannel. The channel may comprise at least one turn. In some instances, the reactor comprises at least one channel, wherein the at least one channel comprises at least about two turns, at least about three turns, at least about four turns. The turn may provide at least about a 15 degree, at least about a 20 degree, at least about a 25 degree, at least about a 30 degree, at least about a 35 degree, at least about a 40 degree, or at least about a 45 degree change in a direction of flow through the channel. The turn may provide at least about a 65 degree, at least about a 70 degree, at least about a 75 degree, at least about a 80 degree, at least about a 85 degree, or at least about a 90 degree change in a direction of flow through the channel. In some instances, the reactor does not comprise a channel that has a turn or angle. In some instances, the system only has a channel that is straight. In some instances, the reactor only has a channel that is straight. In some instances, the system only has channels that are straight. In some instances, the reactor only has channels that are straight.

[0122] Channels may have varying lengths. In some instances, the length of at least one channel is about is about lmm to about 1 cm. In some instances, the length of at least one channel is about is about 5 mm to about 500 mm. In some instances, the length of at least one channel is about is about 5 mm to about 100 mm. In some instances, the length of at least one channel is about is about 5 mm to about 50 mm. At least a portion of the channel may have a length of about 5 mm to about 1 cm. In some instances, the total length of the channel is not greater than 5 cm. In some instances, the system has multiple channels. In some instances, the total volume of the channel(s) in the reactor is about 0.5 nL to about 5 mL. In some instances, the system has multiple channels. In some instances, the total volume of the channel(s) in the reactor is about 0.5 nL to about 1 mL. In some instances, the system has multiple channels. In some instances, the total volume of the channel(s) in the reactor is about 0.5 nL to about 500 pL. In some instances, the total volume of the channel(s) in the reactor is about 0.5 nL to about 100 pL.

[0123] In some instances, at least two of a channel, an inlet, an outlet, a trap, a mixer and a detector are present on two different z-planes relative to the top and bottom of the device. A z- plane may be described as a plane perpendicular to the direction of flow. In some instances, systems comprise at least two channels, wherein the two channels are located on different z- planes. In some instances, systems comprise at least two traps, wherein the two traps are located on different z-planes.

[0124] Various fluids and solutions described herein may be flowed, pumped or drawn through the reactor at a given flow rate. In some instances, the flow rate is about lpL/min to about 25 mL/min. In some instances, the flow rate is about 1 pl/min to about 1 mL/min. By way of non limiting example, methods may comprise flowing reagents at around 15-300 pl/min through a 642.5 mih x 642.5 mih channel size with 200 mih recesses for trapping regions. However, that rate may decrease substantially with a smaller channel size or smaller reactor volume.

[0125] In general, the inlet allows one or more fluids exiting the fluid logic to enter the reactor. As exemplified in FIG. 8, the inlet may be designed to accept a standard tubular interconnect.

The inlet may be designed to accept a standard tubular interconnect via a ¼-28 fitting. In some instances, the inlet is described as a chamber of the reactor. In some instances, the inlet is simply an opening that is sealed to a tube or probe that is compatible with solution reagents. The inlet may be an extension of the reactor in the shape of a tube/probe.

[0126] Fluids or reagents generally flow through the inlet into a trap. In some instances, they pass directly from the inlet into the trap. In some instances, they pass through another reactor component before they enter the trap. Disperse microbeads are generally immobilized in the trap, creating a reaction site for EIA chemistries as other fluids are cycled through the trap.

[0127] Generally, reactors disclosed herein comprise a trap. Often, the trap is described as having a greater dimension than an adjacent channel in the direction perpendicular to flow. See, e.g., FIGS. 6-12, 14, 23, and 25. This may create a lip or edge that defines the area of a bead layer that accumulates in the trap. The trap may be lipped to prevent lateral escape of beads (e.g. due to diffusion or magnetic field fringe. In some instances, the trap is not lipped. In some instances, the trap is a magnetic trapping device. In some instances, the magnetic trapping device comprises a chamber for holding magnetic beads. In some instances, the magnetic trapping device comprises magnetic beads. In some instances, the magnetic beads are superparamagnetic microbeads that are capable of being captured by magnetophoresis. As exemplified in FIG. 11, the trap may expand slightly in some sections of the trap such that a thin wall separates it from two sockets that accept magnets. Magnets compel paramagnetic microbeads to migrate towards the wall and pack within the expanded sections of the trap, thereby forming an immobilized layer on which EIA chemistries will occur. Magnets may be slid into the sockets. As a non-limiting example of the scale of these systems, 1/16” magnets may fitted into the sockets of the trap.

[0128] In some instances, the system comprises at least two magnets. In some instances, the at least two magnets oppose one another. In some instances, the at least two magnets do not oppose one another. In some instances, the respective poles of the at least two magnets are N-N or S-S.

In some instances, the respective poles of the at least two magnets are N-S. In some instances, the respective poles are N-S to maintain the magnets in place through particle layer formation or particle layer movement.

[0129] In some instances, systems support formation of a layer of beads with super high surface area-to-volume ratio. This is useful because drag forces limit total number of beads that can be trapped. In some instances, the system comprises a magnet to be used with the magnetic beads. The magnet may be a constant magnet, actuating magnetophoresis by proximity to the system. The magnet may be an electro-magnet. Electromagnetic switching may be used to mix or homogenize the magnetic beads in the fluid example. See, e.g., FIG. 19. In some instances, homogenization occurs when flow is stopped. In some instances, homogenization occurs when flow is in progress.

[0130] In some instances, systems disclosed herein comprise a trap for trapping magnetic beads. Non-limiting examples of trapping are provided in Examples 5 and 7. In some instances, methods comprise flowing beads over a trap in the reactor. The trap may be subjected to magnetic forces, such that the beads are attracted to a wall of the trap due to the inhomogeneity in the magnetic field strength (magnetophoresis). The wall may also be characterized as a membrane. An immobilized monolayer of beads may be formed against the wall. The immobilized monolayer may conduct magnetic field lines, thereby creating regions of extreme local inhomogeneity in the magnetic field strength, enhancing magnetophoresis. More beads may be added to the monolayer, forming dendrite structures. The resulting sponge-like super high surface area to volume ratio of the monolayer with dendrite structures enables a high degree of binding for target and affinity reagents in a limited exposure time and volume. In some instances, methods comprise adjusting the flow rates and volumes of reagents, solutions, beads and samples so that the process reagents efficiently and optimally infiltrate the monolayer. In some instances, the flow rates and volumes are adjusted to create a thicker layer that captures more target, although the infiltration time may be longer. This may be useful when attempting to detect an analyte present in very small amounts. In some instances, methods further comprise removing the magnet and running cleaning solutions at high flow rates to clear the trap for the next assay or reaction.

