EP4630814A1 - Paper-based multi-well depletion elisa - Google Patents

Abstract

The present disclosure provides for a paper-based well sheet functionalized with a recognition element, the paper-based well sheet comprising hydrophobic lines demarcating at least two columns having a plurality of wells on the paper-based well sheet, wherein the plurality of wells form a grid comprising at least two rows and at least two columns, wherein the paper-based well sheet is folded such that the at least two rows of wells are disposed on one another to result in one folded row of wells; a flow controller; a blotting paper, wherein the paper-based well sheet is disposed between the blotting paper and the flow controller; and a case, wherein the paper-based well sheet, the blotting paper, and the flow controller are together contained within the case. Further provided herein are methods of using thereof.

Description

Docket No.10034-227WO1 PAPER-BASED MULTI-WELL DEPLETION ELISA CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to, and the benefit of, U.S. Provisional Application No. 63/431,004 filed on December 7, 2022, the disclosure of which is hereby expressly incorporated by reference herein in its entirety. BACKGROUND Serological tests measure antibody expressions (in the blood, serum, and plasma) induced against pathogenic agents such as viruses or bacteria. Typically used to identify past infections rather than an active one, serological tests are relevant for a variety of applications including the study of immune responses in a qualitative/quantitative manner, serosurveys to determine the precise rate of infection, identification of potential convalescent donors for serum/plasma therapeutics, evaluation of vaccine efficacy, and assessment of protective immunity from reinfection. As such, different serological testing methods have been developed to address the need with common assay formats utilizing lateral flow assays (LFAs) or enzyme-linked immunosorbent assays (ELISAs). LFAs are the current assay format for point-of-care (POC) testing due to their low cost and convenient operation. In LFAs, samples are spontaneously wicked into a porous substrate (e.g., paper) via capillary action, and the presence of target antibodies in samples is visually reported in the form of colored bands as test and control lines. Although the LFA test format remains a common solution to detect the presence of specific antibodies outside of clinical settings, the method’s convenience comes at the expense of quantitative results. The binary colorimetric output of LFAs cannot report the antibody titer—a parameter that is closely correlated with the degree of immunoreactivity against a pathogen or a transplant. ELISA is the gold standard method for reliable and quantitative measurement of the antibody levels in a sample. In a typical ELISA test, the antibody titer is measured on a sample serially diluted across a well plate. The presence of the target antibody leads to an optical signal through an enzymatic reaction in the wells. The specific well where the optical signal falls below a reference threshold determines the titer, i.e., the amount of dilution wherein the original sample for the analyte concentration drops to an undetectable level. While an established test, antibody titer determination by ELISA is a labor-intensive assay involving multiple steps conventionally performed by trained staff in clinical laboratories. Combined with the fact that the sample has Docket No.10034-227WO1 to be serially diluted and distributed over an array of wells, the overall complexity of the process makes it challenging to adapt ELISA assays to tests amenable for point-of-care use. The methods disclosed herein address these and other needs. SUMMARY In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to an ELISA assay. Thus, in one example, a device is provided, including a paper-based well sheet functionalized with a recognition element, the paper-based well sheet comprising hydrophobic lines demarcating at least two columns having a plurality of wells on the paper- based well sheet, wherein the plurality of wells form a grid comprising at least two rows and at least two columns, wherein the paper-based well sheet is folded such that the at least two rows of wells are disposed on one another to result in one folded row of wells; a flow controller; a blotting paper, wherein the paper-based well sheet is disposed between the blotting paper and the flow controller; and a case, wherein the paper-based well sheet, the blotting paper, and the flow controller are together contained within the case. In a further example, a method of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is provided, including contacting a sample with a device as disclosed herein. Additionally, a method of detecting IgG antibody, IgM antibody, or any combination thereof is provided, including contacting a sample with a device as disclosed herein. Further, a method of detecting cardiac injury is provided, including contacting a sample with a device as disclosed herein. Also provided herein is a method of detecting troponin I, including contacting a sample with a device as disclosed herein. Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Docket No.10034-227WO1 BRIEF DESCRIPTION OF THE DRAWINGS The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. FIG. 1A – FIG. 1F show a paper-based multi-well depletion ELISA device. Figure 1A shows an illustration of the depletion ELISA using vertically stacked paper layers with discrete wells demarcated by water-insoluble ink. The layers were functionalized with a recognition element (e.g., antigen). The target analyte (e.g., antibody) applied on the top layer is gradually depleted by immunocapture, effectively diluting the analyte during flow to create a concentration gradient. The presence of immunocaptured analyte on layers is transduced into an optical signal and the antibody titer can be determined by counting the number of colored wells. Figure 1B shows photographs showing the paper based 96-well sheet. Applying four dye solutions to individual wells confirmed complete isolation between wells with no leakage observed between adjacent wells. Figure 1C shows photographs showing (left) 96-well plate unfolded without application of dyed solutions, (middle) after dyed solutions were introduced to every other stacked well on the folded device and (right) after the 96-well plate was unfolded following application of dyed solutions. The images illustrate successful isolation during vertical transit through the folded well-sheet. Figure 1D shows a schematic showing the procedure to operate the assay. Sample and ELISA reagents were simultaneously introduced from different inlets on the device case enclosing the components. Assay results were read out by unfolding the 96-well sheet. Figure 1E shows photographs of the case embedded with the folded 96-well sheet sandwiched between the flow controller and the blotting paper. Figure 1F shows time-lapse images showing the automated routing of ELISA reagents by the programmed flow controller. The images confirmed as-programmed sequential delivery of washing buffer (yellow), HRP-conjugated secondary antibody (green), another washing buffer (red), and TMB substrate (blue) with preset incubation times to execute the ELISA protocol. FIG.2A – FIG. 2D show characterization of the depletion ELISA. Figure 2A shows images (left) and plots of the measured optical signal from individual wells (right) of an unfolded well sheet, where the row was functionalized at a different concentration to investigate the effect of the surface-immobilized antigen concentration on the detection coverage of antibody titers. Results from assaying both high (top) and low (bottom) titer samples of IgG antibodies on the analytical device functionalized with recombinant SARS- CoV-2 N protein are shown. Figure 2B shows an image (left) of the unfolded 96-well sheet, Docket No.10034-227WO1 functionalized with 1 μg/ml of N protein in wells after it was used to analyze samples with different IgG antibody titers. A plot (right) shows the number of observed colored wells as a function of antibody titers. Figure 2C shows an image of the 96-well plate used to perform the conventional ELISA titer assay on samples matched with those tested with this device in Figure 2B. Figure 2D shows a plot showing the estimated effective IgG concentration in the wells calculated from measured color intensity by employing the Hill equation. The estimated antibody concentrations from cascaded wells in a row are overlaid with 3-fold dilution lines as reference. Error bars in panels represent the standard deviation (n = 3). FIG. 3A – FIG. 3E show the multiplexed depletion ELISA. Figure 3A shows an illustrative plot showing the typical expression profile of IgM and IgG antibodies throughout the infection based on previous reports. Adapted from. Figure 3B shows a schematic of the layout of the 48-well sheet designed to analyze different types of SARS-CoV-2 antibodies: SARS-CoV-2 spike protein (S)-IgM (1st row from top), nucleocapsid protein (N)-IgM (2nd row), S-IgG (3rd row) and N-IgG (4th row). Images of the unfolded well sheet and the associated plot showing measured color intensity as a function of well location in the rows. They represent assay results for six different samples prepared to contain controlled amounts (titers) of different antibodies as a mixture of S-IgM (1:2187) + N10 IgM (1:81) (left), N- IgM (1:2187) + S-IgM (1:81) (right) in Figure 3C; S-IgG (1:2187) + N-IgG (1:81) (left), N- IgG (1:2187) + S-IgG (1:81) (right) in Figure 3D; S-IgM/IgG (1:2187) + N-IgM/IgG (1:2187) (left), S-IgM/IgG (1:81) + N-IgM/IgG (1:81) (right) in Figure 3E. FIG.4A – FIG. 4E show a calibrated depletion ELISA for troponin I measurements. Figure 4A shows a schematic illustrating troponin I release into the bloodstream due to cardiac injury and the introduced method of quantifying troponin I concentration with the depletion ELISA. Figure 4B shows an image of the unfolded 96 well sheet that shows the results from the analysis of control samples with varying concentrations of troponin I (6.5 pg/ml to 60 pg/ml) with this assay. Figure 4C shows a calibration curve that relates the number of colored wells from the assay with troponin I concentration in the sample was established based on results from the control experiments. Figure 4D shows the assay results corresponding to 8 blinded samples. Samples #5 (12 colored wells), #6 (10 colored wells), and #7 (11 colored wells) were identified as those with abnormal levels (> 30 pg/ml) of troponin I concentration by this assay. Figure 4E shows a plot showing the actual troponin I concentration versus the estimated concentration value from this assay for an expanded set of blinded samples. Docket No.10034-227WO1 FIG. 5A – FIG.5F show a schematic showing the developed process to fabricate the paper-based multi-well sheet: Figure 5A shows that cellulose paper was patterned by drawing lines with a water-insoluble ink. The patterned sheet was perforated along vertical lines for easy alignment when folding. Figure 5B The well sheet was folded to stack wells. Figure 5C shows that stacked wells were functionalized by loading a controlled amount of capture elements followed by 2 hours of incubation at room temperature. Figure 5D shows that the well stack was washed 3 times and dried. Figure 5E shows that the well sheet was folded again, and a blocking buffer was applied to the well stack followed by 2 hours of incubation. Figure 5F shows that the well stack was washed 3 times before being unfolded to dry. FIG.6 shows a schematic of the detailed design of the 3D printed case from different views. The dimensions of various features are marked on the drawings. FIG. 7A – FIG.7I show a series of photos showing the device assembly process and the device operation. Figure 7A demonstrates gathering of prepared components: a 3D case, a flow controller, a blotting paper, and a paper-based 96-well sheet. Figure 7B shows folding of the 96-well sheet. Figure 7C shows aligning and placing the folded 96-well sheet on the blotting paper. Figure 7D shows placement of the flow controller on top of the folded well sheet after alignment. Figure 7E shows insertion of the grouped components into the 3D case. Figure 7F shows deposition of the samples on designated inlets. Figure 7G shows deposition of the ELISA reagents to the designated inlets. Figure 7H shows extraction of the folded well sheet from the device case after the completion of the assay after ~35 mins. Figure 7I shows unfolding of the 96-well well sheet for reading the assay results. FIG.8A – FIG.8C show the design of the capillary flow controllers employed in this work. Photos of the paper-based flow controllers built to deliver multiple reagents to the reaction to perform the ELISA assay for Figure 8A shows a 96-well sheet and Figure 8B shows a 48-well sheet. The scale bar is 1 cm. Both photos show the imprinted features for capillary flow manipulation. Figure 8C shows time-lapse images of the sequential delivery of different reagents by the flow controller designed to perform ELISA for the 48-well sheet. The silver lines guided the capillary flow, and the green lines were used as timers to stall the flow for the desired duration. The volume of the washing buffer and TMB substrate was 300 μl and the volume of secondary anti-IgM-HRP and anti-IgG-HRP was 150 μl. DETAILED DESCRIPTION The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiments. Many modifications and other Docket No.10034-227WO1 embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As can be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. Docket No.10034-227WO1 It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure. Definitions As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.” As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a disorder”, includes, but is not limited to, two or more such compounds, compositions, or disorders, and the like. It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed. When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the Docket No.10034-227WO1 disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer Docket No.10034-227WO1 to an amount that is then about 1 % by weight or less, e.g., less than about 0.5 % by