Whole blood CMP in a DMF device without dilution

Abstract

Aspects of the present disclosure provide an analysis cartridge that allows analyzing clinical chemistry or cell types in a fluid sample, such as a blood sample. In certain embodiments, the analysis cartridge comprises a comprehensive metabolic panel (CMP) analysis module. Additional embodiments may further include a complete blood count (CBC) module for analyzing cells. In many embodiments, digital microfluidics (DMF) is used to move samples through the analysis cartridge. Additional embodiments define additional cuvette types and arrangements for detection. Also provided are systems and methods of analyzing fluid samples in the analysis cartridges provided herein.

Description

WHOLE BLOOD CMP IN A DMF DEVICE WITHOUT DILUTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 612,715, filed December 20, 2023, which application is incorporated herein by reference in its entiretyINTRODUCTION

[0002] Analyses of biological samples, such as blood samples often involve testing cellular components as well as chemical components. Analyses of cellular and chemical components are typically performed in separate cartridges and / or analyzers. Such separate analysis increases costs and may cause inconsistencies in the results. For example, for the same sample, different storage, or processing in different cartridges and / or analyzers may produce different results.

[0003] Moreover, analyses of biological samples, such as blood samples, are limited by the availability of the samples. Therefore, analysis of the sample using the smallest possible amount is desirable.SUMMARY

[0004] This summary is meant to provide some examples and is not intended to be limiting of the scope of the invention in any way. For example, any feature included in an example of this summary is not required by the claims, unless the claims explicitly recite the features. Various features and steps as described elsewhere in this disclosure may be included in the examples summarized here, and the features and steps described here and elsewhere can be combined in a variety of ways.

[0005] Some embodiments describe an analysis cartridge, including an input chamber for receiving a sample, one or more reaction lanes in fluid communication with the input chamber, each of the one or more reaction lanes including a first reaction chamber and a detection chamber, where the first reaction chamber is configured to perform a chemical reaction to detect an analyte within the sample, where the first reaction chamber includes a digital microfluidic (DMF) electrode to move a portion of the sample into the reaction chamber, and where the detection chamber is configured to allow detection of the chemical reaction.

[0006] In some additional embodiments, each of the one or more reaction lanes further includes a filling chamber located between and in fluid communication with the first reaction chamber and the detection chamber, such that a reacted sample is moved from the reaction chamber to the filling chamber using a DMF electrode.

[0007] In some additional embodiments, the reacted sample moves into the detection chamber via a method selected from diffusion, convection, pumping, airlift pumping, siphoning, applied pressure, gravity-driven flow, density gradients, temperature gradients, chemical gradients, pressure gradients (positive or negative), pneumatic pressure, gas-producing chemical reactions, centrifugal flow, capillarypressure, wicking, electric field-mediated, electrode-mediated, electrophoresis, dielectrophoresis, magnetophoresis, magnetic fields, magnetically driven flow, optical force, chemotaxis, phototaxis, surface tension gradient driven flow, Marangoni stresses, hermos-capillary convection, surface energy gradients, acoustophoresis, surface acoustic waves, electroosmotic flow, thermophoresis, electrowetting, and optoelectrowetting.L0008J In some additional embodiments, the DMF electrode is coated with a reagent for a particular chemical reaction.

[0009] In some additional embodiments, the detection chamber includes an agglutination reagent to induce aggregation within the sample.

[0010] In some additional embodiments, the chemical reaction is a colorimetric reaction and where the detection chamber is configured to optically detect the component based on a colorimetric reaction.

[0011] In some additional embodiments, the detection chamber is configured to electrochemically detect the component.

[0012] In some additional embodiments, each reaction lane further includes a second reaction chamber in fluid communication with the first reaction chamber and including a DMF electrode to move the portion of the sample from the first reaction chamber into the second reaction chamber, where movement from the first reaction chamber into the second reaction chamber mixes the reacted sample.

[0013] In some additional embodiments, the second reaction chamber of at least one reaction lane includes a second reagent for its chemical reaction.

[0014] In some additional embodiments, each reaction lane includes at least three reaction chambers, where each reaction chamber is in fluid communication with every other reaction chamber, where movement between the reaction chambers mixes the reacted sample.

[0015] In some additional embodiments, at least one reaction chamber includes an affinity matrix capable of isolating a component from the sample.

[0016] In some additional embodiments, the affinity matrix is included of a bead with a conjugated antibody or conjugated antigen.

[0017] In some additional embodiments, the bead is magnetic, paramagnetic, or susceptible to a magnetic field.

[0018] In some additional embodiments, the sample is a blood sample.

[0019] In some additional embodiments, the blood sample is selected from whole blood, peripheral blood, arterial blood, venal blood, and capillary blood.

[0020] In some additional embodiments, the sample further includes an anticoagulant.

[0021] In some additional embodiments, the anticoagulant is selected from heparin, coumadin, dabigatran, rivaroxaban, apixaban, edoxaban, enoxaparin, dalteparin, fondaparinux, argatroban, bivalirudin, betrixaban, hirudin, ethylenedi aminetetraacetic acid (EDTA), sodium citrate, oxalate, citrate - phosphate-dextrose, acid-citrate-dextrose, sodium fluoride, potassium EDTA, and sodium heparin.

[0022] In some additional embodiments, the chemical reaction for each reaction lane is configured to detect one or more of the following glucose, calcium, sodium, potassium, carbon dioxide, chloride,albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.

[0023] In some additional embodiments, the plurality of reaction lanes includes 14 reaction lanes, where the chemical reaction of each reaction lane is configured to detect one of glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.

[0024] In some additional embodiments, the detection chamber includes a top surface constructed of a plastic that is optically clear between approximately 340 nm to approximately 850 nm.

[0025] In some additional embodiments, the top surface is constructed of polyethylene terephthalate, polymethyl methacrylate, polycarbonate, polystyrene, cyclo-olefin copolymer, polysulfone, polyetherimide, polyvinyl chloride, and polyolefin.

[0026] In some additional embodiments, each reaction chamber has a width of between approximately 1 mm and 5 mm.

[0027] In some additional embodiments, each reaction chamber has a length of between approximately 1 mm and 5 mm.

[0028] In some additional embodiments, each reaction chamber has a width of approximately 2 mm and a length of approximately 2 mm.

[0029] In some additional embodiments, each reaction chamber has a height sufficient to contain approximately 0.7 pL of volume.

[0030] In some additional embodiments, each reaction chamber has a height of between approximately 0.050 mm and 0.500 mm.

[0031] In some additional embodiments, each reaction chamber has a height of approximately 0.175 mm.

[0032] In some additional embodiments, each optical chamber has a width of approximately 2 mm and a length of approximately 6 mm.

[0033] In some additional embodiments, each optical chamber has a height sufficient to contain between approximately 0.6 pL to approximately 2.4 pL of volume.

[0034] In some additional embodiments, each optical chamber has a height of between approximately 50 pm to approximately 200 pm.

[0035] Some additional embodiments also include at least one reaction lane that is configured to determine one or more of a red blood cell count, a white blood cell count, a differential white blood cell count, a platelet count, a hemoglobin level, and a hematocrit level.

[0036] In some additional embodiments, a differential white blood cell count determines a level of at least two of the following white blood cell types: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

[0037] In some additional embodiments, a differential white blood cell count determines a level of at least the following white blood cell types: neutrophils, lymphocytes, and monocytes.

[0038] In some additional embodiments, the analysis cartridge includes fourteen reaction lanes in fluid communication with the input chamber, twelve of the fourteen reaction lanes include two reaction chambers, a filling chamber, and a detection chamber, where one of the two reaction chambers includes a reagent to perform a chemical reaction on the sample, and two of the fourteen reaction lanes include at least 4 reaction chambers, a filling chamber, and a detection chamber, where two of the four reaction chambers include a reagent to perform a chemical reaction on the sample and are separated by at least one reaction chamber, such that each reaction occurs in sequence.

[0039] In some additional embodiments, each reaction lane is configured to detect one of glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.

[0040] Various embodiments describe a method of analyzing a fluid sample, including loading the fluid sample in any one of the analysis cartridges described above and analyzing the fluid sample.

[0041] Some additional embodiments also include analyzing between 0.1 pL to 40 pL of the fluid sample.

[0042] Some embodiments describe a method of analyzing a fluid sample in the analysis cartridge as described above, the method including loading a fluid sample into the input chamber, applying an electrical charge to at least one DMF electrode to move a portion of the fluid sample into a reaction chamber, and detecting at least one component in the portion of the fluid sample based on the chemical reaction.

[0043] In some additional embodiments, where detecting at least one component includes detecting at least one component selected from a glucose level, a calcium level, a sodium level, a potassium level, a carbon dioxide level, a chloride level, an albumin level, a total protein level, an alkaline phosphatase level, an alanine transaminase level, an aspartate aminotransferase level, a bilirubin level, a blood urea nitrogen level, a creatinine level, a red blood cell count, a white blood cell count, a platelet count, a hemoglobin level, a hematocrit level, a neutrophil count, a lymphocyte count, a monocyte count, an eosinophil count, and a basophil count.

[0044] In some additional embodiments, detecting at least one component includes imaging the detection chamber of the analysis cartridge.

[0045] In some additional embodiments, the fluid sample is a blood sample.

[0046] In some additional embodiments, the blood sample is selected from whole blood, peripheral blood, arterial blood, and venal blood.

[0047] Additional embodiments can also include mixing an anticoagulant with the blood sample.

[0048] In some additional embodiments, the anticoagulant is selected from heparin, coumadin, dabigatran, rivaroxaban, apixaban, edoxaban, enoxaparin, dalteparin, fondaparinux, argatroban, bivalirudin, betrixaban, hirudin, ethylenediaminetetraacetic acid (EDTA), sodium citrate, oxalte, citrate - phosphate-dextrose, acid-citrate-dextrose, sodium fluoride, potassium EDTA, and sodium heparin.

[0049] Additional embodiments can also include segmenting the image to identify cellular components and plasma components of the sample.

[0050] In some additional embodiments, segmenting includes utilizing an image processing algorithm.

[0051] Additional embodiments describe an analysis cartridge including one or more cuvettes, where each cuvette includes a reflectively coated upper surface, a reflectively coated lower surface bottom, a light entry pore, and a light exit pore, where the upper surface, the lower surface, the light entry pore, and the light exit pore are positioned such that light entering the light entry pore at an incident angle reflects off of the upper surface and the lower surface and exits the light exit pore.

[0052] In some additional embodiments, the light entry pore and the light exit pore are positioned within the upper surface.

[0053] In some additional embodiments, the reflective coating on the upper surface is a ground layer and the reflective coating on the lower surface is a digital microfluidic (DMF) electrode.

[0054] In some additional embodiments, the cuvette further includes a dielectric layer disposed on the DMF electrode.

[0055] In some additional embodiments, the incident angle is approximately 45°.

[0056] In some additional embodiments, the upper surface and the lower surface are positioned approximately 0.175 mm apart.