[0131] In some instances, systems disclosed herein comprise a trap that does not require a magnet. For instances, frits or filters may be used to capture broad classes of microbeads. See, e.g., FIGS. 15 and 16. Frit capture, also referred to as a dam, acts as an effective porous dam to catch particles. Particles do not have to be magnetic. Instead particles may be polymeric. Frits may have a polymer sealing ring. The porosity of a frit may be varied to accommodate for particle size. By way of non-limiting example, frits may be manufactured from polyether ether ketone, stainless steel, ultra high molecular weight polyethylene, or titanium. In some instances, systems comprise multiple frits. In some instances, methods comprise moving beads and particles back and forth between the multiple frits. This may aid in homogenizing beads, exposing beads to analytes/reagents, and reducing bead concentration. See, e.g., FIGS. 18 and 19. Alternatively or additionally, electrodes may be used to capture dielectric microbeads. Chemically coated traps or structures therein, may also be used to capture affinity-tagged beads, wherein the beads can be removed at a later time.

[0132] In some instances, systems disclosed herein comprise a single trap, not more than one trap. In some instances, systems disclosed herein comprise a plurality of traps. In some instances, systems disclosed herein comprise two traps. In some instances, systems disclosed herein comprise three traps. In some instances, systems disclosed herein comprise between one trap and ten traps. In some instances, systems disclosed herein comprise between two traps and ten traps. In some instances, systems disclosed herein comprise at least one trap. In some instances, systems disclosed herein comprise at least two traps. In some instances, systems disclosed herein comprise at least three traps. In some instances, systems disclosed herein comprise not more than ten traps. In some instances, systems disclosed herein comprise not more than twenty traps. In some instances, systems disclosed herein comprise not more than a hundred traps. In some instances, systems disclosed herein comprise not more than a thousand traps. In some instances, systems disclosed herein comprise not more than a million traps.

[0133] Following the trap, fluids and reagents generally flow into a mixer. The mixer may function as a homogenizer. For example, after an indicator substrate is introduced to the trap(s), the detectable signal from the indicator substrate may not be homogenously distributed across the fluid stream. The mixer functions to mix the stream and improve linearity and/or well-behavior of the final signal. Micromixing generally involves mixing of solutions in a microchannel of the reactor. The microchannel may be helical. The microchannel may comprise one or more changes in direction. These turns may slow solution flow or provide turbidity that aids in mixing solutions, reagents or components thereof. In some instances, micromixing may be made possible by lamination. Lamination may comprise arranging inlet co-flows of different reagents in alternating lanes across a channel. Lamination may comprise constructing interdigitated inlets to a single mixing channel or by splitting co-flows and reassembling them. In some instances, micromixing comprises chaotic advection. Mixing flows with chaotic advection comprises stretching and folding flow lines through engineered channel geometries or making channels with three-dimensional architecture. Micromixing may be useful for homogenizing solutions before optical detection. Homogenization may reduce reagent consumption.

[0134] Following the mixer, fluids and reagents generally flow into a detector. The detector may be a subcomponent for detecting intrinsic properties of fluid streams in the reactor, such as the presence of a detectable signal from the indicator substrate. Typically, detection is performed optically, wherein the absorbance/transmittance, fluorescence, or luminescence of a detectable signal is measured. Often, reactors disclosed herein comprises an optical fiber port. See, e.g., FIG. 22. Optical fiber ports may connect a light source or lamp to the system. Optical fiber ports may enable measurement of spectral absorbance, spectral transmittance, fluorescence, luminescence, or any combination thereof. As exemplified in FIG. 22, the detector

subcomponent may accept SMA connector style optical fibers, one of which leads to a light source and the other of which leads to a spectrometer. Detecting may also be performed using a mass spectroscopy system, a thermal sensor, or a non-fiber based optical system (e.g, free space optics).

[0135] In some instances, a reactor comprises a channel in connection with a detection region, wherein the channel supports a first direction of flow that is not parallel to a second direction of flow through the detection region. At least one portion of the channel may support a direction of flow that is different than the direction of flow through the detection region. In some instances, the direction of flow through the detection region is along an axis, and optics of the device are positioned at the ends of the detection region along the axis. Due to this configuration, light may be streamed down this axis (into and throughout the detection region) from a first end, and/or a resulting signal may be detected form a second end. In some instances, the distance along this axis (into and throughout the detection region, e.g. , from first end to second end) is about 1 mm to about lOmm. Due to this configuration, the volume of the detection region that is subjected optics is increased, and therefor signal may be maximized. The dimension of the detection region along the axis of flow may range from about 10 pm to about 100 pm. This is particularly useful for samples with low amounts of analyte or systems with weak substrate-analyte binding (direct or indirect), or weak substrate activity. A Z-shaped channel, such as that exemplified in FIG. 22, may enable linear signal collection. For example, a microfluidic channel inside the

subcomponent may be roughly“Z” shaped so that parallel/normal rays of light travel along a length of the microfluidic channel bearing a stream effectively acting as an aperture (see, e.g., FIG. 231)

[0136] Systems disclosed herein may comprise a plurality of reactors. Generally, each reactor has its own channel. Often, the system has a single detector for detecting a signal in each reactor. In some instances, the channels of the system comprise a different volume or different length so that signal occurs in the detection region at different time points. This allows for a single detector to detect signals individually because signals from the different channels do not interfere with one another. Each channel may be pulled at the same flow rate but has a different path length to a multi-channel, single piston syringe pump. In some instances, flow may be stopped so each channel can be interrogated individually. This eliminates the need for an optical multiplexer, free-space optics, or positioner/aligner systems. By way of non-limiting example, a system may have multiple reactors with varying trap volumes or microchannel volumes causing delays in the transit of fluid volumes (see FIGS. 25A and 25B), eliminating the need for an optical multiplexer, free space optics, or position/alignment of optics.