weight, less than about 0.1 % by weight, less than about 0.05 % by weight, or less than about 0.01 % by weight of the stated material, based on the total weight of the composition. Devices Provided herein is a device comprising: a paper-based well sheet functionalized with a recognition element, the paper-based well sheet comprising hydrophobic lines demarcating at least two columns having a plurality of wells on the paper-based well sheet, wherein the plurality of wells form a grid comprising at least two rows and at least two columns, wherein the paper-based well sheet is folded such that the at least two rows of wells are disposed on one another to result in one folded row of wells; a flow controller; a blotting paper, wherein the paper-based well sheet is disposed between the blotting paper and the flow controller; and a case, wherein the paper-based well sheet, the blotting paper, and the flow controller are together contained within the case. A paper-based well sheet is an alternative to a well plate wherein a paper-based material comprises wells arranged in a grid. The wells are demarcated using ink, wherein the ink is hydrophobic, hydrophilic, or any combination thereof. In some examples, the well sheet comprises, in addition to or instead of paper, a porous material including but not limited to a porous polymer, porous plastic, or hydrogel. In further examples, the well sheet comprises polytetrafluoroethylene (PTFE), polyethylene (PE) foam sheets, polypropylene (PP) foam sheets, ceramic foam sheets, metallic foam sheets, anodic aluminum oxide membrane, agarose gel, polyethylene glycol (PEG) hydrogel, or any combination thereof. In some examples, the well sheet is made of other materials such as, for example, plastic, bamboo, or any combination thereof. In some examples, the wells in the at least two columns are fluidically connected to one another. The biological recognition element is present in the system to capture the analyte of interest, i.e., target analyte, as used herein, an antibody. In some examples, the recognition element is selected from the group comprising an antigen, enzyme, aptamer, ion, drug, toxin, nucleic acid (primers), protein, cell or even an antibody. Herein, the biological recognition element is bound to the vertically stacked paper layers of the device, to create an immunocapture path for the analyte. The recognition element is covalently immobilized on Docket No.10034-227WO1 the paper surface. In some examples, the covalent binding technique can be modified to modify the immunocapture rate and specificity. IN some examples, the hydrophobic lines comprise hydrophobic ink, wherein the ink repels and/or lacks an affinity for water, such that the samples in the wells across columns are not fluidically connected. The hydrophobicity of the lines separates the samples in their respective columns from contact and/or combining with one another. The wells are arranged in a grid such that there are columns of wells and rows of wells. (Figure 1B; Figure 1C; Figure 2A; Figure 2B) The paper-based well sheet is folded such that the rows of wells in the grid are disposed on one another to result in one folded row of results. As shown in Figure 1C, an example paper-based well sheet with 8 columns and 12 rows was folded such that the 12 rows are disposed on one another, and one row and 8 wells are visible on the resulting folded paper-based well sheet. The wells within the individual columns are fluidically connected such that a sample placed in the first row of each column can move between wells within each respective column. A flow controller is a platform for the quantification and detection of analytes in a sample. In some examples, the flow controller comprises at least one timer, wherein the timer is delaminating such that it allows for movement of a reagent through the controller based on the rate of delamination of the timer. In further examples, multiple delaminating timers are used in the flow controller to allow for further control of the movement of reagents through the controller. As shown in Figure 1F, the strategic use of delaminating timers in the flow controller allows for controlled and strategic movement of the ELISA reagents through the example flow controller. In Figure 1F, the first delaminating timer allows for movement of the washing buffer to the samples first, then the 2nd Ab-HRP, followed by another washing buffer, and lastly a TMB substrate. The reagents moved through the flow controller in the prescribed order as demonstrated because of the placement of timers. Furthermore, the use of timers eliminates the need for a user to administer the reagents one after another over the span of the 35 minutes process. Figure 8C shows the use of delaminating timers such that washing buffer moves through the controller to the sample first, the anti-IgM-HRP and the Anti-IgG-HRP move through the flow controller next, followed by an additional washing buffer, and lastly a TMB substrate, all over the span of 30 minutes. Again, this is able to be performed without the intervention of a user. Docket No.10034-227WO1 As shown in Figure 7D, an example flow controller is disposed on the folded paper- based well sheet before insertion into the case. Figure 8A and Figure 8B show further examples of flow controller configurations, wherein the flow controllers comprise sample inputs, ELISA reagent inlets, channels, and delaminating timers. Blotting paper is an absorbent sheet of paper used to absorb and/or transfer materials in laboratory processes. As shown in Figure 7A – Figure 7F, an example blotting paper is folded to fit underneath the folded paper-based well sheet, which is then plugged into the case along with the flow controller. The case encapsulates the blotting paper, flow controller, and paper-based well sheet. In some examples, the case is manufactured via 3D printing. In some examples, the case comprises plastic. In further examples, the case comprises glass, metal, bamboo, composite materials, or any combination thereof. In some examples, the paper-based well sheet has a dimension of from 70 mm to 90 mm by from 120 mm to 140 mm. In some examples, the paper-based well sheet has a dimension of from 70 to 75 mm, 75 to 80 mm, 80 to 85 mm, or 85 to 90 mm; 70 to 72 mm, 72 to 74 mm, 74 to 76 mm, 76 to 78 mm, 78 to 80 mm, 80 to 82 mm, 82 to 84 mm, 84 to 86 mm, 86 to 88 mm, or 88 to 90 mm; 70 to 80 mm or 70 to 85 mm; or 70 to 74 mm, 70 to 76 mm, 70 to 78 mm, 70 to 80 mm, 70 to 82 mm, 70 to 84 mm, 70 to 86 mm, or 70 to 88 mm; by from 120 to 125 mm, 125 to 130 mm, 130 to 135 mm, or 135 to 140 mm; 120 to 122 mm, 122 to 124 mm, 124 to 126 mm, 126 to 128 mm, 128 to 130 mm, 130 to 132 mm, 132 to 134 mm, 134 to 136 mm, 136 to 138 mm, or 138 to 140 mm; 120 to 130 mm or 120 to 135 mm; or from 120 to 124 mm, 120 to 126 mm, 120 to 128 mm, 120 to 130 mm, 120 to 132 mm, 120 to 134 mm, 120 to 136 mm, or 120 to 138 mm. In further devices, the wells have a dimension of from 5 to 7 mm by from 5 to 7 mm. In some examples, the wells have a dimension of from 5 to 5.5 mm, 5.5 to 6 mm, 6 to 6.5 mm, or 6.5 to 7 mm; 5 to 5.2 mm, 5.2 to 5.4 mm, 5.4 to 5.6 mm, 5.6 to 5.8 mm, 5.8 to 6.0 