[0057] In some additional embodiments, the light entry pore and the light exit pore are positioned approximately 7.07 mm apart.

[0058] In some additional embodiments, the light entry pore and the light exit pore are each approximately 0.8 mm to approximately 1 mm in width.

[0059] Additional embodiments can also include a plurality of DMF electrodes configured to move a sample into each of the one or more cuvettes.

[0060] Additional embodiments describe an analysis system including an analysis cartridge as described above, a light source, and a detector, where the analysis cartridge, the light source, and the detector are positioned such that light emitted from the light source enters the light entry pore and exits from the light exit pore of each cuvette and impinges on the detector.

[0061] Additional embodiments can also include an optical element to collimate the light emitted by the light source.

[0062] In some additional embodiments, the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.

[0063] In some additional embodiments, the detector includes an array of pixels.

[0064] In some additional embodiments, the array of pixels is selected from a one dimensional array and a two dimensional array.

[0065] Additional embodiments describe an analysis system including an analysis cartridge including a plurality of cuvettes, each cuvette including an upper surface and a lower surface, where the upper surface includes a ground layer and the lower surface includes a digital microfluidic (DMF) electrode, a light source, and a detector, where the analysis cartridge, the light source, and the detector are positioned suchthat light emitted from the light source enters the cuvette along its length, passes through the sample, and impinges on the detector.

[0066] Additional embodiments can also include a first optical element to route the light from the analysis cartridge to the detector.

[0067] Additional embodiments can also include a second optical element to route the light from the light source to the analysis cartridge.

[0068] In some additional embodiments, the first and second optical element are each selected from a lens and a mirror.

[0069] Additional embodiments can also include an optical element to collimate the light emitted by the light source.

[0070] In some additional embodiments, the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.

[0071] In some additional embodiments, the detector includes an array of pixels.

[0072] In some additional embodiments, the array of pixels is selected from a one dimensional array and a two dimensional array.

[0073] Additional embodiments can also include a set of pinholes to minimize stray light.

[0074] In some additional embodiments, the set of pinholes have a thickness of approximately 10 mm to approximately 30 mm, and the pinholes have a diameter from approximately 0.5mm to approximately 1.0.

[0075] In some additional embodiments, the set of pinholes arc located between the light source and the analysis cartridge or between the analysis cartridge and the detector.

[0076] Additional embodiments can also include two sets of pinholes to minimize stray light, where a first set of pinholes are located between the light source and the analysis cartridge and a second set of pinholes are located between the analysis cartridge and the detector.

[0077] Additional embodiments describe an analysis cartridge, including two or more cuvettes, where each cuvette includes a central through hole, a cuvette region, and a capillary region, where the cuvettes are stacked such that the central through holes are aligned and in fluid communication with each other to form a sample reservoir, and where the capillary region can move a portion of a sample to the cuvette region.

[0078] In some additional embodiments, the two or more cuvettes are formed as alternating layers of a hydrophilic film and a pressure sensitive adhesive.

[0079] In some additional embodiments, the central through holes have a diameter ranging from 2 mm to 4 mm.

[0080] In some additional embodiments, the cuvette regions have a constant width.

[0081] In some additional embodiments, the cuvette regions have a variable width.

[0082] In some additional embodiments, the cuvette regions have a width ranging from 2 mm to 4 mm.

[0083] Additional embodiments describe an analysis system, including an analysis cartridge as described above, a light source, and a detector, where the light source, the analysis cartridge, and the detector are arranged, such that a light emitted from the light source passes through the width of the cuvette region of each analysis cartridge and impinges on the detector.

[0084] Additional embodiments can also include an optical element to route the light from the analysis cartridge to the detector.

[0085] In some additional embodiments, the optical element is a mirror.

[0086] In some additional embodiments, the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.

[0087] In some additional embodiments, the detector includes an array of pixels.

[0088] In some additional embodiments, the array of pixels is selected from a one dimensional array and a two dimensional array.BRIEF DESCRIPTION OF THE FIGURES

[0089] FIGs. 1A-1C illustrate examples of the principles underlying digital microfluidics (DMF) in an open (FIG. 1A) and a closed (FIG. IB) chip, while FIG. 1C provides an example of how a droplet within a DMF array may be manipulated, in accordance with various embodiments.

[0090] FIGs. 2A-2C provide illustrations of an exemplary analysis cartridge or chip for a fluid sample, where FIG. 2A illustrates an exemplary analysis cartridge with fourteen reaction lanes, each with one or more reaction chambers; FIG. 2B illustrates a branched arrangement of reaction chambers that allows for selective removal of a component within a sample; and FIG. 2C illustrates a side view of an exemplary analysis cartridge showing a reaction chamber, a filling chamber, and a detection chamber, in accordance with various embodiments.

[0091] FIGs. 3A-3B provide micrographs of non-agglutinated (FIG. 3A) and agglutinated (FIG. 3B) blood samples in accordance with various embodiments.

[0092] FIGs. 4A-4C illustrate various views of an exemplary analysis cartridge and system that uses internal reflectance to increase light pathlength in accordance with various embodiments.

[0093] FIGs. 5A-5C illustrate various views of an exemplary analysis cartridge and system that uses a linear dimension of a cuvette to increase light pathlength in accordance with various embodiments.

[0094] FIGs. 6A-6C illustrate various views of an exemplary analysis cartridge and system that uses a stacked arrangement in accordance with various embodiments.

[0095] FIG. 7 illustrates an exemplary setup using front and back pinholes to minimize crosstalk between microcuvettes in accordance with various embodiments.

[0096] FIG. 8 provides tables of exemplary data for various blood component measurements made using microcuvettes and setups in accordance with various embodiments.DETAILED DESCRIPTIONF0097] Certain aspects of the present disclosure provide an analysis cartridge that allows analyzing cellular as well as chemical components of a fluid sample, such as a blood sample (including undiluted whole blood). In certain embodiments, the analysis cartridge comprises a CMP module and a CBC module for analyzing cells. The CBC module comprises an imaging chamber comprising a top panel and a bottom panel separated by a suitable distance to produce a monolayer of cells between the panels. The CBC module also comprises a mechanism to deliver into the imaging chamber a small volume of fluid sample, for example, between 0.1 and 40 pL of fluid sample. In certain embodiments, the CMP module is configured to analyze, for example, electrochemically or optically, multiple metabolites in a fluid sample, for example, a blood sample. Also provided herein are methods of analyzing a fluid sample, such as a blood sample (including undiluted whole blood) in the analysis cartridges provided herein.

[0098] Before the devices, methods, and systems of the present disclosure are described in greater detail, it is to be understood that the devices, methods, and systems are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the devices, methods, and systems will be limited only by the appended claims.DEFINITIONS

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0100] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0101] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0102] An “amount” as used herein refers to a quantity specified (e.g., high or low) or a number e.g., where the number is a level, such as a position on a real or imaginary scale of amount or quantity, or aconcentration, such as, for example, a relative amount of a given substance contained within a solution or in a particular volume of space, e.g., the amount of solute per unit volume of solution .

[0103] "Analyte", "target analyte", "analyte of interest" as used interchangeably herein, refers to a substance, material or chemical constituent the presence, absence and / or amount of which is being analyzed in a sample (e.g., capillary blood sample and / or venous blood sample) obtained from a subject. In some aspects, the analyte is a biomolecule. Non-limiting examples of biomolecules include macromolecules such as proteins, lipids, and carbohydrates. In certain instances, the analyte may be hormones, antibodies, growth factors, cytokines, enzymes, receptors (e.g., neural, hormonal, nutrient, and cell surface receptors) or their ligands, cancer markers (e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g., troponin, creatine kinase, Creatinine kinase-cardiac muscle biomarker (CK-MB), and the like), toxins, drugs (e.g., drugs of addiction), metabolic agents (e.g., including vitamins), and the like. Non-limiting examples of protein analytes include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, or the like.

[0104] “Component,” “components,” or “at least one component,” refer generally to a capture antibody, a detection or conjugate a calibrator, a control, a sensitivity panel, a container, a buffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, a detection reagent, a pretreatment reagent / solution, a substrate (e.g., as a solution), a stop solution, and the like that can be included in a kit for assay of a test sample, such as a patient urine, whole blood, serum or plasma sample, in accordance with the methods described herein and other methods known in the art. Some components can be in solution or lyophilized for reconstitution for use in an assay.

[0105] As used herein the term “hydrophobic”, such as in reference to a “hydrophobic material” (e.g., membrane, film, etc.) refers to those materials having a water contact angle greater than about 80 degrees.

[0106] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non- human. In some embodiments, the subject is a human. The subject or patient may be undergoing other forms of treatment.DEVICES

[0107] As summarized above, certain aspects of the present disclosure provide analysis cartridges for analyzing chemical components of fluid samples, such as blood samples, including whole and / or undiluted blood. Also, certain aspects of the disclosure provide analysis cartridges that allow analysis of small volumes of fluid samples.

[0108] A suitable fluid sample analyzed in the analysis cartridges disclosed herein can be blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, feces, or the like. Additional fluid samples that could beanalyzed in the analysis cartridges disclosed herein can be readily identified by a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0109] In certain embodiments, the analysis cartridge comprises a CMP module, a CBC module, a lipid module, an HbAlc module, an immunoassay module, and any combination thereof — e.g., CMP and CBC, CMP and lipid, CBC and HbAlc, etc.

[0110] In many instances, such modules utilize digital micro fluidics (DMF) to actively move portions of a sample around an analysis cartridge. Figures 1A-1B provide a schematic illustration of the principles of DMF. Specifically, Figure 1 A illustrates an example of an “open” DMF system, that does not possess a top layer or film, while Figure IB provides a “closed” DMF system that includes a top panel or layer. As shown, many instances include a substrate 102 upon which the remaining components are installed. In many instances, the substrate is inert and / or non-conducting. Suitable materials can include glass, plastics, ceramics, and / or any other suitable material. Within the substrate 102, a patterned array of individually controllable electrodes 104, 106 are implanted. Electrodes 104, 106 can be patterned in any desirable path or pattern to allow movement of a droplet. Such patterns can be a one dimensional (e.g., a singular linear path), two dimensional (e.g., moving along two linear directions), and potentially three dimensional. The electrodes 104, 106 can be separated with a dielectric layer 108 to act as an insulator between the electrodes and / or to build up charges and electrical field gradients on the chip. Suitable dielectric materials can include polyethylene, polypropylene, Teflon, mica, ceramics, glass, silicon dioxide, silicon nitride, epoxy resins, ceramic -polymer composites, cyanoethyl pullulan (CEP), and / or other suitable materials that can operate as a dielectric. In many instances, the substrate 102 and electrodes 104, 106 include a hydrophobic layer 110 disposed on its surface to decrease the surface energy and possibly assist in movement of an aqueous sample (e.g., blood). In closed systems — e.g., Figure IB — a top layer may also be constructed of a substrate 102 with a hydrophobic layer 110. The top layer may further include a ground layer 112.