[0137] Systems disclosed herein may comprise a plurality of reactors with a single particle manipulator. In some instances, systems comprise a single fixture of at least one magnet, wherein the single fixture is capable of manipulating magnetic particles in the plurality of reactors simultaneously, thereby providing consistent active bead homogenization between channels. In some instances, the magnet is an electromagnet and the single fixture is configured to remain stationary. In some instances, the magnet is not an electromagnet and the single fixture is configured to physically move. In general, all reactors are subject to the same magnetic forces. Although in some instances, it may be desirable to subject one or more reactors to a different magnetic force than that of the other reactors.

[0138] In general, reactors disclosed herein comprise an outlet. Fluids, reagents, and waste may exit the reactor through the outlet. In some instances, the outlet is connected to a waste reservoir, or waste management subsystem.

Fluid Logic Unit

[0139] Systems and methods disclosed herein may comprise a fluid logic unit or a use thereof. For simplicity, the fluid logic unit may simply be referred to herein as a“fluid logic.” In general, the fluid logic functions to route one or more fluids into at least one reactor. In general, the fluid logic draws the fluids through the reactor. Thus, the fluid logic may be connected to the outlet of a reactor. The fluid logic may route fluids through the reactor either simultaneously or sequentially. The fluid logic may comprise a motorized gantry, a motorized mover, an array of valves, a rotary shear valve, or a combination thereof. In general, the fluid logic may be described as a digitally controlled unit that selects one or more fluids to be drawn through the reactor. In some instances, the fluid logic draws a first fluid through the reactor at a first time point and draws a second fluid through the reactor at a second time point. In some instances, the fluid logic draws a first fluid and a second fluid through the reactor at the same time.

[0140] In some instances, the fluid logic draws a fluid through the reactor with a rotary sheer valve. Rotary sheer valves are described in greater detail herein. In some instances, the fluid logic selects one or more fluids to be drawn through the reactor. The one or more fluids may be selected with a robotic gantry and at least one descendible probe. The one or more fluids may be selected with a robotic turntable and at least one descendible probe. The descendible probe may be capable of holding or dispensing selected volumes of solutions and liquids. Non-limiting examples of robotic gantries suitable for handling small volumes of liquids are provided by Teledyne CETAC Technologies. Gantry and/or probe activity may be automated.

Valves

[0141] Disclosed herein are systems and methods that comprise a valve or a use thereof. A valve may allow for selection of one or several reagent streams entering the reactor. A valve may allow for a waste stream to exit the reactor. A valve may allow for a product of a reaction that occurs in the reactor to exit the reactor. Valves, as used in the systems disclosed herein, should have minimal holdover, minimal dead volume, and minimal displacement volume. By way of non limiting example, valves may be actuated electromechanically, hydraulically, or pneumatically.

[0142] In some instances, systems disclosed herein comprise a rotary sheer valve. The rotary sheer valve may provide for zero dead volume. The rotary valve may provide for low pressure e.g ., less than about 125 psi, 9 bar). In some instances, the system comprises an array of rotary sheer valves. The array may allow for switching between rotary sheer valves in the system, as needed. The array may allow for switching between connections to rotary sheer valves in the system, as needed. In some instances, the array limits selection to only one fluid at a time. A non limiting example of an array of rotary shear valves is Idex Health and Science’s Titan Ex™. In some instances, the fluid logic draws a fluid through the reactor with an array of 2-way and/or 3- way valves. The array may comprise a network of valves that are integrated with microfluidic channels, millifluidic channels, or a combination thereof.

[0143] In some instances, a valve disclosed herein is a solenoid valve. In some instances, the system comprises a normally closed (n.c.) solenoid valve which minimizes heat generation. See, e.g., FIG. 3. Solenoid valves may have very small dead volumes, operate rapidly, and can be manufactured with inert parts. In some instances, a system disclosed herein comprises a valve array of interfacing valve-compatible tubing to the reactor and running that tubing into and out of solenoid valves of the valve array. In some instances, the solenoid valve has at least two ports. In some instances, the solenoid valve has a series of ports (e.g., 3 or more ports). This,

advantageously, allows one to take full advantage of the high-quality characteristic of the solenoid valves while operating them in-line with reactor flow. In some instances, the solenoid valve is magnetically latched, therefore requiring very little power to operate and producing very little heat. In some instances, solenoid valves require brief (10-30 ms) pulses of current to switch to and remain in in each flow state. A lower power requirement makes devices ideal for point of care. [0144] In some instances, systems comprise one or more valves that take fluids that have been flowed into the reactor and redirects them to one or more waste containers. These valves may be referred to as waste management valves. The waste container can optionally be emptied and reused. For parallel operation of multiple reactors, multiple waste valves may be operated, one for each reactor. A fluid that has contacted surfaces inside of the reactor may be considered waste and may be delivered to a waste container. See FIG. 6 for an example EIA binding curve dataset that demonstrates the effectiveness of this procedure.

Optics

[0145] Disclosed herein are systems and methods that comprise energy source or a use thereof. The energy source and any external detection equipment may be used with a reactor’s detector disclosed herein. By way of non-limiting example, the light source may be a lamp. Other non limiting examples of light sources are a laser and a light emitting diode (LED). By way of non limiting example, external detection equipment may comprise an optic fiber. As exemplified in FIGS. 1 & 2, an optical fiber carries light from a lamp or LED source into the reactor’s detector while another fiber carriers light from the reactor’s detector to a spectrometer with digital output. This setup can be used to detect colorimetric, fluorescent, or luminescent indicator substrates or detectable signals thereof. In the latter two cases, a variety of filters or other optical components may be required in the optical circuit.