mm, 6.0 to 6.2 mm, 6.2 to 6.4 mm, 6.4 to 6.6 mm, 6.6 to 6.8 mm, or 6.8 to 7 mm; 5 to 6 mm or 5 to 6.5 mm; or 5 to 5.4 mm, 5 to 5.6 mm, 5 to 5.8 mm, 5 to 6 mm, 5 to 6.2 mm, 5 to 6.4 mm, 5 to 6.6 mm, or 5 to 6.8 mm; by from 5 to 5.5 mm, 5.5 to 6 mm, 6 to 6.5 mm, or 6.5 to 7 mm; 5 to 5.2 mm, 5.2 to 5.4 mm, 5.4 to 5.6 mm, 5.6 to 5.8 mm, 5.8 to 6.0 mm, 6.0 to 6.2 mm, 6.2 to 6.4 mm, 6.4 to 6.6 mm, 6.6 to 6.8 mm, or 6.8 to 7 mm; 5 to 6 mm or 5 to 6.5 mm; or 5 to 5.4 mm, 5 to 5.6 mm, 5 to 5.8 mm, 5 to 6 mm, 5 to 6.2 mm, 5 to 6.4 mm, 5 to 6.6 mm, or 5 to 6.8 mm. Docket No.10034-227WO1 In certain examples, the wells hold from 3 to 5 µL of a sample prior to saturation of the paper-based well sheet. In some examples, the wells hold from 3 to 3.5 µL, 3.5 to 4 µL, 4 to 4.5 µL, or 4.5 to 5 µL. In further examples, the wells hold from 3 to 3.2 µL, 3.2 to 3.4 µL, 3.4 to 3.6 µL, 3.6 to 3.8 µL, 3.8 to 4 µL, 4 to 4.2 µL, 4.2 to 4.4 µL, 4.4 to 4.6 µL, 4.6 to 4.8 µL, or 4.8 to 5 µL. In certain examples, the wells hold from 3 to 4 µL or 3 to 4.5 µL. In specific examples, the wells hold from 3 to 3.4 µL, 3 to 3.6 µL, 3 to 3.8 µL, 3 to 4 µL, 3 to 4.2 µL, 3 to 4.4 µL, 3 to 4.6 µL, 3 to 4.8 µL, or 3 to 5 µL. In some examples, the wells have a volume of from 3 to 200 µL. In further examples, the wells have a volume of from 3 to 10 µL, 10 to 20 µL, 20 to 30 µL, 30 to 40 µL, 40 to 50 µL, 50 to 60 µL, 60 to 70 µL, 70 to 80 µL, 80 to 90 µL, 90 to 100 µL, 100 to 110 µL, 110 to 120 µL, 120 to 130 µL, 130 to 140 µL, 140 to 150 µL, 150 to 160 µL, 160 to 170 µL, 170 to 180 µL, 180 to 190 µL, or 190 to 200 µL. In further examples, the wells have a volume of from 3 to 20 µL, 3 to 30 µL, 3 to 40 µL, 3 to 50 µL, 3 to 60 µL, 3 to 70 µL, 3 to 80 µL, 3 to 90 µL, 3 to 100 µL, 3 to 110 µL, 3 to 120 µL, 3 to 130 µL, 3 to 140 µL, 3 to 150 µL, 3 to 160 µL, 3 to 170 µL, 3 to 180 µL, or 3 to 190 µL. In certain examples, the wells have a volume of from 3 to 25 µL, 25 to 50 µL, 50 to 75 µL, 75 to 100 µL, 100 to 125 µL, 125 to 150 µL, 150 to 175 µL, or 175 to 200 µL. In specific examples, the wells have a volume of from 3 to 75 µL, 3 to 125 µL, or 3 to 175 µL. In some examples, the wells have a volume of from 3 to 75 µL, 75 to 125 µL, 125 to 175 µL, or 175 to 200 µL. Saturation refers to the point at which the paper-based well sheet, and by extension each well in the paper-based well sheet, cannot absorb any further material. In specific examples, the recognition element is an antigen. An antigen is a molecule or moiety comprising a protein, peptide, polysaccharide, lipid, or nucleic acid. In some examples, it is a foreign particulate matter or an allergen, such as pollen. An “epitope” or “antigenic determinant” refers to the part of an antigen, a molecular structure, or foreign particulate that can bind to a specific antibody or T-cell receptor. The presence of antigens or epitopes of antigens within a host can illicit an immune response against said molecule, moiety, foreign particulate matter, or allergen. In some examples, the antigen is a viral antigen. A viral antigen is a toxin or other substance produced by a virus which elicits an immune response in its host. In some examples, the viral antigen is a protein encoded by the viral genome. Some examples of viral antigens include but are not limited to Cytomegalovirus Glycoproteins I and III, Hepatitis B Docket No.10034-227WO1 surface antigen, Hepatitis C E2 and NS3 antigens, Hepatitis D HBsAg (S-HBsAg), medium- HBsAg (M-HBsAg) and large-HBsAg (L-HBsAg) envelope proteins, Hepatitis E ORF2 and ORF3 proteins, Herpes Simplex virus glycoprotein D (gD) antigen, Epstein Barr virus nuclear antigen 1, Respiratory syncytial virus F protein, Chikungunya E1/E2 envelope protein antigens, Human Immunodeficiency virus gp41 and gp120 antigens, Measles virus glycoproteins, the hemagglutinin (H) and the fusion (F) protein, Varicella-Zoster gE glycoprotein. In further examples, the viral antigen comprises a SARS-CoV-2 antigen or an influenza antigen. SARS-CoV-2 antigen or Severe-acquired respiratory syndrome-coronavirus-2 antigen comprises of four proteins that contribute to the overall structure of all coronaviruses: the spike (S), envelope (E), membrane (M) and nucleocapsid (N). The type I glycoprotein, the spike (S) that forms the peplomers on the virion surface, giving the virus its corona- or crown-like morphology; the membrane (M) protein, a protein that spans the membrane three times and has a short N-terminal ectodomain and a cytoplasmic tail; and small membrane protein (E), a short ectodomain, a transmembrane domain, and a cytoplasmic tail. The genome RNA of Coronaviruses is complexed with the basic nucleocapsid (N) protein to form a helical capsid found within the viral membrane. Influenza antigen comprises the Hemagglutinin (HA) and Neuraminidase (NA) proteins. Influenza Hemagglutinin (HA) is a homotrimeric glycoprotein found on the surface of influenza viruses and is integral to its infectivity. HA is responsible for binding the Influenza virus to sialic acid on the surface of target cells and the fusion of the viral envelope with the late endosomal membrane once exposed to low pH. The neuraminidase (NA) helps viruses to be released after budding from the plasma membrane of a host cell. Viral neuraminidase cleaves terminal sialic acid residues from glycan structures on the surface of the infected cell. This promotes the release of progeny viruses and the spread of the virus from the host cell to uninfected surrounding cells. Neuraminidase also cleaves sialic acid residues from viral proteins, preventing aggregation of viruses. In certain examples, SARS-CoV-2 spike proteins are disposed in the columns with odd numbers and the SARS-CoV-2 nucleocapsid proteins are disposed in the columns with even rows. SARS-CoV-2 spike protein (S) or E2 glycoprotein is highly immunogenic. Antibodies against spike glycoprotein are found in patients recovered from SARS and COVID-19. The function of the spike glycoprotein is to mediate entry of the Docket No.10034-227WO1 virus into the host cell by first interacting with receptor molecules on the exterior surface of the host cells and then fusing the viral and cellular membranes. The spike protein is 1273 amino acid residues long with SEQ ID NO.1. It is a single-pass transmembrane protein with a short C-terminal tail on the interior of the virus, a transmembrane helix, and a large N- terminal ectodomain exposed on the virus exterior. SARS-CoV-2 nucleocapsid protein is a protein that packages the positive-sense RNA genome of coronaviruses to form ribonucleoprotein structures enclosed within the viral capsid. The N protein is the most highly expressed of the four major coronavirus structural proteins. In addition to its interactions with RNA, N forms protein- protein interactions with the coronavirus membrane protein (M) during the process of viral assembly. N also has additional functions in manipulating the cell cycle of the host cell. The N protein is highly immunogenic and antibodies to N are found in patients recovered from COVID-19. The N protein is composed of two main protein domains connected by an intrinsically disordered region (IDR) known as the