[0111] Figures 1A-1B further illustrate the active movement of a droplet. In such situations, a voltage is applied to a first electrode 106, which is adjacent to the droplet while simultaneously deactivating a second electrode 104 upon which the droplet sets. With this action, the droplet moves to the first electrode 106. This process allows for a droplet to be moved in multiple directions and / or cover various pathways by activating a desired electrode. Additionally, electrode activation patterns can be varied to allow for mixing, perturbing, merging, splitting, and / or another action to the droplets. For example, Figure 1C illustrates top and side views of a process of splitting a droplet into two individual droplets. As illustrated on the left panels of the diagrams, a droplet sets on an initial electrode 114. By activating adjacent electrodes 116, the droplet may be split into two smaller droplets, each setting on adjacent electrodes 116. This splitting process may be reversed by activating electrode 114 to recombine the droplets. In certain instances, the splitting process may not be taken to completion (e.g., middle panel), before allowing the droplet to return to its original form, such as by activating electrode 114. With this agitation or perturbation functionality, individual droplets can be used as distinct microreactors to conduct specific reactions.

[0112] In various embodiments substrate 102 may be of any thickness that provides strength, structure, or other property to a cartridge. In many instances, the substrate is between approximately 0.100mm and approximately 0.500 mm, such as 0.100 mm, 0.125 mm, 0.150 mm, 0.175 mm, 0.200 mm, 0.250 mm, 0.300 mm, 0.350 mm, 0.400 mm, 0.450 mm, or 0.500 mm. IN embodiments using a colorimetric or other analytical method using light transmittance, the substrate may also be optically clear within at least some range of wavelengths, as will be discussed below.

[0113] Turning to Figure 2A, an example of an analysis cartridge of many embodiments. The illustrated embodiment includes an input chamber 202 for receiving a sample and at least one (or one or more) reaction lane 204, 204’ in fluid communication with the input chamber 202. In many embodiments an input chamber 202 is capable of accepting whole blood. In various instances, the whole blood is undiluted. Without dilution, these cartridges may be used directly without additional steps or processes that can be susceptible to contamination, spillage, or other spoilage of the sample. Reaction lanes 204, 204’ can be a single reagent reaction lane 204 or a multi-reagent reaction lane 204’. Each reaction lane 204, 204’ includes at least one reaction chamber 206 and a detection chamber 208. Single reagent reaction lanes 204 include a single reaction chamber 206 with a reagent (•), while multi-reagent reaction lanes 204’ include multiple reaction chambers 206 with a reagent (•). In many instances, the reagent or reagents are necessary for a reaction to detect an analyte within a sample. Such reagents may be applied to a surface within the reaction chamber, such as to an electrode. The reagent(s) can be deposited as a desiccated, lyophilized, and / or otherwise dry application within the reaction chamber. In some instances, the reagent(s) is provided under a disintegrating coating to prevent mixing, contamination, loss, and / or other complication. In coated embodiments, the underlying reagent can be wet or dry. While multi-reagent reaction lanes 204’ arc illustrated with reaction chambers 206 between reaction chambers 206 with a reagent (•), in certain instances, the reaction chambers 206 with a reagent (•) may be consecutive or adjacent.

[0114] As illustrated in Figure 2A, each of the one or more reaction lane may comprise additional reaction chambers 206 without a reagent. These additional reaction chambers can be used to mix, agitate, or otherwise perturb a sample to assist in the reaction, such as illustrated in Figure 1C. Thus, each of the one or more reaction lanes 204, 204’ can include any number of reaction chambers 206 to add a reagent and mix the sample. Various instances, may have as few as 1 reaction chamber 206 up to approximately 10 reaction chambers 206, including 1 reaction chamber, 2 reaction chambers, 3 reaction chambers, 4 reaction chambers, 5 reaction chambers, 6 reaction chambers, 7 reaction chambers, 8 reaction chambers, 9 reaction chambers, or 10 reaction chambers. The reaction chambers 206 can be configured in a linear or non-linear pattern to provide the desired properties (e.g., to mix and / or for a particular form factor). As described above, the patterning can be one dimensional, two dimensional, or three dimensional.

[0115] Certain embodiments may include a configuration of reaction chambers 206 that can allow for separation and / or removal of certain components (e.g., certain cell types, certain cells displaying a particular antigen(s), etc.). Figure 2B provides a diagram of a branched arrangement of reaction chambers 2Q6a-d. In this example, a sample 212 is combined with an affinity matrix 214 in reaction chamber 206a. The affinity matrix 214 can be static (e.g., bound to the analysis chamber) or free, such as beads, particles or other unattached feature. The affinity matrix 214 can acquire its affinity by being conjugated to anantibody (e.g., to remove specific antigens and / or cells displaying such antigens) or an antigen or ligand to remove particular antibodies or cells with receptors for the particular ligand. When the affinity matrix 214 is comprised of beads, the beads can be magnetic, paramagnetic, and / or susceptible to a magnet to allow for removal of the beads with an application of a magnetic field. If the affinity matrix 214 is comprised of beads that are susceptible to a magnetic field (e.g., magnetic, paramagnetic, otherwise susceptible to a magnet, etc.), a magnetic field 216 may be applied to reaction chamber 206a to isolate the beads.(01161 If the purpose of the affinity matrix 214 is to remove a component, the sample 212 can be moved to a downstream reaction chamber 2066 for a subsequent reaction or detection. However, in certain instances, the goal is to isolate a desired component using the affinity matrix 214. In these instances, the unbound sample 218 can be moved to a waste chamber 206c. Subsequently, a solvent, eluent, or wash 220 can be moved from a reservoir chamber 206d to reaction chamber 206a to elute the bound sample. This eluted sample can be moved to a downstream reaction chamber 2066 for a subsequent reaction or detection.

[0117] The detection chamber 206 allows a portion of the sample to undergo a reaction, the results of which can be observed, measured, or otherwise analyzed in the detection chamber 208. In certain instances, the reaction results are colorimetric, chromatographic, electrochemical, and / or any other form of chemical reaction. Depending upon the reaction type (e.g., colorimetric, electrochemical, etc.), the detection chamber 208 can be configured to detect the particular results. For example, detection chamber 208 can be an optical chamber to detect a colorimetric reaction. In some instances, the reaction may be detected via electrical conductance, such as for electrochemical reactions.

[0118] In numerous embodiments, a colorimetric reaction is measured based on optical density that is measured through the detection chamber via one or both of reflective illumination or transmitted illumination. Specifically, optical density can be detected or measured based on the Beer-Lambert Law: A — logWhere A is optical density or absorbance, Z is light intensity through the chemical component, and Io is the incident light intensity, £ is the molar absorptivity constant, £ is the path length, and c is the analyte concentration. Given the above, the equation can be rearranged to:A c=eWhere can be calibrated for each analyte.

[0119] In many instances, DMF — as previously described — is used to move a sample droplet through the at least one reaction chambers 206. However, DMF may not be suitable to move the sample into the detection chamber 208, where an electrode may obscure or prevent proper determination of an analyte, such as when absorbance, transmittance, and / or another form of photometry is used to detect and / or determine the analyte. Various methods may be used to move the sample into detection chamber 208, including diffusion, convection, pumping, airlift pumping, siphoning, applied pressure, gravity-driven flow, density gradients, temperature gradients, chemical gradients, pressure gradients (positive or negative), pneumatic pressure, gas-producing chemical reactions, centrifugal flow, capillary pressure,wicking, electric field-mediated, electrode-mediated, electrophoresis, dielectrophoresis, magnetophoresis, magnetic fields, magnetically driven flow, optical force, chemotaxis, phototaxis, surface tension gradient driven flow, Marangoni stresses, hermos-capillary convection, surface energy gradients, acoustophoresis, surface acoustic waves, electroosmotic flow, thermophoresis, electrowetting, opto-electrowetting, a pipette, a peristaltic pump, a syringe pump, a pressure-drive flow control pump, and / or any other method or device to move a sample into a detection chamber 208. When capillary action is used to move the sample into the detection chamber 208, such capillary action may include passive or active feedback. To fill the detection chamber 208 via capillary action, certain embodiments include a filling chamber 210 located between the at least one reaction chamber 206 and the detection chamber 208 and in fluid communication with both. Figure 2C illustrates a side view of a reaction chamber 206, detection chamber 208, and filling chamber 210. As can be seen in this side view, the term “chamber” refers to the region of the analysis cartridge, rather than individual or sectioned off boxes. In many instances, the filling chamber 210 is similar to a reaction chamber 206 (e.g., includes an electrode); however, the filling chamber 210 interfaces with the detection chamber 208 via a descending top surface, which allows capillary action to move the sample 212 into the detection chamber 208 once the sample is moved into a filling chamber 210. The capillary forces can follow the Young-Laplace equation, and the chamber height can be adjusted to for a particular or desired force. In many instances, analysis cartridges can include one or more spacing pillars 222 within the detection chamber 208. Spacing pillars 222 can ensure a fixed height of a detection chamber 208 to prevent any distortion, variable pathlength, and / or any other situation that can interfere with or alter detection. The spacing pillars 222 can be placed at regularly (e.g., patterned at specific dimensions) or irregularly (e.g., random). In certain instances, spacing pillars 222 may have a variable density, such that more spacing pillars 222 have a higher density in a specified area in one region and are lower density in other regions. While described as pillars, spacing pillars 222 can be of any shape so long as the distance between a top and bottom surface is the same. Such shapes can be a cylinder, a column, a cube, a cuboid, a sphere, any other regular or irregular shaped, and combinations thereof.

[0120] Returning to Figure 2A, the sizes of the chambers within a reaction lane 204 (i.e., reaction chamber 206, detection chamber 208, filling chamber 210) are sufficient to allow an accurate reaction, mixing, and / or observation of the reaction. In various instances, each reaction lane 204 has a uniform width w. In other instances, width w can vary. Variation in width w can have various purposes, including to mix or agitate a sample, to alter the form factor and / or any other reason. Chamber height (perpendicular to plane of Figure 2) and length / may vary based on desired volume and / or function for each chamber. For example, chamber dimensions may be formed to replicate a smear between a top and bottom panel — i.e., a thin separation of approximately the diameter of one cell in distance. In such instances, volume may be achieved by increasing chamber length and decreasing chamber height.