[0146] The light source may be necessary to execute absorbance or colorimetric based assays. In some instances, the light source emits light of a single wavelength. In some instances, the light source is capable of emitting light of a single wavelength. In some instances, the light source emits light of multiple wavelengths. In some instances, the light source emits light in all wavelengths of the visible light spectrum. In some instances, the light source emits light in all wavelengths of the ETV spectrum. In some instances, the light source is a halogen light source. In some instances, the light source is a deuterium light source. In some instances, the light source is a combination halogen/deuterium lamp, which covers the visible light spectrum and ETV spectrum.

[0147] Systems disclosed herein generally comprise a detector. The detector may be capable of receiving, detecting, processing, or quantifying light. The detector may comprise a camera. In some instances, the detector comprises a spectrophotometer. A spectrophotometer may also be referred to as a spectrometer. In some instances, a spectrophotometer is used to quantify the analyte in absorbance or colorimetric based assays. The spectrophotometer may cover the visible light spectrum. The spectrophotometer may cover the UV spectrum (-200-400 nm). Other optical techniques may allow for lower background and higher sensitivity. In some instances, the system is fluorescence based, wherein the indicator or substrate undergoes a change in fluorescent excitation wavelength or emission wavelength upon reacting with the enzyme-linked antibody.

In some instances, the system is luminescence-based, wherein the indicator or substrate photochemically emits light upon reaction with the enzyme.

[0148] In some instances, systems disclosed herein comprise a detector and an indicator substrate or detectable signal that is not light-based. For example, the detector may be capable of detecting a nanoparticle ( e.g ., quantum dot), an electronic signal or a radioactive signal.

[0149] In some instances, optics are configured to detect multiple analytes simultaneously. In some instances, optics are configured to detect multiple signals simultaneously. For example, a system may comprise multiple reactors, wherein each reactor receives an indicator substrate or detectable signal that is detectable at a different wavelength. In some instances, the optics are configured to detect and quantify signals at different wavelengths. In some instances, methods comprise using a camera and illumination source, such as in a microscope, to visualize all channels simultaneously despite operating in parallel.

[0150] In some instances, methods disclosed herein comprise adjusting an amount of captured target to put a detectable signal, corresponding to the captured target, in a detectable range.

Traditional microtiter plate-based and microbead-based EIA kits have a strict range of detectable concentrations. This is set by the density of capture ligand bound to surfaces in wells or on beads, various incubation times in the procedure, and other parameters prone to intrinsic and operator motivated errors. Many biomarker targets have a wide range of naturally occurring

concentrations in biological fluids. Thus, if the concentration of target in a sample lies outside of the detectable range of a traditional EIA kit, it cannot be quantified. As a result, the sample may need to be retested after dilution or enrichment. In contrast, systems disclosed herein may contain a variety of sites (e.g., multiple traps, as exemplified in FIGS. 8 & 11) on which a target may be bound to immobilized microbeads and subsequent EIA chemistries may be performed. If too much target is bound and causes the resulting assay signal to be beyond the detectable range, magnets may be disengaged from specific sites to wash away excess target and effectively move the signal range. This eliminates the need to retest samples under new preparation conditions.

[0151] In traditional EIA procedures, titrations of positive control analytes are measured in different wells to create a binding curve. The binding curve then acts as a reference for calculating the concentration of target in a sample. In research applications, the binding curve itself may have immediate value in assessing the affinity and selectivity on novel binding ligands. Variation in the capture ligands used to bind these control analytes or operator errors can cause the binding curve to be imprecise. For example, a traditional microtiter plate may have dried down captured antibody on each well, each well with different drying patterns (e.g.,“coffee ring” effect) and levels of coverage, creating inconsistent patterns from well to well. As a result, many points on the binding curve are subject to differing binding conditions. In turn, this may cause inaccuracies in the determination of target concentration in sample or the affinity and selectivity of experimental ligands. In contrast, methods disclosed herein often comprise sequentially exposing solutions with increasing concentrations of positive control analyte to the same binding sites and detecting the positive control analyte, largely eliminating the sources of error present in traditional EIA. Methods disclosed herein may comprise trapping a set of particles, flowing a low concentration of reagent and/or purified analyte, building up a layer of beads, taking a measurement, subsequently flowing a higher concentration of reagent and/or purified analyte, and repeating layer formation and measurement, and repeating in order to build a binding curve. In some instances, a layer is formed without analyte for negative control. In some instances, a layer is formed with a high concentration of analyte for positive control.

Relative differences in signal from various concentrations of analyte eliminates variation.

Relative to previously known methods, this flowing-capturing-measuring methods with beads cuts down on reagent and artifacts, e.g. , excessive reagent drying patterns.

[0152] In some instances, systems and methods comprise a processor, automated feedback control, or a use thereof, that provides real-time feedback process control. Real-time feedback from an optical sensor may allow for immediate and/or continuous detection of a disturbance in an optical light path. Methods disclosed herein may comprise detecting a signal (e.g., from the indicator substrate) at any step during the EIA process. In some instances, methods comprise detecting a signal continuously throughout each step of the EIA process. Disturbances in the light path may cause the spectrometer integration time (exposure time) to be out of range. By way of non-limiting example, disturbances include bubbles, debris, and window fouling. If there is a disturbance, the computer or computer program may instruct the system to clear a channel or replace a reactor. For example, contamination of a given reagent may be quantified by spectrophotometry. The user may then be alerted of a bad reagent or system internal failure.

Small bubbles can often cause failures in microfluidic devices. The presence of such bubbles can be monitored through active optical detection during flows in the detector subcomponent of the reactor. If bubbles get trapped in the microchannel, a spike in the pressure or flow rate can dislodge them and restore correct operation of the reactor. Similarly, flow rates and residence times for flows can be actively optimized through detection and feedback controls. See FIG. 5 for a flow diagram that exemplifies real-time feedback process control.

Particles/ Beads

[0153] Disclosed herein are systems and methods that comprise particles or a use thereof. The term,“particle,” as used herein may refer to a bead, often a microparticle ( e.g ., a microbead). The terms particles, beads, microbeads and microparticles may be used interchangeably, unless otherwise specified. The particle may be spherical. The particle may not be spherical. Particles used herein may comprise a polymer. Particles disclosed herein may comprise a metal. Particles disclosed herein may be magnetic. Particles may be microbeads, wherein microbeads have a greatest width or diameter ranging from about 20 nm to about 1000 micrometers. Non-limiting examples of beads are Dynabeads™ MyOne™ beads and Sphere™ magnetic particles.