linker region, with additional disordered segments at each terminus. A third small domain at the C-terminal tail appears to have an ordered alpha helical secondary structure and may be involved in the formation of higher-order oligomeric assemblies. In SARS-CoV-2, the causative agent of COVID-19, it is 419 residues long with SEQ ID NO.2. In specific examples, SARS-CoV-2 spike proteins comprise spike protein IgM and spike protein IgG antibodies. In some examples, the SARS-CoV-2 nucleocapsid proteins comprise nucleocapsid protein IgM antibodies and nucleocapsid protein IgG antibodies. In some examples, the antibody is an Immunoglobulin G (IgG) and in some examples it is an Immunoglobulin M (IgM). Immunoglobulin G (IgG) is the main type of glycoprotein antibody found in human serum, secreted by B cells. It is found in blood and comprises four polypeptide chains, made of two identical 50KDa γ heavy (H) chain and two identical 25KDa κ or λ light (L) chains. There chains are inter-linker by di-sulfide bonds. IgG exists in four isotypes IgG1, IgG2, IgG3, and IgG4. IgG has the longest serum half-life compared to other immunoglobulins. Immunoglobulin M (IgM) is also a glycoprotein produced by B cells and they regulate the physiology and growth of the human B cell reserve. IgM is a high molecular weight protein that has five to six subunits. IgM monomers are made of two heavy chains and two light chains connected by a disulfide bond. In further examples, the antigen is related to a disorder. In certain examples, the disorder is a cardiovascular disorder, disease, or cancer. Docket No.10034-227WO1 Cardiovascular disease includes, but is not limited to coronary artery disease, high/low blood pressure, cardiac arrest/heart failure, congestive heart failure, congenital heart defects/diseases (including, but not limited to atrial septal defects, atrioventricular septal defects, coarctation of the aorta, double-outlet right ventricle, d-transposition of the great arteries, Ebstein anomaly, hypoplastic left heart syndrome, and interrupted aortic arch), arrhythmia, peripheral artery disease, stroke, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathies, hypertensive heart disease, pulmonary heart disease, cardiac dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, eosinophilic myocarditis, valvular heart diseases, rheumatic heart diseases, and other related cardiovascular diseases. The term “cancer” is used to address any neoplastic disease and is not limited to epithelial neoplasms (surface and glandular cancers; such a squamous cancers or adenomas). It is used here to describe both solid tumors and hematologic malignancies, including epithelial (surface and glandular) cancers, soft tissue and bone sarcomas, angiomas, mesothelioma, melanoma, lymphomas, leukemias and myeloma. In specific examples, the device comprises 48 wells. In some examples, the grid comprises 4 columns and 12 rows. (Figure 2A; Figure 3B; Figure 3C-Figure 3E) In further examples, the device comprises 96 wells. In certain examples, the grid comprises 8 columns and 12 rows. (Figure 1B; Figure 1C; Figure 2B; Figure 2C; Figure 4B; Figure 4D) In specific examples, IgM is disposed in the first two columns and IgG in the last two columns. (Figure 3B) In some examples, the wells are coated with a capture antibody. A “capture” antibody is immobilized on the surface of the wells of the plate. The “capture” antibody binds and retains analyte from the sample. The remaining matrix is rinsed away. An enzyme conjugated “detector” antibody, raised against a different epitope on the analyte is added to the plate. A standard colorimetric detection method is used to detect and quantify analytes in the sample. In some examples, the capture antibody is a monoclonal antibody. In further examples, the flow controller comprises a sample input, wherein the number of sample inputs is equal to the number of columns. As shown in Figure 8A and Figure 8B, sample inputs are present on the flow controller. In the example flow controller of Figure 8A, there are 8 sample inputs, while the example flow controller in Figure 8B has 4 sample inputs. As can be seen in Figure 7A, in some examples, the number sample inputs in the flow controller (8) matches the number of rows in the well sheet and the number of sample inputs Docket No.10034-227WO1 in the case, which allow for access to the sample inputs in the flow controller upon assembly of the device. In certain examples, the flow controller comprises at least one ELISA reagent inlet. In specific examples, the flow controller comprises four ELISA reagent inlets. As shown in Figure 8A and Figure 8B, the flow controller has ELISA reagent inlets. In the example flow controller of Figure 8A, there are 4 ELISA reagent inlets: one for washing buffer, one for TMB substrate, one for additional washing buffer, and one for secondary Ab-HRP. As can be seen in Figure 7A, in some examples, the number of ELISA reagent inlets in the flow controller (4) matches the number of ELISA reagent inlets in the case, which allows for access to the ELISA reagent inlets in the flow controller upon assembly of the device. ELISA reagents comprise of a washing buffer, primary and secondary detection antigen or antibody, ELISA coating buffer, protein stabilizer, sample or assay diluents, blocking buffer, signal enhancer, recognition element, in-solution stabilizers or conjugate stabilizers, a detection substrate and a stop solution, or any combination thereof. In some examples, the washing buffer is Phosphate Buffered Saline (PBS) with 0.1% detergent. In some examples, the blocking buffer is 5% Bovine serum albumin solution in PBS, or a 5% non-fat skimmed milk solution in PBS. In some examples, the ELISA reagent includes a washing buffer, 3,3’,5,5’- Tetramethylbenzidine (TMB) substrate, Ab-HRP, Anti-IgM-HRP, Anti-IgG-HRP, or any combination thereof. 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate is a chromogenic substrate used as a visualizing reagent in enzyme-linked immunosorbent assays (ELISA). TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase (HRP). The resulting one-electron oxidation product is a diimine- diamine complex, which causes the solution to take on a blue color. This color change can be read on a spectrophotometer at the wavelengths of 370 and 650 nm. The reaction can be halted by addition of acid or another stop reagent. Using sulfuric acid turns TMB yellow, with a peak absorbance of 450 nm. The amount of converted TMB may be indexed by the amount of 450 nm light it absorbs. Antibody-Horseradish peroxidase (Ab-HRP) is an enzyme that catalyzes the conversion of chromogenic substrates (e.g., TMB, DAB, ABTS), which are oxidized by HRP using hydrogen peroxide as the oxidizing agent, to yield a characteristic color change that is detectable by spectrophotometric methods. In some examples the HRP can produces light when acting on chemiluminescent substrates (e.g. Enhanced Chemiluminescence Docket No.10034-227WO1 by luminol). Anti-IgM-HRP and anti-IgG-HRP are purified IgM and IgG antibodies, respectively, conjugated to horseradish peroxidase (HRP). In further examples, the flow controller comprises a first channel