[0121] In various instances, reaction lane width (and reaction chamber widths) may be between 1 mm and 5 mm, such as a size selected from approximately 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm. In some preferred instances, lane and chamber width is 2 mm. As noted above, height and length dimensions may be altered to provide a desirable sample volume. In some instances a desirable sample volume for eachreaction is between 0.5 |iL and 40 pL, including approximately 0.5 pL, 0.7 pL, 1.0 pL, 1.25 pL, 1.5 pL,1.75 pL, 2.0 pL, 2.5 pL, 3.0 pL, 3.5 pL, 4.0 pL, 4.5 pL, 5.0 pL, 7.5 pL, 10.0 pL, 12.5 pL, 15 pL, 17.5 pL, 20 pL, 22.5 pL, 25 pL, 30 pL, 35 pL, or 40 pL. In some preferred embodiments, the volume is approximately 0.7 pL. To achieve this volume, reaction chamber length and height may be altered accordingly. In various instances, the length may be selected to match the width as described above, while the height is adjusted for volume. In various instances, reaction lane height may be between 0.050 mm and 0.500 mm, such as a size selected from approximately 0.050 mm, 0.075 mm, 0.100 mm, 0.125 mm, 0.150 mm, 0.175 mm, 0.200 mm, 0.250 mm, 0.300 mm, 0.350 mm, 0.400 mm, 0.450 mm, or 0.500 mm. In some instances, if the width and length are each approximately 2 mm, the height can be approximately 0.175 mm to provide a desired volume of approximately 0.7 pL.

[0122] Regarding the detection chamber 208, the height of the detection chamber 208 may be lower height to allow for capillary action to fill the detection chamber 208. Additionally, a lower height may improve imaging and / or measurement of the sample. In such instances, the height may be between approximately 2 pm and approximately 200 pm. For low resolution imaging, larger sizes can be used, such as approximately 50 pm and approximately 200 pm, such as approximately 50 pm, 55 pm, 60 pm, 65 pm, 70 pm, 75 pm, 80 pm, 85 pm, 90 pm, 95 pm, 100 pm, 110 pm, 120 pm, 125 pm, 130 pm, 140 pm, 150 pm, 175 pm, or 200 pm. Low resolution imaging may be sufficient for clinical chemistry and / or CMP assays. For high resolution imaging, which can be used for CBC assays, including WBC differential assays, the height of the detection chamber may be reduced, such as to between approximately 2 pm and approximately 5 pm, including approximately 2 pm, 2.25 pm, 2.5 pm, 2.75 pm, 3 pm, 3.25 pm, 3.5 pm,3.75 pm, 4 pm, 4.25 pm, 4.5 pm, 4.75 pm, 5 pm. With such factors, the length may be the desirable aspect to alter in order to provide the desired volume for the detection chamber. In many instances, the desired length is approximately 2 mm to approximately 30 mm, depending on height. As such, the chamber length can be approximately 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 12.5 mm, 15 mm, 17.5 mm, 20 mm, 22.5 mm, 25 mm, 27.5 mm, or 30 mm. Additionally depending on the type of reaction or detection, the volume of the detection chamber is approximately 0.3 pL to 3.0 pL of volume (e.g., approximately 0.3 pL, 0.6 pL, 0.8 pL, 1.0 pL, 1.2 pL, 1.4 pL, 1.6 pL, 1.8 pL, 2.0 pL, 2.2 pL, 2.4 pL, 2.6 pL, 2.8 pL, or 3.0 pL). As a nonlimiting example, a reaction from a CMP test may utilize a length of approximately 6 mm and volume of approximately 0.6 pL to approximately 2.4 pL, while the detection chamber for an assay in a CBC test may have a volume of approximately 0.3 pL and a length of approximately 30mm. One of skill in the art will understand how to manipulate the dimensions of length, height, and width as necessary for a particular ability to detect an assay and / or utilize a specified volume. For example, a detection chamber for an assay in a CBC test may have an increased width as compared to a reaction chamber. For example, if the reaction chambers have a width of approximately 2 mm, the detection chamber may have a width of approximately 6 mm — in such instances a filling chamber can have the same width as the reaction chamber (2 mm) or the filling chamber can act as a transition, where the width increases across its length.

[0123] As noted previously, the reaction may be colorimetric or otherwise optically detectable. To accomplish this, the detection chamber (which may be considered an “optical chamber” or “imaging chamber” in these instances) may be optically transparent within the visible light spectrum. In certain instances, the detection / optical chamber is optically clear between UV (e.g. approximately 340 nm) and near infrared (NIR; e.g. approximately 850 nm). The optical clarity may be on just a single surface (e.g., either top or bottom surface) or on both top and bottom surfaces, depending on if the measurement technique is based on reflectance (transparent only a single surface) or transmittance (transparent on both surfaces). The transparency can be provided by the particular component used to construct the surfaces of the detection chamber. Such components can include certain types of glass or plastics. A non-limiting list of examples of suitable plastics include polyethylene terephthalate, polymethyl methacrylate, polycarbonate, polystyrene, cyclo-olefin copolymer, polysulfone, polyetherimide, polyvinyl chloride, and polyolefin.

[0124] In various instances, the reagents are specific to detect at least one component for a CMP analysis. In instances that perform a CMP, each lane of the bioassay chip is configured to detect one or more component selected from: glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine. Certain embodiments include fourteen reaction lanes, where each lane is configured to detect only one of the following components: glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine. Additional embodiments can include one or more lanes to perform a CBC analysis. Similar to above, various reagents may be added to reaction chambers to assist in a red blood cell count, a white blood cell count (including differential counting of neutrophils, lymphocytes, monocytes, eosinophils, and basophils), a platelet count, determining hemoglobin level, and determining hematocrit level.

[0125] Each reaction to detect at least one component for a CMP and / or CBC test can be quantitative, qualitative, and / or semi-quantitative. A quantitative result would provide a specific value for each component (e.g., glucose = 85 mg / dL, calcium = 9.5 mg / dL, red blood cell = 5.35 X 1012cells / liter, etc.). Qualitative results can provide a binary indicator of a component, such as yes-no, present- bsent, positivenegative, pregnant-not-pregnant, and / or another combination of terms for qualitative results. Additional instances may provide semi-quantitative results, such as low-normal-high or another set of semi- quantitative indicators. Certain instances may provide a combination of quantitative and qualitative (or semi-quantitative) results. For example, certain instances can provide glucose = 130 mg / dL “high” for the ease of comprehension.

[0126] Additionally, cells (e.g., blood cells) may interfere with the ability to detect reaction products within some embodiments. Such interference can be caused by any present cells absorbing, diffracting, and / or deflecting light. To assist in detection, certain instances provide an agglutination reagent to the sample. Similar to the reagents as described above, agglutination reagents can be deposited as a desiccated, lyophilized, and / or otherwise dry application within a reaction chamber 206, a filling chamber 210, and / ora detection chamber 208. In some instances, the reagent(s) is provided under a disintegrating coating to prevent mixing, contamination, loss, and / or other complication. In coated embodiments, the underlying reagent can be wet or dry. In various embodiments, the agglutination reagent can be selected from one or more of the following: antibodies against surface antigens (e.g., anti-A, anti-B, anti-Rh(D) antibodies), anti-human globulin reagents, Coombs reagents, lectins, antisera for direct antiglobulin test (DAT), antiplatelet antibodies, any other compound capable of agglutinating a sample, and combinations thereof. Figure 3A provides an exemplary image of a non-agglutinated sample, while Figure 3B provides an example of an agglutinated sample. As demonstrated in Figures 3A-3B, the agglutinated sample allows for detection between the cellular aggregates. As can be seen in these figures, cells cluster in the agglutinated sample, which allows the plasma region to be more obvious and unobstructed. A resulting image can be segmented to identify and / or discriminate between cellular components (clustered cells) and plasma regions. Segmentation can occur either manually or via an image processing algorithm. A colorimetric measurement can be obtained from the sample at a particular wavelength based on the specific component (e.g., cellular or plasma) for the particular assay. By adjusting the detection chamber height, the pathlength of optical density measurement can be optimized for the dynamic range of the specific assay. In many instances, a pathlength of 0.1 mm can yield an optical density within the linearity region of many spectrophotometers without requiring dilution of a blood sample.

[0127] In lieu of or in addition to an agglutinating reagent, certain embodiments may instead remove the cellular component using an affinity matrix, such as described above. Removing the cellular component can allow for a spectroscopic analysis, such as absorbance or transmittance without image segmentation or interference caused by the presence of cells. To obtain a quality measurement of absorbance, increasing a pathlength of such the light beam can be helpful. But, long pathlengths can create a complication when attempting small form factors. Many embodiments design the detection chamber(s) to increase pathlength without increasing overall size of an analysis cartridge.

[0128] A first strategy to increase pathlength is to use internal reflection through the sample to increase pathlength. Figures 4A-4C provide illustrations of exemplary embodiments that utilize internal reflection. Figure 4 A provides a side view of an exemplary cuvette 400 (which could be a detection chamber 208 such as illustrated in Figure 2) using reflective coatings 402 to increase pathlength. As illustrated, reflective coatings 402 are applied to the upper and lower surfaces of the cuvette 400. One surface further includes a light entry pore 404 and a light exit pore 406. A light source 408 can be angled to enter the cuvette 400 at an incident angle that allows the light path 410 to internally reflect. The reflection may reflect off the upper surface and lower surfaces. The internal reflection can reflect off the upper and lower surfaces numerous times to increase the effective pathlength. Similarly, a detector 412 can be placed at an angle such that the light exiting light exit pore 406 impinges on the detector 412. This methodology can allow for capillary action and / or DMF electrodes to be used to move a sample into the cuvette 400. In certain instances, the DMF electrodes and a ground layer form the reflective coatings 402 on the bottom surface and top surface, respectively. A dielectric layer 414 may be applied on the DMF electrodes to prevent contact with a liquid sample. A dielectric layer 414 may be comprised of any suitablematerial, including (but not limited to) indium tin oxide (ITO), gold, platinum, graphene, conductive polymers, copper, aluminum, and / or any other suitable material. Additionally, the surfaces of cuvette 400 may be coated with one or more reagents, such as described above.

[0129] The sizes of light entry pore 404 and a light exit pore 406 may be any applicable size to allow sufficient light to enter and exit the cuvette 400. These pores may be different sizes to allow for beam spreading. In various embodiments, the light entry pore 404 and the light exit pore 406 are approximately 0.8 mm to approximately 1 mm, such as 0.8 mm, 0.9 mm, or 1.0 mm. Additionally the distance d between the light entry pore 404 and the light exit pore 406 should be such that the light path 410 will pass through the light exit pore 406. Ray tracing programs may be used to help determine distances between a light entry pore 404 and a light exit pore 406 based on height h, angle of entry (incident angle), and / or any other relevant parameter. Additionally, such ray tracing programs may assist in calculating a total pathlength. For example, for a cuvette having a height of 0.175 mm and an incident angle of 45°, a 10 mm light path can be obtained with a length / of approximately 7.072 mm. Of note, formation of a meniscus, such as caused by a capillary effect, may affect light transmittance through a sample. Thus, in many instances, the sample should be of ample enough volume to cover light entry power 404 and light exit pore 406.