[0154] In some instances, particles are magnetic. Particles may be responsive to magnetic forces in the range of (1-999) pN to (1-999) nN of. Magnetic force should exceed the forward drag force (dependent on flow rate) carrying the particle in the flowing solution in order to capture the particles against an internal surface of the system. In some instances, particles are dielectric. In some instances, particles are inert. In some instances particles are not magnetic. In some instances particles are not dielectric. In some instances particles are not dielectric or magnetic.

[0155] In some instances, particles disclosed herein are coated, or capable of being coated, with a binding moiety that binds the target analyte. In some instances, systems and methods disclosed herein comprise a binding moiety or a use thereof. The binding moiety binds to the target analyte. The binding moiety may comprise an antibody or antigen binding antibody fragment, a target analyte substrate, a small molecule, a peptide, an aptamer, or a combination thereof. In some instances, particles are pre-coated with the binding moiety. In some instances, the binding moiety is provided in a solution and methods disclosed herein comprise applying the binding moiety to the particles. In some instances, the binding moiety is an antibody or antigen binding antibody fragment. As used herein, the term "antigen binding antibody fragment " refers to forms of an antibody other than the full-length form. Antigen binding antibody fragments herein include smaller components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, and antigen-binding portions thereof. Unless specifically noted otherwise, statements and claims that use the term "antibody" or "antibodies" may specifically include "antigen-binding antibody fragment" and " antigen-binding antibody fragments." [0156] In some instances, particles comprise a mixture magnetic and plastics, or similar composite, structures. Magnetic beads may comprise a shell around a magnetic material, wherein the shell is capable of linking to an antibody. Non-limiting examples of shell components are polystyrene, polycarbonate, poly(lactic-co-glycolic acid), gold, and silica. By way of non limiting example, a capture antibody may be covalently linked to the shell or linked to the shell with a streptavidin-biotin linkage. These modifiable beads enable systems disclosed herein to work with a wide variety of EIA formats, reagents, and sample types, thus producing a modular system overall.

Solutions

[0157] Systems and methods disclosed herein may comprise one or more solutions. Liquid solutions are critical to running systems disclosed herein, and may include blocking/passivation solutions, wash buffers, ligand solutions, calibration target solutions, biological sample solutions, microbead dispersions, and indicator substrate solutions. Table 1 indicates typically necessary reagents for a variety of EIA formats.

Table 1. EIA Liquid Reagents

[0158] In addition, systems, kits and methods may comprise a cleaning fluid or use thereof.

These maybe used for sterilizing a system disclosed herein. The cleaning fluid may also be referred to as a sterilization solution or a wash solution. The cleaning fluid may be capable of removing one or more components of a blocking buffer, a reagent, a target analyte, a particle, or any combination thereof. In some instances, the cleaning fluid leaves one or more components of a blocking buffer in place on the interior surface of the system. Non-limiting examples of cleaning fluids include acid solutions, detergent solutions, and deionized filtered water.

Analytes

[0159] Systems and methods disclosed herein are useful for detecting a wide array of analytes. Analytes include, but are not limited to, proteins, nucleic acids, and viral particles. In some instances, the protein is a cell surface protein. Non-limiting examples of cell-surface proteins are cancer cell surface proteins and fetal cell surface proteins. In some instances, the protein is part of a protein aggregate, e.g., a Lewy body, an amyloid plaque. Non-limiting examples of viral particles are a viral capsid and a viral envelope. Non-limiting examples of nucleic acids are DNA and RNA. Although many examples provided herein are directed to proteins and antibodies, one of skill in the art understands that such similar systems could be used for nucleic acids with oligonucleotide probes conjugated to detectable labels.

[0160] Often, systems and methods disclosed herein comprise an indicator substrate or use thereof, wherein the indicator substrate is processed by an enzyme, thereby providing a signal that indicates whether or not an analyte is present. For simplicity, the indicator substrate may also be referred to herein simply as a“substrate.” In some instances, the substrate comprises an optically detectable molecule. In some instances, the substrate releases or generates an optically detectable molecule upon enzyme processing. Optically detectable molecules provide optically detectable signals or readouts. Optically detectable signals include colorimetric signals, fluorescent signals, luminescent signals, electrochemical signal, and electrochemiluminescent signals. In some instances, systems or methods amplify the signal.

System Manufacturins

[0161] Reactors disclosed herein may be manufactured as multiple subcomponents and assembled and together. In some instances, the multiple subcomponents are sealed together. By way of non-limiting example, multiple subcomponents may be sealed together with ultrasonic welding, thermal bonding, laser welding, glue, epoxy, or a combination thereof. In some instances, multiple subcomponents are not sealed together. In some instances, multiple subcomponents are not sealed together permanently. For instances, subcomponents may be assembled with interference fits or screws and gaskets. In some instances, multiple

subcomponents are assembled under a microscope. Alternatively, the entire reactor is manufactured as a single piece (e.g., monolithic). The monolithic may comprise a microchannel. The monolithic may comprise a compartment, e.g., an inlet, trap, detector, outlet. In either case, some or all components or subcomponents may be manufactured using additive techniques such as stereolithography. In some instances, one or more components or subcomponents are manufactured using fused deposition printing. In some instances, one or more components or subcomponents are manufactured using a combination of additive and classic machining techniques.

[0162] Systems disclosed herein, or components thereof, may be manufactured by a variety of methods. In some instances, systems, or a component thereof, are manufactured by

stereolithographic 3D printing. In some instances, systems, or a component thereof, are manufactured by polyjet 3D printing. In some instances, systems, or a component thereof, are manufactured by fused deposition. In some instances, systems, or a component thereof, are manufactured by laser sintering. In some instances, systems, or a component thereof, are manufactured by injection molding. In some instances, systems, or a component thereof, are manufactured by micromachining. In some instances, systems, or a component thereof, are manufactured by a combination of sterolithographic 3D printing, polyjet 3D printing, fused deposition, laser sintering, injection molding, and micromachining.