comprising a first delaminating timer, wherein the first channel comprises a first washing buffer, a second channel comprising a second delamination timer, wherein the second channel comprises a secondary Ab-HRP, a third channel comprising a third delamination timer and a fourth delamination timer, wherein the third channel comprises the washing buffer; and a fourth channel comprising a fifth delamination time, a sixth delamination timer, a seventh delamination timer, and an eighth delamination timer, wherein the fourth channel comprises a TMB substrate, wherein the washing buffer leaves the first channel first, the secondary Ab- HRP leaves the second channel second, the washing buffer leaves the third channel third, and the TMB substrate leaves the fourth channel fourth. In further examples, the flow controller has the configuration of the example device in Figure 8A. In certain examples, the flow controller comprises a first channel and a second channel both comprising a washing buffer, a third channel comprising a first delamination timer, wherein the third channel comprises a secondary Anti-IgM-HRP, a fourth channel comprising a second delamination timer, wherein the fourth channel comprises a secondary Anti-IgG- HRP, a fifth channel comprising a third delamination timer and a fourth delamination timer, wherein the fifth channel comprises a TMB substrate, and a sixth channel comprising a fifth delamination timer, a sixth delamination timer, and a seventh delamination timer, wherein the sixth channel comprises a washing buffer, wherein the washing buffer leaves the first channel and the second channel first, the secondary Anti-IgM-HRP leaves the third channel second, the secondary Anti-IgG-HRP leaves the fourth channel third, the TMB substrate leaves the fifth channel fourth, and the washing buffer leaves the fourth channel fifth. In further examples, the flow controller has the configuration of the example device in Figure 8B. Methods Method of Detecting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV- 2) The present disclosure, in one aspect, provides for a method of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising contacting a sample with a device provided herein. Docket No.10034-227WO1 SARS-CoV-2 is a strain of coronavirus that causes COVID-19, the respiratory illness and shares more than 70% genetic similarity with SARS-CoV-1 the causative agent of Severe acute respiratory syndrome (SARS). The virus is of zoonotic origin. It is a positive-sense single-stranded RNA (+ssRNA) virus, with a single linear RNA segment is approximately 30,000 bases in length. Each SARS-CoV-2 virion is 60 to 140 nanometers (2.4×10⁻⁶ to 5.5×10⁻⁶ in) in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. In some examples, the device comprises 48 wells. In further examples, the device has a flow controller with the configuration of the example device in Figure 8B. In a further example, a method of detecting a recognition element is provided, including contacting a sample with a device as disclosed herein. In some examples, the recognition element is selected from the group comprising an antigen, enzyme, aptamer, ion, drug, toxin, nucleic acid (primers), protein, cell or even an antibody. In some examples, a method of detecting an antigen is provided, including contacting a sample with a device as disclosed herein. In some examples, a method of detecting a protein is provided, including contacting a sample with a device as disclosed herein. In some examples, a method of detecting an antibody is provided, including contacting a sample with a device as disclosed herein. Method of Detecting IgG Antibody, IgM Antibody, or Any Combination Thereof Also provided herein is a method of detecting IgG antibody, IgM antibody, or any combination thereof comprising contacting a sample with a device provided herein. In some examples, the device comprises 48 wells. In further examples, the device has a flow controller with the configuration of the example device in Figure 8B. Method of Detecting a Cardiac Injury Further provided herein is a method of detecting a cardiac injury comprising contacting a sample with a device provided herein. A cardiac injury can range from clinically silent, transient arrhythmias to fatal cardiac rupture. Histologically, it is characterized by a contused myocardium with hemorrhagic infiltration, localized necrosis, and/or edema. further described by specific injuries or observed dysfunction. Patients with abnormal findings should be admitted for continuous Docket No.10034-227WO1 cardiac monitoring; and subjected to Electrocardiogram measurements with levels of cardiac troponin I (cTnI) measured 24 hours later. In some examples, the device comprises 96 wells. In further examples, the device has a flow controller with the configuration of the example device in Figure 8A. Method of Detecting Troponin I in a Sample Also provided herein is a method of detecting troponin I in a sample comprising contacting the sample with a device provided herein. Troponin I or cardiac troponin I, (cTnI), is presented in cardiac muscle tissue by a single isoform with a molecular weight of 23.9 kDa. It consists of 209 amino acid residues which comprises SEQ ID NO. 3. cTnI differs from other troponins due to its N-terminal extension of 26 amino acids. This extension contains two serines, residues 23 and 24, which are phosphorylated by protein kinase A in response to beta-adrenergic stimulation, which is the increase of an ionic current through the calcium, potassium and chloride channels of the heart muscles. A significant part of cTnI released into the patient’s blood stream is phosphorylated. cTnI is a reliable marker of cardiac muscle tissue injury. In some examples, troponin I levels are increased in subjects with chronic kidney failure, heart failure, subarachnoid hemorrhage and pulmonary embolus compared to controls. In some examples, the device comprises 96 wells. In further examples, the sample comprises blood, urine, semen, saliva, or any combination thereof. In further examples, the device has a flow controller with the configuration of the example device in Figure 8A. A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. EXAMPLES The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of Docket No.10034-227WO1 all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions. Example 1: Paper-Based Multi-Well Depletion ELISA Enzyme-linked immunosorbent assay (ELISA) is employed for detecting target molecules in bioassays including the serological assays that measure specific antibody titers. However, ELISA tests are limited to centralized laboratories staffed with trained personnel as the assay workflow comprises multiple steps to be performed in a specific sequence. Disclosed herein, is a dipstick ELISA test that automates this otherwise laborious process and reports the titer of a target molecule in a digital manner without an external instrument or operator. The assay measures titer by gradually immuno-depleting the target analyte from a flowing sample effectively diluting the residual target – a process previously achieved through serially diluting the whole sample in numerous, time-consuming