[0130] Figure 4B illustrates an overhead view of this arrangement and how it can be applied to multiple cuvettes 400 simultaneously. In this illustration, DMF electrodes 416 (which could also be reaction chambers 206, as illustrated in Figure 2A) can be used to move a sample (e.g., plasma after removal of cellular components) into cuvettes 400. Incoming light may enter a light entry pore 404 on each cuvette 400, and the light can exit a light exit pore 406 on each cuvette 400. Figure 4C illustrates the side view of this larger arrangement, where incoming light source 408 emits light 410 (e.g., collimated light) that impinges on cuvettes 400. The light eventually exits cuvettes 400 to impinge on a detector 412. A detector used in such an embodiment can include a microscope, a CMOS sensor, a CCD sensor, and / or any other applicable type of sensor. In some embodiments, a detector is arrayed as a two dimensional array (e.g., linear sensor) or a three dimensional array (e.g., a grid). In some embodiments, the detector 412 is a camera. In certain instances, a camera can include a telecentric lens.

[0131] Another strategy to increase pathlength is to use the length I of a cuvette 500 (or detection chamber 206) rather than transmitting light through a cuvette (e.g., passing through height li) . Figures 5A- 5B illustrate exemplary schematics of this strategy, where Figure 5A uses an optical element 502 (e.g., mirror, lens, etc.) to route light path 504 through the sample, while Figure 5B illustrates edge-light illumination to route light path 504 through the sample. In both instances, an optical element 502 is used to route light path 504 to a detector (or sensor) 506. Similar to the embodiments illustrated in Figures 4A- 4C, capillary action and / or DMF electrodes 508 can be used to move a sample into the cuvette 500. In such embodiments, a substrate can include a ground layer 510 and a dielectric layer 512 may be included. In the embodiments of Figures 5A-5B, the pathlength can be a dimension of the cuvette 500. Using a detection chamber as illustrated in Figure 2, the pathlength of such a cuvette can be approximately 2 mm (e.g., width w) or approximately 6 mm (e.g., length I). As described above, the actual dimension can vary depending on various factors; thus, the pathlength can be anywhere between approximately 2 mm andapproximately 10 mm, including approximately 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm. It should be noted that a meniscus formed at an open end of a cuvette (e.g., a meniscus caused by a capillary effect) may refract light. Thus, light path 504 may pass through closed sides of cuvette 500, rather than an open face.

[0132] Figure 5C illustrates an overhead view of the embodiments exemplified in Figures 5A-5B. a light source 514 produces light, which can be collimated by an optical element 516, such as a mirror or condenser lens. Collimated light can pass through cuvettes 500 to impinge on an optical element 502 to route the light to a detector (or sensor) 506. Additional embodiments can include one or both of a front set of pinholes 518 and a back set of pinholes 520 to narrow the light angle and / or minimize the stray light. The front set of pinholes 518 are located between a light source 514 and sample to limit stray light or narrow the light angle of light entering the cuvette, while the back set of pinholes 520 may be located between the sample and the detector 506 to limit stray light or narrow the light angle of light exiting the cuvette. Pinhole diameter and / or thickness can be adjusted on one or both of a front set of pinholes 518 and a back set of pinholes 520 to limit stray light. As such, pinholes may be of any diameter from approximately 0.25 mm to approximately 1.5 mm, such as 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, 0.45 mm, 0.5 mm, 0.55 mm, 0.6 mm, 0.65 mm, 0.7 mm, 0.75 mm, 0.8 mm, 0.85 mm, 0.9 mm, 0.95 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, or 1.5 mm. Thickness or depth of the pinholes can also be adjusted. The thickness of pinholes in many embodiments can range from approximately 10 mm to approximately 35 mm, such as 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm, 26 mm, 27 mm, 28 mm, 29 mm, 30 mm, 31 mm, 32 mm, 33 mm, 34 mm, or 35 mm. In some preferred embodiments, a 25.4 mm thickness and 0.8 mm diameter limited crosstalk to less than 0.0062%. Similar to Figure 4B, DMF electrodes 522 (or reaction chambers 206 from Figure 2A) may be used to move a sample into cuvettes 500. Additionally, the detector used in such embodiments can include a microscope, a CMOS sensor, a CCD sensor, and / or any other applicable type of sensor. In some embodiments, a detector is arrayed as a one dimensional array (e.g., linear sensor) or a two dimensional array (e.g., a grid). In some embodiments, the detector 506 is a camera. In certain instances, a camera can include a telecentric lens.

[0133] While the foregoing embodiments generally illustrate the analysis chamber arranged as a horizontal array of reaction lanes, various embodiments can provide the reaction lanes as a vertical stack. Figures 6A-6C provide an illustration for a vertical stack. Specifically, Figure 6A illustrates a top view of a single layer 600 with a central through hole 602 that is in fluid communication with a capillary segment 604. A cuvette region 606 is distal from the central through hole 602 to allow a sample input into the central through hole 602 to fill the cuvette region 606 via capillary action from the capillary segment 604. Figure 6B shows a perspective view of a stack of layers 600. As provided, the central through holes 602 of each layer 600 are in fluid communication with each other and part of a central plasma collection reservoir. As such, a plasma sample may be provided to the central plasma collection reservoir, which then feeds into the cuvette region 606 of each layer 600. Figure 6C provides a side view of the stack, showing a path length of each cuvette region 606 in each layer 600. As shown in Figures 6B-6C, as light608 passes through the cuvette regions 606, it can impinge on a mirror 610 to route the light to a detector. Alternatively, the mirror 610 can be replaced with a detector (such as described above) the light can impinge directly on a detector. Additionally, one or more DMF electrodes 612 may be placed above, below, or within the central through hole 602 to assist with movement of a sample.

[0134] In the embodiments represented by Figures 6A-6C, each layer may be constructed of a lower hydrophilic film and an upper hydrophilic film laminated to a double sided pressure sensitive adhesive (PSA). In a stack, a single hydrophilic layer may form the lower hydrophilic film of one layer is the upper hydrophilic film of the next layer — in such a way, the entire stack is constructed of alternating layers of hydrophilic film and double sided PSA, where the topmost and bottom-most layers are both hydrophilic film. Additionally, the central through holes 602 may be concentric and aligned. The diameter of the central through holes 602 may form a sample reservoir sufficient to provide a sample for every layer to be analyzed, when the volume of the central through holes 602 are combined. The diameter may be approximately 2 mm to approximately 5 mm, such as approximately 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm,4.5 mm, or 5 mm. Some preferred embodiments possess a central through hole 602 with a diameter of approximately 3 mm. Additionally, the cuvette region 606 may have a width w sufficient to provide an adequate pathlength for detection of one or more analytes. In various instances, the cuvette region may have a width w between approximately 2 mm and approximately 10 mm, including approximately 2 mm,2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, or 10 mm. In certain instances, the cuvette region 606 may be variable in width, such that multiple pathlengths may be measured simultaneously. Such variable cuvette regions 606 can have a stepped shape with multiple sections, where each section has a different width. Alternatively, a cuvette region 606 may have a triangular shape, such that the width is constantly increasing over distance. As can be appreciated to one in the art, each layer 600 may alternate in direction, such that the cuvette regions 606 will effectively be spaced apart from a cuvette region 606 of an adjacent layer 600. Alternatively, each layer 600 may have a second capillary segment 604 and a second cuvette region 606, where thee second capillary segment 604 and a second cuvette region 606 can be opposite the second capillary segment 604 and a second cuvette region 606 illustrated in these figures. Such an arrangement may increase throughput in an analysis cartridge of the same size. Alternatively, such channels may be used as a duplicate or redundant test, in case of a failure or fault.METHODS

[0135] Using analysis cartridges as described herein can include adding a liquid sample into an input chamber 202. In many instances the liquid sample is blood, such as whole blood, peripheral blood, arterial blood, venal blood, capillary blood, and / or any other applicable blood sample. In various instance, the blood has been mixed with an anticoagulant to prevent coagulation or clotting within the sample prior to analysis. Many anticoagulants are known in the art, including (but not limited to) heparin, coumadin, dabigatran, rivaroxaban, apixaban, edoxaban, enoxaparin, dalteparin, fondaparinux, argatroban, bivalirudin, betrixaban, hirudin, ethylenediaminetetraacetic acid (EDTA), sodium citrate, oxalte, citrate-phosphate-dextrose, acid-citrate-dextrose, sodium fluoride, potassium EDTA, sodium heparin, and combinations thereof.

[0136] The chip may be added to a device that interfaces with the chip on a preprogrammed or automatic process or via manual control. In many instances, the device applies an charge to select electrodes to move a droplet of the sample into a reaction chamber in each reaction lane. This movement can be via DMF as described herein. Additional charges may be applied to assist in mixing and / or movement of the droplets through one or more additional reaction chambers. Such mixing and movement may be solely in a single direction (e.g., from input chamber to optical chamber), bidirectional (e.g., allowing movement toward both the optical chamber and toward the input chamber), and / or multiple directions simultaneously, which may allow deformation of a droplet that can help with mixing. As noted previously, loading of the optical chambers can occur via DMF and / or capillary action.