[0163] Systems disclosed herein, or components thereof, may be manufactured with a variety of materials. Systems disclosed herein, or components thereof, may be manufactured with a single material. In some instances, the material comprises a polymer. Non-limiting examples of polymers are acrylonitrile butadiene styrene, polycarbonate, polystyrene, polyethylene, etc. In some instances, the material comprises a metal. Non-limiting examples of metal are steel and aluminum. In some instances, the material comprises glass. In some instances, the material comprises fused silica. In some instances, the material comprises quartz.

[0164] Systems disclosed herein are often manufactured for use at point of care. It is ideal for systems to be handheld and mobile. Systems disclosed herein may weigh less than about five pounds. Systems disclosed herein may weigh less than about ten pounds. Systems disclosed herein may weigh less than about two pounds. Systems disclosed herein may weigh less than about one pound. Systems disclosed herein may weigh less than about twelve ounces. Systems disclosed herein may weigh less than about eight ounces.

III. Kits

[0165] Disclosed herein, in some aspects, are kits for use with systems disclosed herein. In some instances, kits comprise particles disclosed herein. In some instances, kits comprise one or more reagents disclosed herein. In some instances, kits comprise one or more binding moieties disclosed herein. In some instances, kits comprise reagents required for an immunosorbent assay. In some instances, kits comprise reagents required for an EIA. In some instances, the EIA is ELISA. The ELISA may be a direct assay, an indirect assay, a sandwich assay, or a competitive assay. In some instances, kits comprise at least one antibody.

[0166] In some instances, kits comprise a first solution in a first container and a second solution in a second container. In some instances, the kit comprises a container for receiving a sample containing a target analyte. The first solution or second solution may comprise the particles. The first solution or second solution may comprise the target analyte. The first solution or second solution may comprise an antibody to the target analyte. In some instances, kits are compatible with a fluid logic of the system, such that the fluid logic is capable of drawing up the first solution and the second solution and delivering them to the reactor.

[0167] Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to the fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES

[0168] The examples and embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the claims provided herein. Various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1. EIA Systems

[0169] An exemplary system for EIA is shown in FIG. 1. Fluid reservoirs shown at the far left are connected to a fluid logic. The fluid logic allows for one or more fluids to be selected and drawn into the system at any time. One or more channels allow a syringe pump (shown at the far right) draw the one or more fluids into the reactor, also referred to in this instance as the“rapid EIA reactor.” Non-limiting examples of the internal components of the reactor are shown in FIGS. 6- 11. In general, the reactor at least comprises a trap or a mixer/homogenizer, as described herein. In some instances, the reactor comprises a trap and magnetic beads are sent into the reactor. Magnets on either side of the trap are alternately applied to multiple sides of the trap to homogenize the beads in solution, coating the beads comprehensively with a solution comprising a target analyte and/or reagents such as antibodies and indicator substrates. In this exemplary system, there is a lamp that illuminates the contents of the reactor through an optic fiber. Another optic fiber is connected to a spectrophotometer or other optics device for detection of fluorescence,

luminescence, color, or other optical signal from the indicator substrate. A valve may be used to direct waste to a waste reservoir (not shown) after detection is performed. A designated line to the waste reservoir may optionally be used to flush the valve. A computer or microcontroller is connected to the syringe pump, valve, fluid logic, and optics so that all steps performed by the system are automated and monitored.

[0170] FIG. 2 shows a system with multiple reactors. The system may include any of the components shown in FIG. 1. Note that a single fluid logic may be used to dispense solutions, reagents and samples into the multiple reactors. Although not shown, a single computer or microcontroller can operate any combination of valves, syringe pumps, and fluid logic.

Example 2 EIA System with Constant Pressure Regulator

[0171] FIG. 3 shows an EIA system comprising fluid reservoirs, an optional fluid logic, a reactor, detection equipment (lamp & optics), a computer/microcontroller, and a constant pressure (CP) regulator/source. Note that this system does not require syringes or valves. Fluid reservoirs are connected to the CP regulator/source as well as the fluid logic. However, the fluid logic may not be needed if the reservoirs are independently controlled. Note that the CP regulator/source is providing positive pressure at the fluid reservoirs and negative pressure at the waste reservoir, creating a pressure gradient that draws solutions (of analytes and reagents) through the reactor.

[0172] Similar to the system described in Example 1, there is a lamp that illuminates the contents of the reactor through an optic fiber, and another optic fiber is connected to a spectrophotometer or other optics device for detection of fluorescence, luminescence, color, or other optical signal from the indicator substrate. A computer or microcontroller is connected to CP regulator/source, fluid logic, and optics so that all steps performed by the system are automated and monitored.

Example 3 Reactor

[0173] FIGS. 6-8 show exemplary reactors used in devices disclosed herein. These reactors comprise an inlet, a trap, a mixer, a detector, and an outlet (as shown from bottom to top).

Alternatively, these reactors may not comprise a mixer, as the trap may provide a sufficient means for mixing the beads in solutions, such as reagent solutions and wash solutions, thereby homogenizing the beads and bead surfaces. In FIGS. 7 and 8, there is more than one trap. A series of traps may increase homogenization, thereby maximizing reagent binding and signal. This is especially useful when dealing with low sample amounts, low analyte concentrations and weak antibody binding.

Example 4 Operational Workflow for sandwich ELISA

[0174] To initiate a sandwich ELISA using a system disclosed herein, a solution comprising a first primary antibody to the analyte of interest is drawn into the reactor using a system disclosed herein. Next, a solution of beads is drawn into the reactor, where the beads become coated with the first primary antibody. Optionally, the beads are mixed in the reactor in an effort to coat all bead surfaces with the first primary antibody. For example, the beads may be magnetic, and mixing may comprise alternating the presence of a magnet on various sides of the reactor to move the beads around. Once beads are coated, a wash buffer is drawn through the reactor to wash away any excess first primary antibody that is not bound to the beads. Following the wash, a solution containing a target analyte (or at least suspected of containing the target analyte) is drawn into the reactor. The first primary antibody on the beads binds the target analyte and again the reactor is subjected to a wash solution to wash away any unwanted substances, such as non -target analytes which may create signal noise. A second primary antibody to the target analyte is then drawn through the reactor. Alternatively, a ligand or other binding moiety of the target analyte is drawn through the reactor. In some instances, another wash is performed followed by drawing a secondary antibody through the reactor that binds the second primary antibody. The second primary antibody, secondary antibody, ligand or other binding moiety may be conjugated to an enzyme, enzymatic substrate or detectable signal. Throughout the flow of the reagents and sample, the reactor of the system is being monitored for an optically detectable signal, appears at this point if there is target analyte in the sample. Throughout the run, the system is also being monitored for contaminants, bubbles, and anything else that may disrupt the signal or precise

detecti on/ quanti ficati on thereof.