pipetting steps performed manually. Furthermore, the execution of the depletion ELISA process is automated by a built-in flow controller which sequentially delivers different reagents with preset delays. The technology is applied to develop assays measuring (1) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers (IgM/IgG antibodies to nucleocapsid and spike protein) and (2) troponin I, a cardiac biomarker. To perform ELISA assays in mobile settings, miniaturized ELISA assays implemented on microfluidic devices are disclosed herein. These devices have previously utilized miniature multi-well plates patterned on a polymer [e.g., Polydimethylsiloxane (PDMS), Polymethylmethacrylate (PMMA), or polycarbonate (PC)] or paper substrate and can be used to measure antibody titers while still relying on manual operation and pipetting, potentially hindering their use in some POC settings. On the other hand, attempts to automate Docket No.10034-227WO1 these miniaturized systems, either require unscalable sophisticated designs that rely on external instruments for operation or can only provide a snapshot measurement that cannot be used for antibody titer measurement or multiplexed analysis. Shown herein is a paper imprinted LFA that can automatically perform a multi-well ELISA assay for titer measurements. Instead of serial dilution and manual dispensing of the sample into microwells, this approach relies on gradual dilution of the target analyte as it is depleted from the sample by immobilized capture elements during flow to create a concentration gradient for titer measurement. To automate the sequence and timing of applying different reagents used to perform ELISA, a lateral flow device is equipped with an imprinted flow controller programmed with delaminating timer gates that eliminated the use of manual supervision of the assay. To demonstrate practical applications of the developed technology, lateral flow devices were created to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers (both IgG and IgM to spike and nucleocapsid protein) and troponin I, a cardiac biomarker, with a device having the sample and ELISA reagents to loaded for operation. Results Paper-based multi-well depletion ELISA device This lateral flow device design strategy centered on accomplishing two main tasks: (1) to serially dilute the target analyte and distribute it to individually isolated wells spontaneously with no external sample manipulation and (2) to automatically perform the delivery of reagents in a programmed manner with preset incubation times to execute an ELISA protocol. Because conventional serial dilution and dispensing of those diluted samples into wells would require manipulation of samples with pipetting, instead the aim was to decrease the target analyte concentration by gradually depleting it from the sample through immunocapture. This in-situ process, referred to as depletion ELISA in this work, leads to sample dilution factors varying along the flow path simultaneously achieving both serial dilution and spatial separation of those serially diluted samples. To create an immunodepletion path for the analyte, this device uses vertically stacked paper layers, functionalized with a relevant recognition element (e.g., antigen). The designed layout was realized as a paper strip with discrete wells demarcated by water-insoluble ink and was folded over itself along the well boundaries (Figure 1A). In operation, the sample applied on the top layer is wicked towards the bottom layer and the analyte is gradually depleted by immunocapture leaving the analyte at a lower concentration for the subsequent layer. For a high titer sample, analytes can reach the bottom layer after passing through the stack with a Docket No.10034-227WO1 gradual decrease in concentration, while a low titer sample is already depleted of analytes when it reaches the lower layer because the upper few layers can rapidly consume the limited amount of analytes. When mixed with conventional ELISA reagents, the presence of immunocaptured antibodies on layers is transduced into an optical signal. The optical signal from individual layers or wells can then be observed on the unfolded strip and the titer can be determined by counting the number of colored wells as in a conventional titer assay. Based on the depletion ELISA strategy, a device was created that reports its results in a multi-well configuration similar to a plastic 96-well plate. A paper-based 96-well sheet was first created by simply patterning the cellulose paper with water-insoluble ink containing a hydrophobic resin (Figure 5A-Figure 5F). The size of the 96-well sheet was purposely designed to be 80 mm by 127 mm – a footprint similar to that of a plastic 96-well plate. At this size, an individual well measures 6 mm by 6 mm and was observed to hold ~ 4 μl prior to saturation. Applying dye solutions to individual wells on the paper confirmed complete isolation between wells with no leakage observed between adjacent wells (Figure 1B). The 96-well sheet was then folded along vertical lines effectively creating a column of 83D wells, formed by a vertical stack of 12 wells fluidically connected from the top to bottom layer with a combined volume of ~ 48 μl. Demarcating hydrophobic lines aligned in a layer ensures the flow to be in a vertical direction with no crosstalk between them (Figure 1C). To couple the 96-well sheet to the paper-based flow controller and create inlets for samples and reagents, a device case was designed (Figure 6) and manufactured it with a 3D printer. The folded 96- well sheet was sandwiched between a paper-based flow controller and a blotting paper and plugged into the 3D-printed device case (Figure 1D and 7). The blotting paper underneath the folded 96-well sheet served both to act as a fluidic sink to drive vertical capillary flow and to mechanically compress the folded 96-well sheet to ensure contact between different layers within a well (Figure 1E). The section of the case securing the whole paper stack was designed to be 9 mm in height. Considering the uncompressed thicknesses of the folded 96-well sheet (12-layer stack) and blotting papers are 3 mm and 7 mm, respectively, the case resulted in a 10% compression in height and ensured physical contact with consistent force between stacked wells. To automatically perform ELISA reactions on the lateral flow device, the imprinted flow controller, contained within the device, ensured the delivery of reagents to wells in a programmed sequence with incubation periods. The paper-based flow controller utilized timer gates that relied on the delamination of the wetted paper from a laminating tape to coordinate different flows. The controller was created by imprinting patterns on paper with different types of inks that produced different levels of adhesion between the paper and the Docket No.10034-227WO1 laminating tape (Figure 8A). After optimizing the ELISA reaction time on the paper substrate, the controller was programmed to automate the protocol. For this assay, a 5-minute incubation of serially diluted samples with antibodies was sufficient as diminishing gains in signal strength were observed