[0137] Tn certain instances, a higher reaction volume may be desirable for one reaction. To increase reaction volume for such a reaction, one or more additional droplets may be moved into one reaction lane 204 or 204’. Each droplet may be individually mixed with a reagent (•). The individual droplets may be moved to filling chamber 210 and / or detection chamber 208 individually and consecutively or the individual droplets may be homogenized by merging and / or mixing prior to moving into a filling chamber 210 and / or a detection chamber 208. The movement, merging, and / or mixing can occur in accordance with the patterns illustrated in Figures 1 A-IC.When the sample has filled the optical chambers, any applicable measurement system may be utilized, including a single sensor capable of measuring reflectance and / or absorbance, a one-dimensional sensor capable of creating a tiled array of measurements across a lane, and / or a two-dimensional sensor capable measuring the entire chip simultaneously. Regardless of sensor arrangement (single, ID, 2D, etc.). Sensors may be monochromatic and / or polychromatic.For purposes of completeness, various aspects of the present disclosure are set out in the following numbered clauses.Aspect 1. An analysis cartridge, comprising: an input chamber for receiving a sample; one or more reaction lanes in fluid communication with the input chamber, each of the one or more reaction lanes comprising a first reaction chamber and a detection chamber, wherein the first reaction chamber is configured to perform a chemical reaction to detect an analyte within the sample, wherein the first reaction chamber comprises a digital microfluidic (DMF) electrode to move a portion of the sample into the reaction chamber, and wherein the detection chamber is configured to allow detection of the chemical reaction.Aspect 2. The analysis cartridge of Aspect 1, wherein each of the one or more reaction lanes further comprises a filling chamber located between and in fluid communication with the first reaction chamber and the detection chamber, such that a reacted sample is moved from the reaction chamber to the filling chamber using a DMF electrode.Aspect 3. The analysis cartridge of Aspect 1 or 2, wherein the reacted sample moves into the detection chamber via a method selected from: diffusion, convection, pumping, airlift pumping, siphoning, applied pressure, gravity-driven flow, density gradients, temperature gradients, chemical gradients, pressure gradients (positive or negative), pneumatic pressure, gas-producing chemical reactions, centrifugal flow, capillary pressure, wicking, electric field-mediated, electrode-mediated, electrophoresis, dielectrophoresis, magnetophoresis, magnetic fields, magnetically driven flow, optical force, chemotaxis, phototaxis, surface tension gradient driven flow, Marangoni stresses, hermos-capillary convection, surface energy gradients, acoustophoresis, surface acoustic waves, electroosmotic flow, thermophoresis, electrowetting, opto-electrowetting.Aspect 4. The analysis cartridge of any one of Aspects 1-3, wherein the DMF electrode is coated with a reagent for a particular chemical reaction.Aspect 5. The analysis cartridge of any one of Aspects 1-4, wherein the detection chamber comprises an agglutination reagent to induce aggregation within the sample.Aspect 6. The analysis cartridge of any one of Aspects 1-5, wherein the chemical reaction is a colorimetric reaction and wherein the detection chamber is configured to optically detect the component based on a colorimetric reaction.Aspect 7. The analysis cartridge of any one of Aspects 1-6, wherein the detection chamber is configured to electrochemically detect the component.Aspect 8. The analysis cartridge of any one of Aspects 1-7, wherein each reaction lane further comprises a second reaction chamber in fluid communication with the first reaction chamber and comprising a DMF electrode to move the portion of the sample from the first reaction chamber into the second reaction chamber, wherein movement from the first reaction chamber into the second reaction chamber mixes the reacted sample.Aspect 9. The analysis cartridge of Aspect 8, wherein the second reaction chamber of at least one reaction lane comprises a second reagent for its chemical reaction.Aspect 10. The analysis cartridge of any one of Aspects 1-9, wherein each reaction lane comprises at least three reaction chambers, wherein each reaction chamber is in fluid communication with every other reaction chamber, wherein movement between the reaction chambers mixes the reacted sample.Aspect 11. The analysis cartridge of any one of Aspects 1-10, wherein at least one reaction chamber comprises an affinity matrix capable of isolating a component from the sample.Aspect 12. The analysis cartridge of Aspect 11, wherein the affinity matrix is comprised of a bead with a conjugated antibody or conjugated antigen.Aspect 13. The analysis cartridge of Aspect 13, wherein the bead is magnetic, paramagnetic, or susceptible to a magnetic field.Aspect 14. The analysis cartridge of any one of Aspects 1-13, wherein the sample is a blood sample.Aspect 15. The analysis cartridge of Aspect 14, wherein the blood sample is selected from whole blood, peripheral blood, arterial blood, venal blood, and capillary blood.Aspect 16. The analysis cartridge of Aspect 14 or 15, wherein the sample further comprises an anticoagulant.Aspect 17. The analysis cartridge of Aspect 16, wherein the anticoagulant is selected from heparin, coumadin, dabigatran, rivaroxaban, apixaban, edoxaban, enoxaparin, dalteparin, fondaparinux, argatroban, bivalirudin, betrixaban, hirudin, ethylenediaminetetraacetic acid (EDTA), sodium citrate, oxalte, citrate-phosphate-dextrose, acid-citrate-dextrose, sodium fluoride, potassium EDTA, and sodium heparin.Aspect 18. The analysis cartridge of any one of Aspects 1-17, wherein the chemical reaction for each reaction lane is configured to detect one or more of the following: glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.Aspect 19. The analysis cartridge of any one of Aspects 1-18, wherein the plurality of reaction lanes comprises 14 reaction lanes, wherein the chemical reaction of each reaction lane is configured to detect one of glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.Aspect 20. The analysis cartridge of any one of Aspects 1-19, wherein the detection chamber comprises a top surface constructed of a plastic that is optically clear between approximately 340 nm to approximately 850 nm.Aspect 21. The analysis cartridge of Aspect 20, wherein the top surface is constructed of polyethylene terephthalate, polymethyl methacrylate, polycarbonate, polystyrene, cyclo-olefin copolymer, polysulfone, polyetherimide, polyvinyl chloride, and polyolefin.Aspect 22. The analysis cartridge of any one of Aspects 1-21, wherein each reaction chamber has a width of between approximately 1 mm and 5 mm.Aspect 23. The analysis cartridge of any one of Aspects 1-22, wherein each reaction chamber has a length of between approximately 1 mm and 5 mm.Aspect 24. The analysis cartridge of any one of Aspects 1-23, wherein each reaction chamber has a width of approximately 2 mm and a length of approximately 2 mm.Aspect 25. The analysis cartridge of any one of Aspects 1-24, wherein each reaction chamber has a height sufficient to contain approximately 0.7 pL of volume.Aspect 26. The analysis cartridge of any one of Aspects 1-25, wherein each reaction chamber has a height of between approximately 0.050 mm and 0.500 mm.Aspect 27. The analysis cartridge of any one of Aspects 1-26, wherein each reaction chamber has a height of approximately 0.175 mm.Aspect 28. The analysis cartridge of any one of Aspects 1-27, wherein each optical chamber has a width of approximately 2 mm and a length of approximately 6 mm.Aspect 29. The analysis cartridge of Aspect 28, wherein each optical chamber has a height sufficient to contain between approximately 0.6 pL to approximately 2.4 L of volume.Aspect 30. The analysis cartridge of Aspect 28 or 29, wherein each optical chamber has a height of between approximately 50 pm to approximately 200 pm.Aspect 31. The analysis cartridge of any one of Aspects 1-30, further comprising at least one reaction lane that is configured to determine one or more of a red blood cell count, a white blood cell count, a differential white blood cell count, a platelet count, a hemoglobin level, and a hematocrit level.Aspect 32. The analysis cartridge of Aspect 31, wherein a differential white blood cell count determines a level of at least two of the following white blood cell types: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.Aspect 33. The analysis cartridge of Aspect 31 or 32, wherein a differential white blood cell count determines a level of at least the following white blood cell types: neutrophils, lymphocytes, and monocytes.Aspect 34. The analysis cartridge of any one of Aspects 1-33: wherein the analysis cartridge comprises fourteen reaction lanes in fluid communication with the input chamber, wherein twelve of the fourteen reaction lanes comprise two reaction chambers, a filling chamber, and a detection chamber, wherein one of the two reaction chambers comprises a reagent to perform a chemical reaction on the sample, and wherein two of the fourteen reaction lanes comprise at least 4 reaction chambers, a filling chamber, and a detection chamber, wherein two of the four reaction chambers comprise a reagent to perform a chemical reaction on the sample and are separated by at least one reaction chamber, such that each reaction occurs in sequence.Aspect 35. The analysis cartridge of Aspect 34, wherein each reaction lane is configured to detect one of: glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.Aspect 36. A method of analyzing a fluid sample, comprising loading the fluid sample in any one of the analysis cartridges of Aspects 1 to 35 and analyzing the fluid sample.Aspect 37. The method of Aspect 36, comprising analyzing between 0.1 pL to 2 pL of the fluid sample.Aspect 38. A method of analyzing a fluid sample in the analysis cartridge of any one of Aspects 1- 35, the method comprising: loading a fluid sample into the input chamber; applying an electrical charge to at least one DMF electrode to move a portion of the fluid sample into a reaction chamber; anddetecting at least one component in the portion of the fluid sample based on the chemical reaction.Aspect 39. The method of Aspect 38, wherein detecting at least one component comprises detecting at least one component selected from: a glucose level, a calcium level, a sodium level, a potassium level, a carbon dioxide level, a chloride level, an albumin level, a total protein level, an alkaline phosphatase level, an alanine transaminase level, an aspartate aminotransferase level, a bilirubin level, a blood urea nitrogen level, a creatinine level, a red blood cell count, a white blood cell count, a platelet count, a hemoglobin level, a hematocrit level, a neutrophil count, a lymphocyte count, a monocyte count, an eosinophil count, and a basophil count.Aspect 40. The method of Aspect 38 or 39, wherein detecting at least one component comprises imaging the detection chamber of the analysis cartridge.Aspect 41 . The method of any one of Aspects 38-40, wherein the fluid sample is a blood sample.Aspect 42. The method of Aspect 41, wherein the blood sample is selected from whole blood, peripheral blood, arterial blood, and venal blood.Aspect 43. The method of Aspect 41 or 42, further comprising mixing an anticoagulant with the blood sample.Aspect 44. The method of Aspect 43, wherein the anticoagulant is selected from heparin, coumadin, dabigatran, rivaroxaban, apixaban, edoxaban, enoxaparin, dalteparin, fondaparinux, argatroban, bivalirudin, betrixaban, hirudin, ethylenediaminetetraacetic acid (EDTA), sodium citrate, oxalte, citrate -phosphatc-dcxtrosc, acid-citratc-dcxtrosc, sodium fluoride, potassium EDTA, and sodium heparin.Aspect 45. The method of Aspect 43 or 44, further comprising segmenting the image to identify cellular components and plasma components of the sample.Aspect 46. The method of Aspect 45, wherein segmenting comprises utilizing an image processing algorithm.Aspect 47. An analysis cartridge comprising: one or more cuvettes, wherein each cuvette comprises a reflectively coated upper surface, a reflectively coated lower surface bottom, a light entry pore, and a light exit pore, wherein the upper surface, the lower surface, the light entry pore, and the light exit pore are positioned such that light entering the light entry pore at an incident angle reflects off of the upper surface and the lower surface and exits the light exit pore.Aspect 48. The analysis cartridge of Aspect 47, wherein the light entry pore and the light exit pore are positioned within the upper surface.Aspect 49. The analysis cartridge of Aspect 47 or 48, wherein the reflective coating on the upper surface is a ground layer and the reflective coating on the lower surface is a digital microfluidic (DMF) electrode.Aspect 50. The analysis cartridge of Aspect 49, wherein the cuvette further comprises a dielectric layer disposed on the DMF electrode.Aspect 51. The analysis cartridge of any one of Aspects 47-50, wherein the incident angle is approximately 45°.Aspect 52. The analysis cartridge of any one of Aspects 47-51, wherein the upper surface and the lower surface are positioned approximately 0.175 mm apart.Aspect 53. The analysis cartridge of any one of Aspects 47-52, wherein the light entry pore and the light exit pore are positioned approximately 7.07 mm apart.Aspect 54. The analysis cartridge of any one of Aspects 47-53, wherein the light entry pore and the light exit pore are each approximately 0.8 mm to approximately 1 mm in width.Aspect 55. The analysis cartridge of any one of Aspects 47-54 further comprising a plurality of DMF electrodes configured to move a sample into each of the one or more cuvettes.Aspect 56. An analysis system comprising: the analysis cartridge of any one of Aspects 47-55; a light source; and a detector; wherein the analysis cartridge, the light source, and the detector are positioned such that light emitted from the light source enters the light entry pore and exits from the light exit pore of each cuvette and impinges on the detector.Aspect 57. The analysis system of Aspect 56, further comprising an optical element to collimate the light emitted by the light source.Aspect 58. The analysis system of Aspect 56 or 57, wherein the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.Aspect 59. The analysis system of any one of Aspects 56-58, wherein the detector comprises an array of pixels.Aspect 60. The analysis system of Aspect 59, wherein the array of pixels is selected from a one dimensional array and a two dimensional array.Aspect 61. An analysis system comprising: an analysis cartridge comprising a plurality of cuvettes, each cuvette comprising an upper surface and a lower surface, wherein the upper surface comprises a ground layer and the lower surface comprises a digital microfluidic (DMF) electrode; a light source; and a detector; wherein the analysis cartridge, the light source, and the detector are positioned such that light emitted from the light source enters the cuvette along its length, passes through the sample, and impinges on the detector.Aspect 62. The analysis system of Aspect 61, further comprising a first optical element to route the light from the analysis cartridge to the detector.Aspect 63. The analysis system of Aspect 61 or 62, further comprising a second optical element to route the light from the light source to the analysis cartridge.Aspect 64. The analysis system of Aspect 62 or 63, wherein the first and second optical element are each selected from a lens and a mirror.Aspect 65. The analysis system of any one of Aspects 61-64, further comprising an optical element to collimate the light emitted by the light source.Aspect 66. The analysis system of any one of Aspects 61-65, wherein the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.Aspect 67. The analysis system of any one of Aspects 61-66, wherein the detector comprises an array of pixels.Aspect 68. The analysis system of Aspect 67, wherein the array of pixels is selected from a one dimensional array and a two dimensional array.Aspect 69. The analysis system of any one of Aspects 61-68, further comprising a set of pinholes to minimize stray light.Aspect 70. The analysis system of Aspect 69, wherein the set of pinholes have a thickness of approximately 10 mm to approximately 30 mm, and wherein the pinholes have a diameter from approximately 0.5mm to approximately 1.0 mm.Aspect 71. The analysis system of Aspect 69 or70, wherein the set of pinholes are located between the light source and the analysis cartridge or between the analysis cartridge and the detector.Aspect 72. The analysis system of any one of Aspects 61-71, further comprising two sets of pinholes to minimize stray light, wherein a first set of pinholes are located between the light source and the analysis cartridge and a second set of pinholes arc located between the analysis cartridge and the detector.Aspect 73. An analysis cartridge, comprising: two or more cuvettes, wherein each cuvette comprises a central through hole, a cuvette region, and a capillary region, wherein the cuvettes are stacked such that the central through holes are aligned and in fluid communication with each other to form a sample reservoir, and wherein the capillary region can move a portion of a sample to the cuvette region.Aspect 74. The analysis cartridge of Aspect 73, wherein the two or more cuvettes are formed as alternating layers of a hydrophilic film and a pressure sensitive adhesive.Aspect 75. The analysis cartridge of Aspect 71 or 74, wherein the central through holes have a diameter ranging from 2 mm to 4 mm.Aspect 76. The analysis cartridge of any of Aspects 73-75, wherein the cuvette regions have a constant width.Aspect 77. The analysis cartridge of any of Aspects 73-75, wherein the cuvette regions have a variable width.Aspect 78. The analysis cartridge of any of Aspects 73-77, wherein the cuvette regions have a width ranging from 2 mm to 4 mmAspect 79. An analysis system, comprisingan analysis cartridge according to any one of Aspects 73-78; a light source; and a detector; wherein the light source, the analysis cartridge, and the detector are arranged, such that a light emitted from the light source passes through the width of the cuvette region of each analysis cartridge and impinges on the detector.Aspect 80. The analysis system of Aspect 46, further comprising an optical element to route the light from the analysis cartridge to the detector.Aspect 81. The analysis system of Aspect 80, wherein the optical element is a mirror.Aspect 82. The analysis system of any one of Aspects 79-81, wherein the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.Aspect 83. The analysis system of any one of Aspects 79-82, wherein the detector comprises an array of pixels.Aspect 84. The analysis system of Aspect 83, wherein the array of pixels is selected from a one dimensional array and a two dimensional array.EXAMPLES