[0175] After signal detection, the system is washed and sterilized for reuse. To sterilize, valves are set to open the reactor and close off the system to the waste reservoir. The reactor inlets are moved to an acid solution. The acid solution is drawn through the reactor. Valves are then set to close off the reactor and open the system to the waste reservoir in order to clear the used acid solution. The used acid solution is discarded. The same sterilization steps are then repeated with detergent instead of acid solution. Finally, the sterilization steps are repeated with water.

Optionally, the sterilization steps are repeated one or more times with acid solution, detergent or water.

Example 5 Trapping Beads

[0176] A dispersion of superparamagnetic beads is flowed into a trap. Beads near the bottom of a microchannel are attracted to the thin wall due to the inhomogeneity in the magnetic field strength (magnetophoresis). An immobilized monolayer of beads is formed above the thin wall/membrane. This is illustrated in FIG. 12. The monolayer conducts magnetic field lines. This creates extreme local inhomogeneities in the field, enhancing magnetophoresis, as exemplified in FIG. 13. More beads are added to the monolayer, forming dendrite structures. Adjacent dendrite structures merge and form an effectively porous network of beads. Before layer formation, shear forces are minimal, see, e.g., FIG. 14. As the porous layer grows, the effective size of the channel reduces and the pressure rises, increasing shear forces on the growing interface. The porous layer saturates in thickness when the shear forces due to flow above it equilibrate with the trapping forces.

Example 6 Multi-frit trapping

[0177] A valve is opened in a 3D microfluidic EIA system. An example of the system is shown in FIG. 18. Beads are loaded into the reactor of the system and trapped on the surface of multiple frits. The valve is then closed, and reagents begin to flow through the reactor. Beads are homogenized in solution. There is maximum exposure of bead surface to analyte/reagents, and bead concentration is reduced. Flow can be managed to run back and forth between the multiple frits to minimize reagent consumption. A given volume of fluid can be used to wash beads back and for the between the frit traps.

Example 7 Active Bead Homogenization for EIA

[0178] FIG. 23 shows an exemplary method comprising active bead homogenization (e.g., coating a collection of beads to a similar extent with reagents and/or analytes). Various solutions are run through the system sequentially, and active bead homogenization may occur in the presence of any of these solutions, including solutions containing a target analytes, solutions containing reagents (antibodies, indicator substrates, etc.), and sterilization solutions.

[0179] FIG. 23A shows microbeads are immobilized on one side of a trap by Magnet B during solution exchange (when solution is actively flowing). The microbeads form a layer of beads as described in Example 5. FIG. 23H shows a 90 degree rotated view of FIG. 23A along the axis of flow. For instance, FIG. 23A may be considered a side view, while FIG. 23H is considered a top view.

[0180] After flow is stopped, magnets may be moved to begin the homogenization of

microbeads. FIG. 23B shows microbeads initially engaged during solution exchange are now disengaged by Magnet B by moving Magnet B away from the trap, and engaged by Magnet A which is moved to be near or against the trap, or at least close enough to exert a magnetic force on the beads. Flow is stopped in FIG. 23B. As the beads move from the side of the trap corresponding to Magnet B to the side of the trap corresponding to Magnet A, the disengaged beads are dispersed in solution, thereby exposing the beads to any reagents (e.g., antibodies, substrates) or analytes. See FIG. 23C. FIG. 23D shows that beads have moved to Magnet A. Disengaging the beads by removing Magnet A and re-engaging with Magnet B will effectively disperse the beads once again before they move back to the side corresponding to Magnet B, thereby providing the beads, and all surfaces thereof, another chance to become exposed to reagents and analytes. See FIG. 23E through FIG. 23G. Throughout the engaging/disengaging process (FIGS. 23B-G), flow is stopped. This process may be repeated multiple times for maximal bead coating and analyte/reagent binding. Engagement and disengagement of magnets are optionally automated. Engagement and disengagement of magnets are optionally

programmed to occur rapidly in order to achieve an accurate result in a relatively short time.

[0181] In addition to active bead homogenization, the system has a detection region and optics arranged for a concentrated signal, therefore providing for ultra-low limits of detection. As shown in FIG. 231, the microchannel has a roughly Z-shaped (or S-shaped) detection region.

This provides a region of the microchannel that is perpendicular to the direction of flow in the remaining microchannel, giving it a region with a second axis of direction (referred to as “aperture section of channel” in FIG. 231). This results in a larger detection region that is exposed to an emitter and detector, than if the microchannel only had one axis of direction ( e.g ., axis of the initial direction of flow). FIG. 23J shows a 90 degree rotated view of FIG. 231 along the axis of initial flow (e.g., flow through the trap). In this example, FIG. 23J is considered a side view and FIG. 231 is considered a top view. As indicated by FIG. 23K, the positions of the optics (emitter and detector) and the direction of the Z-shaped portion of the microchannel may be rotated around the axis of initial flow. However, the aperture section of the channel should line up along its axis with the optic devices for maximized signal detection.

[0182] Optionally, the system has multiple channels with multiple reactors, as shown in FIGS. 25A and 25B. In this example, there is a horizontally mounted optics system relative to the traps shown from a top view. Flow rate is similar in each channel, but each channel has a different path length such that signals from channels are detected separately. Alternatively, flow through each channel is regulated so that signals from each channel are detected individually (e.g.,

sequentially) and do not interfere with one another. Alternatively, flow may be initiated at each channel at a different time point, so that signals from each channel are detected individually (not shown).

[0183] In any instance, magnets may move on a single fixture for consistent active bead homogenization between channels.