[0138] The following examples are offered by way of illustration and not by way of limitation.EXAMPLE 1 : AVOIDING CROSSTALK BETWEEN WELLS USING PINHOLES

[0139] Figure 7 illustrates a schematic of a detection setup to avoid crosstalk between microcuvettes 702. In this schematic, a first array of pinholes 704 was placed below the microcuvettes 702 to act as front pinholes, while a second array of pinholes 706 was placed above the microcuvettes 702 to act as back pinholes. A sensor 708 is placed above the second array of pinholes 706 to image, detect, and / or quantify light or other signal passing through or emanating from the microcuvettes 702.

[0140] In an experimental setup, the microcuvettes were replaced with 1536-well plates. These plates are commonly used and readily available from several manufacturers and generally possess a common shape, size, and spacing to be interchangeable and have commonality or uniformity with 96-well and 384- well plates in terms of spacing and overall size. In the present experiment, Nunc™ 1536-plates (ThermoFisher, Cat # 253614) were obtained as the source of a signal and proxy for a detection chamber. These plates were made of polystyrene and clear in color. Wells within these plates had a diameter of 1.7 mm, a pitch (distance from center to center of neighboring wells) of 2.2 mm, and a height d3 of 7.4 mm.

[0141] To measure crosstalk, one well of the 1536-well plate was obscured with a black and opaque material to prevent a light from emitting from that well — this well acted as the data point to identify any crosstalk from another well impinging on a CMOS sensor. The arrays of pinholes were manufactured to have the same pitch (i.e., 2.2 mm) between pinholes. In the experimental setup, the first array X04 has a depth (or thickness) dl with pinholes having a diameter (j)l, while the second array X06 has a depth d2with pinholes having a diameter 2. The depths and thicknesses can be altered to form a tradeoff between minimizing crosstalk and overall formfactor of a device.

[0142] In a first experiment, both plates had a thickness of 25 mm and the pinholes of both arrays had a diameter of 0.8 mm. This setup resulted in a cross talk of 0.0062%.EXAMPLE 2: ANALYTE MEASUREMENT WITHOUT DILUTION

[0143] Various components were measured using a microcuvette, including components from a CMP panel and a lipid panel without dilution. The table illustrated in Figure 8 provides a summary of the various components measured along with light path, wavelength, and optical densities for particular “low” and “high” concentrations. As seen in Figure 8, the analytes (e.g., total protein, creatinine, TC, etc.) are categorized by their test (e.g., CMP or lipid). Two different protocol types were used (e.g., Protocol #1 and Protocol #2) in the various reactions for each analyte. The light path used for each analyte is illustrated along with its total volume in microliters. The light paths ranged from 0.1 mm to 5.0 mm, with respective volumes of 0.3 L to 15.7 pL. Optical density (OD) ranges are provided for each analyte, including initial OD, final OD, and reaction times, as appropriate for a particular analyte. The small table provides the dynamic range of the sensor used along with the error rate or uncertainty for the range — e.g., loosening error rate from 0.3% to 1.0% allows for a broader dynamic range. The data also shows that the device can provide accurate measurements of CMP and lipids with volumes as low as 0.3 pL per analyte.

[0144] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1. An analysis cartridge, comprising: an input chamber for receiving a sample; one or more reaction lanes in fluid communication with the input chamber, each of the one or more reaction lanes comprising a first reaction chamber and a detection chamber, wherein the first reaction chamber is configured to perform a chemical reaction to detect an analyte within the sample, wherein the first reaction chamber comprises a digital microfluidic (DMF) electrode to move a portion of the sample into the reaction chamber, and wherein the detection chamber is configured to allow detection of the chemical reaction.

2. The analysis cartridge of claim 1, wherein each of the one or more reaction lanes further comprises a filling chamber located between and in fluid communication with the first reaction chamber and the detection chamber, such that a reacted sample is moved from the reaction chamber to the filling chamber using a DMF electrode.

3. The analysis cartridge of claim 1 or 2, wherein the reacted sample moves into the detection chamber via a method selected from: diffusion, convection, pumping, airlift pumping, siphoning, applied pressure, gravity-driven flow, density gradients, temperature gradients, chemical gradients, pressure gradients (positive or negative), pneumatic pressure, gas-producing chemical reactions, centrifugal flow, capillary pressure, wicking, electric field-mediated, electrodemediated, electrophoresis, dielectrophoresis, magnetophoresis, magnetic fields, magnetically driven flow, optical force, chemotaxis, phototaxis, surface tension gradient driven flow, Marangoni stresses, hermos-capillary convection, surface energy gradients, acoustophoresis, surface acoustic waves, electroosmotic flow, thermophoresis, electrowetting, optoelectrowetting.

4. The analysis cartridge of any one of claims 1-3, wherein the DMF electrode is coated with a reagent for a particular chemical reaction.

5. The analysis cartridge of any one of claims 1 -4, wherein the detection chamber comprises an agglutination reagent to induce aggregation within the sample.

6. The analysis cartridge of any one of claims 1-5, wherein the chemical reaction is a colorimetric reaction and wherein the detection chamber is configured to optically detect the component based on a colorimetric reaction.

7. The analysis cartridge of any one of claims 1-6, wherein the detection chamber is configured to electrochemically detect the component.

8. The analysis cartridge of any one of claims 1-7, wherein each reaction lane further comprises a second reaction chamber in fluid communication with the first reaction chamber and comprising a DMF electrode to move the portion of the sample from the first reaction chamber into the second reaction chamber, wherein movement from the first reaction chamber into the second reaction chamber mixes the reacted sample.

9. The analysis cartridge of claim 8, wherein the second reaction chamber of at least one reaction lane comprises a second reagent for its chemical reaction.

10. The analysis cartridge of any one of claims 1-9, wherein each reaction lane comprises at least three reaction chambers, wherein each reaction chamber is in fluid communication with every other reaction chamber, wherein movement between the reaction chambers mixes the reacted sample.

11. The analysis cartridge of any one of claims 1-10, wherein at least one reaction chamber comprises an affinity matrix capable of isolating a component from the sample.

12. The analysis cartridge of claim 11, wherein the affinity matrix is comprised of a bead with a conjugated antibody or conjugated antigen.

13. The analysis cartridge of claim 13, wherein the bead is magnetic, paramagnetic, or susceptible to a magnetic field.

14. The analysis cartridge of any one of claims 1-13, wherein the sample is a blood sample.

15. The analysis cartridge of claim 14, wherein the blood sample is selected from whole blood, peripheral blood, arterial blood, venal blood, and capillary blood.

16. The analysis cartridge of claim 14 or 15, wherein the sample further comprises an anticoagulant.

17. The analysis cartridge of claim 16, wherein the anticoagulant is selected from heparin, coumadin, dabigatran, rivaroxaban, apixaban, edoxaban, enoxaparin, dalteparin, fondaparinux, argatroban, bivalirudin, betrixaban, hirudin, ethylenediaminetetraacetic acid (EDTA), sodium citrate, oxalte, citrate -phosphate-dextrose, acid-citrate-dextrose, sodium fluoride, potassium EDTA, and sodium heparin.