Example 8 Comparing surface to volume ratio of homogenized beads to non-homogenized beads

[0184] A first microfluidic system for analyte detection that employs the use of magnetic beads is run in an effort to detect an analyte of interest in a sample where the analyte is suspected to be present at a very low amount. However, this systems does not allow for bead homogenization. Instead, a layer of beads is formed, in which a resulting layer surface is exposed to analytes and reagents. This does not maximize bead surface area. A second microfluidic system is also employed to detect the analyte of interest. However, the second system provides for bead homogenization, exposing the entire surface area of the beads at multiple stages to reagents and analytes.

[0185] Consider the first system, wherein a layer is formed before exposure to a reagent or analyte. For simplicity, the layer is cubic. Even if all surfaces of the layer are exposed to reagent/ anal yte, the surface to volume ratio of the beads altogether is 6/S, wherein S is the side length S. (The volume is S^A3, and the surface area is 6*S^A2. Hence, the surface area to volume ratio is 6*S^A2/S^A3 = 6/S). Beads inside the layer are not exposed in a similar way as the beads on the outer surface of the layer.

[0186] Now consider the second system, wherein all surfaces of all beads are exposed to analyte/reagent through active homogenization. Provide the beads have a diameter 0. l *S, there are 10 beads per side, 100 beads per face, and 1000 beads in the volume. The surface area of each bead is 4*pi*(0.1 *S)^A2. This means that the total surface area to volume ratio is

l000*4*pi*(. l)^A2*S^A2/(4*S^A3/3) = 94.2478/S.

[0187] In this example, active bead homogenization results in a 15.7 times higher surface area for binding. The analyte is detected with the second system, but not the first system.

Claims

CLAIMS What is claimed is:

1. A microfluidic system for performing an assay for a target analyte in a sample, the

microfluidic system comprising:

a) a reactor for binding a target analyte to particles, the reactor comprising at least one trap or mixer for homogenizing the surface of particles with the target analyte;

b) at least one particle manipulator configured to move the particles from a first internal surface to a second internal surface of the reactor in a solution comprising the target analyte, thereby homogenizing the surface of particles with the target analyte;

c) a flow regulator that draws a solution containing the target analyte through the

system; and

d) at least one of a computer, microcontroller, and detector for detecting the target

analyte.

2. The system of claim 1, wherein the at least one particle manipulator is a magnet and the particles are magnetic.

3. The system of claim 1, wherein the at least one particle manipulator is an electrode and the particles are dielectric.

4. The system of claim 1, wherein the at least one particle manipulator comprises the first pump, first valve, first pressure regulator, a second pump, second valve, second pressure regulator or combination thereof, and the reactor comprises the at least one trap, wherein the at least one trap comprises a frit.

5. The system of claim 4, wherein the at least one trap or mixer comprises an internal

surface, wherein the internal surface comprises a material comprising at least one of a metal, a ceramic, and a plastic.

6. The system of claim 1, wherein the microfluidic system comprises at least one of the computer and microcontroller, and the microfluidic system function is automated by at least one of the computer and microcontroller.

7. The system of claim 1, wherein the flow regulator is selected from a pump, a valve, and a constant pressure regulator.

8. The system of claim 1, comprising a fluid logic configured to draw a solution from a reservoir and delivers it to at least one of the flow regulator and reactor.

9. The system of claim 1, wherein all components of the microfluidic system are integrated into a single device.

10. The system of claim 9, wherein the single device is encompassed by a single housing unit.

11. The system of claim 8, wherein the solution is a self-sterilizing solution, and the fluid logic is configured to draw the self-sterilizing solution from the reservoir and deliver it to at least one of the flow regulator and rector to remove essentially all of target analyte, and optionally all of the particles.

12. The system of claim 1, comprising the detector, wherein the detector comprises an optical fiber, and the microfluidic system is compatible with an optical fiber based detector.

13. The system of claim 1, comprising the detector, wherein the detector comprises a mass spectroscopy system, a thermal sensor, or a non-fiber based optical system.

14. A method of performing an assay for a target analyte in a sample, comprising:

a) flowing a first solution of particles into a microfluidic system, wherein the particles are magnetic particles;

b) contacting the microfluidic system at a first external surface of the microfluidic system with at least one magnet to exert a magnetic force on the magnetic particles, thereby drawing the magnetic particles to a first internal surface of the microfluidic system;

c) flowing a second solution comprising at least one of a reagent and a first target analyte into the microfluidic system;

d) withdrawing the magnetic force; and

e) contacting the microfluidic system at a second external surface of the microfluidic system with the at least one magnet to exert the magnetic force on the magnetic particles, thereby drawing the magnetic particles to a second internal surface of the microfluidic system, wherein the magnetic particles are dispersed in the second solution during movement from the first internal surface to the second internal surface, thereby exposing the magnetic particles surfaces to at least one of the reagent and the first target analyte.

15. The method of claim 13, comprising repeating steps b and d, thereby repeatedly exposing magnetic particle surfaces to at least one of the reagent and the first target analyte.

16. The method of claim 1 or 14, wherein the second solution comprises the first target

analyte, and the method further comprises detecting the first target analyte.

17. The method of claim 15, comprising washing the microfluidic system to remove

essentially all of the first target analyte.

18. The method of claim 16, comprising contacting the microfluidic system with a second target analyte and detecting the second target analyte.

19. A method of performing an assay for a target analyte in a sample, comprising:

a) contacting a microfluidic system with a first sample solution containing a first target analyte, wherein the first target analyte binds a first set of particles in the microfluidic system;

b) detecting the first target analyte;

c) washing the microfluidic system to remove essentially all of the first target analyte, and optionally all of the particles;

d) contacting the microfluidic system with a second sample solution containing a second target analyte, wherein the first target analyte binds a second set of particles in the microfluidic system; and

e) detecting the second target analyte.

20. The method of claim 19, comprising:

a) flowing particles into the microfluidic system thereby exposing particles surfaces to the target analyte, wherein the particles are magnetic particles;

b) contacting the microfluidic system with at least one magnet to draw the magnetic particles to a first internal surface of the microfluidic system;

c) contacting the microfluidic system with the at least one magnet to draw the magnetic particles to a second internal surface of the microfluidic system;