18. The analysis cartridge of any one of claims 1-17, wherein the chemical reaction for each reaction lane is configured to detect one or more of the following: glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.

19. The analysis cartridge of any one of claims 1-18, wherein the plurality of reaction lanes comprises 14 reaction lanes, wherein the chemical reaction of each reaction lane is configured to detect one of glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.

20. The analysis cartridge of any one of claims 1 -19, wherein the detection chamber comprises a top surface constructed of a plastic that is optically clear between approximately 340 nm to approximately 850 nm.

21. The analysis cartridge of claim 20, wherein the top surface is constructed of polyethylene terephthalate, polymethyl methacrylate, polycarbonate, polystyrene, cyclo-olefin copolymer, polysulfone, polyetherimide, polyvinyl chloride, and polyolefin.

22. The analysis cartridge of any one of claims 1-21, wherein each reaction chamber has a width of between approximately 1 mm and 5 mm.

23. The analysis cartridge of any one of claims 1-22, wherein each reaction chamber has a length of between approximately 1 mm and 5 mm.

24. The analysis cartridge of any one of claims 1-23, wherein each reaction chamber has a width of approximately 2 mm and a length of approximately 2 mm.

25. The analysis cartridge of any one of claims 1-24, wherein each reaction chamber has a height sufficient to contain approximately 0.7 pL of volume.

26. The analysis cartridge of any one of claims 1-25, wherein each reaction chamber has a height of between approximately 0.050 mm and 0.500 mm.

27. The analysis cartridge of any one of claims 1-26, wherein each reaction chamber has a height of approximately 0.175 mm.

28. The analysis cartridge of any one of claims 1-27, wherein each optical chamber has a width of approximately 2 mm and a length of approximately 6 mm.

29. The analysis cartridge of claim 28, wherein each optical chamber has a height sufficient to contain between approximately 0.6 ,uL to approximately 2.4 L of volume.

30. The analysis cartridge of claim 28 or 29, wherein each optical chamber has a height of between approximately 50 pm to approximately 200 pm.

31. The analysis cartridge of any one of claims 1-30, further comprising at least one reaction lane that is configured to determine one or more of a red blood cell count, a white blood cell count, a differential white blood cell count, a platelet count, a hemoglobin level, and a hematocrit level.

32. The analysis cartridge of claim 31, wherein a differential white blood cell count determines a level of at least two of the following white blood cell types: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

33. The analysis cartridge of claim 31 or 32, wherein a differential white blood cell count determines a level of at least the following white blood cell types: neutrophils, lymphocytes, and monocytes.

34. The analysis cartridge of any one of claims 1-33: wherein the analysis cartridge comprises fourteen reaction lanes in fluid communication with the input chamber, wherein twelve of the fourteen reaction lanes comprise two reaction chambers, a filling chamber, and a detection chamber, wherein one of the two reaction chambers comprises a reagent to perform a chemical reaction on the sample, and wherein two of the fourteen reaction lanes comprise at least 4 reaction chambers, a filling chamber, and a detection chamber, wherein two of the four reaction chambers comprise a reagent to perform a chemical reaction on the sample and are separated by at least one reaction chamber, such that each reaction occurs in sequence.

35. The analysis cartridge of claim 34, wherein each reaction lane is configured to detect one of: glucose, calcium, sodium, potassium, carbon dioxide, chloride, albumin, total protein, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, bilirubin, blood urea nitrogen, and creatinine.

36. A method of analyzing a fluid sample, comprising loading the fluid sample in any one of the analysis cartridges of claims 1 to 35 and analyzing the fluid sample.

37. The method of claim 36, comprising analyzing between 0.1 pL to 2 pL of the fluid sample.

38. A method of analyzing a fluid sample in the analysis cartridge of any one of claims 1-35, the method comprising: loading a fluid sample into the input chamber; applying an electrical charge to at least one DMF electrode to move a portion of the fluid sample into a reaction chamber; and detecting at least one component in the portion of the fluid sample based on the chemical reaction.

39. The method of claim 38, wherein detecting at least one component comprises detecting at least one component selected from: a glucose level, a calcium level, a sodium level, a potassium level, a carbon dioxide level, a chloride level, an albumin level, a total protein level, an alkaline phosphatase level, an alanine transaminase level, an aspartate aminotransferase level, a bilirubin level, a blood urea nitrogen level, a creatinine level, a red blood cell count, a white blood cell count, a platelet count, a hemoglobin level, a hematocrit level, a neutrophil count, a lymphocyte count, a monocyte count, an eosinophil count, and a basophil count.

40. The method of claim 38 or 39, wherein detecting at least one component comprises imaging the detection chamber of the analysis cartridge.

41. The method of any one of claims 38-40, wherein the fluid sample is a blood sample.

42. The method of claim 41, wherein the blood sample is selected from whole blood, peripheral blood, arterial blood, and venal blood.

43. The method of claim 41 or 42, further comprising mixing an anticoagulant with the blood sample.

44. The method of claim 43, wherein the anticoagulant is selected from heparin, coumadin, dabigatran, rivaroxaban, apixaban, edoxaban, enoxaparin, dalteparin, fondaparinux, argatroban, bivalirudin, betrixaban, hirudin, ethylenediaminetetraacetic acid (EDTA), sodium citrate, oxalte, citrate -phosphate-dextrose, acid-citrate-dextrose, sodium fluoride, potassium EDTA, and sodium heparin.

45. The method of claim 43 or 44, further comprising segmenting the image to identify cellular components and plasma components of the sample.

46. The method of claim 45, wherein segmenting comprises utilizing an image processing algorithm.

47. An analysis cartridge comprising: one or more cuvettes, wherein each cuvette comprises a reflectively coated upper surface, a reflectively coated lower surface bottom, a light entry pore, and a light exit pore, wherein the upper surface, the lower surface, the light entry pore, and the light exit pore are positioned such that light entering the light entry pore at an incident angle reflects off of the upper surface and the lower surface and exits the light exit pore.

48. The analysis cartridge of claim 47, wherein the light entry pore and the light exit pore are positioned within the upper surface.

49. The analysis cartridge of claim 47 or 48, wherein the reflective coating on the upper surface is a ground layer and the reflective coating on the lower surface is a digital microfluidic (DMF) electrode.

50. The analysis cartridge of claim 49, wherein the cuvette further comprises a dielectric layer disposed on the DMF electrode.

51. The analysis cartridge of any one of claims 47-50, wherein the incident angle is approximately 45°.

52. The analysis cartridge of any one of claims 47-51, wherein the upper surface and the lower surface are positioned approximately 0.175 mm apart.

53. The analysis cartridge of any one of claims 47-52, wherein the light entry pore and the light exit pore are positioned approximately 7.07 mm apart.

54. The analysis cartridge of any one of claims 47-53, wherein the light entry pore and the light exit pore are each approximately 0.8 mm to approximately 1 mm in width.

55. The analysis cartridge of any one of claims 47-54 further comprising a plurality of DMF electrodes configured to move a sample into each of the one or more cuvettes.

56. An analysis system comprising: the analysis cartridge of any one of claims 47-55; a light source; and a detector; wherein the analysis cartridge, the light source, and the detector are positioned such that light emitted from the light source enters the light entry pore and exits from the light exit pore of each cuvette and impinges on the detector.

57. The analysis system of claim 56, further comprising an optical element to collimate the light emitted by the light source.

58. The analysis system of claim 56 or 57, wherein the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.

59. The analysis system of any one of claims 56-58, wherein the detector comprises an array of pixels.

60. The analysis system of claim 59, wherein the array of pixels is selected from a one dimensional array and a two dimensional array.

61. An analysis system comprising: an analysis cartridge comprising a plurality of cuvettes, each cuvette comprising an upper surface and a lower surface, wherein the upper surface comprises a ground layer and the lower surface comprises a digital microfluidic (DMF) electrode; a light source; and a detector; wherein the analysis cartridge, the light source, and the detector are positioned such that light emitted from the light source enters the cuvette along its length, passes through the sample, and impinges on the detector.

62. The analysis system of claim 61, further comprising a first optical element to route the light from the analysis cartridge to the detector.

63. The analysis system of claim 61 or 62, further comprising a second optical element to route the light from the light source to the analysis cartridge.

64. The analysis system of claim 62 or 63, wherein the first and second optical element are each selected from a lens and a mirror.

65. The analysis system of any one of claims 61-64, further comprising an optical element to collimate the light emitted by the light source.

66. The analysis system of any one of claims 61-65, wherein the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.

67. The analysis system of any one of claims 61-66, wherein the detector comprises an array of pixels.

68. The analysis system of claim 67, wherein the array of pixels is selected from a one dimensional array and a two dimensional array.

69. The analysis system of any one of claims 61-68, further comprising a set of pinholes to minimize stray light.

70. The analysis system of claim 69, wherein the set of pinholes have a thickness of approximately 10 mm to approximately 30 mm, and wherein the pinholes have a diameter from approximately 0.5mm to approximately 1.0 mm.

71. The analysis system of claim 69 or 70, wherein the set of pinholes are located between the light source and the analysis cartridge or between the analysis cartridge and the detector.

72. The analysis system of any one of claims 61-71, further comprising two sets of pinholes to minimize stray light, wherein a first set of pinholes are located between the light source and the analysis cartridge and a second set of pinholes are located between the analysis cartridge and the detector.

73. An analysis cartridge, comprising: two or more cuvettes, wherein each cuvette comprises a central through hole, a cuvette region, and a capillary region, wherein the cuvettes are stacked such that the central through holes are aligned and in fluid communication with each other to form a sample reservoir, and wherein the capillary region can move a portion of a sample to the cuvette region.

74. The analysis cartridge of claim 73, wherein the two or more cuvettes are formed as alternating layers of a hydrophilic film and a pressure sensitive adhesive.

75. The analysis cartridge of claim 71 or 74, wherein the central through holes have a diameter ranging from 2 mm to 4 mm.

76. The analysis cartridge of any of claims 73-75, wherein the cuvette regions have a constant width.

77. The analysis cartridge of any of claims 73-75, wherein the cuvette regions have a variable width.

78. The analysis cartridge of any of claims 73-77, wherein the cuvette regions have a width ranging from 2 mm to 4 mm79. An analysis system, comprising an analysis cartridge according to any one of claims 73-78; a light source; and a detector; wherein the light source, the analysis cartridge, and the detector are arranged, such that a light emitted from the light source passes through the width of the cuvette region of each analysis cartridge and impinges on the detector.

80. The analysis system of claim 46, further comprising an optical element to route the light from the analysis cartridge to the detector.

81. The analysis system of claim 80, wherein the optical element is a mirror.

82. The analysis system of any one of claims 79-81, wherein the detector is selected from a microscope, a CMOS sensor, a CCD sensor, and a camera.

83. The analysis system of any one of claims 79-82, wherein the detector comprises an array of pixels.

84. The analysis system of claim 83, wherein the array of pixels is selected from a one dimensional array and a two dimensional array.

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