with further increases in the incubation time. Overall, the optimized incubation times for both the sample and the subsequently introduced reagents in this assay were found to be shorter than those in conventional ELISA (30 min to 1 hour). Without wishing to be bound by theory, faster reactions in this device are likely due to the shorter analyte diffusion length for reaching to the immunocapture elements immobilized on fiber surfaces distributed over the volume of the paper in contrast with a conventional well reaction in a 96 well plate. To validate the automated routing of ELISA reagents by the programmed flow controller, dye solutions were introduced, at a 600 μl volume (accounting 75 μl of the solution for each of the 8 rows of stacked 12 wells), from four reagent inlets and observed the flow sequence via snapshot measurements by taking devices out from the case at specific time points (Figure 1F). It was observed that the dye representing the washing buffer (yellow) first flowed to the folded 96well sheet at ~5 min. Then, the dye solution representing the horseradish peroxidase (HRP)-conjugated secondary antibody (2nd Ab-HRP, green) and the dye for another washing buffer (red) sequentially flowed at ~15 min and ~25 min, respectively. Last, the dye solution representing 3, 3’, 5, 5’-tetramethylbenzidine (TMB) substrate (blue) took ~35 min to reach the folded 96-well sheet. Characterization of the depletion ELISA In the characterization experiments, a recombinant SARS-CoV-2 nucleocapsid (N) protein was used as the antigen and recombinant SARS-CoV-2 nucleocapsid IgG antibody (IgG antibody) as the target antibody as a model pair. Rows of stacked wells were functionalized with the N protein at different concentrations (10 ng/ml – 10 μg/ml) and these analytical devices were used to test high (1:6561) and low titer (1:243) samples of the IgG antibody. For a test, the sample and ELISA reagents/buffers were simultaneously (within ~30 s) loaded to the designated inlets on the device and at the completion of the assay, the well sheet was taken out, unfolded, and the color changes were analyzed. Spatial color patterns formed due to the ELISA reaction were observed to be nonuniform across individual wells and also varied from one another (Figure 2A). These nonuniform patterns were due to random patterns of sample wicking into the paper substrate – an issue that can potentially be alleviated by homogenously loading the wells with a dispenser that matches the well size. In this work, Docket No.10034-227WO1 a well was considered as a single entity, and therefore, integrated colorimetric signals across the whole well to quantify the analyte concentration for analysis. To develop an assay that covers a broad range of titers, first investigated were the effect of the surface-immobilized antigen concentration on the detection coverage of antibody titers. While the tested conditions produced gradually decreasing optical signal, suggestive of successful serial dilution of the analyte, immobilized antigen concentration differences were observed to affect assay sensitivity and dynamic range (Figure 2A). Specifically, lower surface antigen concentrations led to reduced amounts of captured IgG antibodies in the wells, and weaker optical signals were observed to fall below visually detectable levels sooner. On the other hand, the efficient immunocapture on wells with high surface antigen concentration (10 μg/ml) led to premature depletion of the limited amount of IgG antibodies in the low titer sample and effectively reduced the dynamic range of the assay. Based on these results, 1 μg/ml was determined as a concentration among the tested conditions for surface functionalization with the goal of matching the coverage of this assay to the titer ranges of practical interest in biomedical applications. To associate the assay output (i.e., the number of colored wells) with specific antibody titers, the results from this study were compared with those from conventional ELISA assay performed on matched samples. Upon testing different dilution factors, it was determined that wells functionalized with the surface-coated N protein at 1 μg/ml produced results consistent with a conventional ELISA titer assay performed by serially diluting the samples 3 times (Figure 2B and Figure 2C). The match between this assay and conventional titer assay across different sample concentrations was evaluated. In testing this device, a 1:19683 titer sample (810 μg/ml in PBS) was first serially diluted 3-fold from 1:19683 to 1:27 titer, and 50 μl aliquots were deposited on different wells along with negative control (NC). The number of the colored wells in a row on the unfolded well sheet was determined in independent experiments (n=3) by measuring color intensities with digital image processing (see Materials and Methods). The number of the colored wells, 10 for the 1:19683 titer sample, was found to decrease by one in a row of 3x diluted antibody titer with the sample of the lowest titer tested (1:27) producing the 4 colored wells (Figure 2B). These results from the depletion ELISA were consistent with the conventional ELISA performed on a plastic 96-well plate by manually 3-fold diluting the identical samples (Figure 2C). Next, the observed antibody dilution factor in this assay was verified by estimating the dilution factor directly from the measured intensity of optical signals. Based on the optical signal amplitude from a well, the effective concentration of IgG in that well for the ELISA reaction was calculated by employing the Hill equation as Docket No.10034-227WO1 where I is the measured color is the maximum color intensity, [L] is the antibody concentration in the corresponding well, [LSO] the antibody concentration corresponding to half of the binding sites occupied, and n is the Hill coefficient. Using the fact that the sample IgG concentration and the resulting color intensity were both known for the first well in each row (i.e., the first column in Figure 2B), it was estimated that the remaining parameters in the equation (Imax, [LSO], n) for the reaction through curve fitting. Through the derived expression, the sample antibody concentration (L) was then estimated in each of the subsequent wells from the measure color intensities (I). The computed well-to- well dilution factors were on average ~ 3.07-fold (STD = 0.21) (Table 1) with the estimated antibody concentrations in cascaded wells decreased consistently following reference lines of 3-fold dilution (Figure 2D), regardless of the tested sample concentration. Table 1. Calculated well-to-well dilution factors observed for the depletion ELISA assay. Calculated dilution factors in each row d n Next, the capability of performing an automated multi-well ELISA in a lateral flow device was utilized to create a multiplexed antibody test for COVID-19. Simultaneous measurement of SARS-CoV-2 spike (S) protein IgM/IgG (S-IgM/S-IgG) and nucleocapsid (N) protein IgM/IgG (N-IgM/N-IgG) antibodies concentrations has recently been one of the applications of the multiplexed ELISA. Throughout the infection, peak expression of these antibodies occurs at different timepoints (Figure 3A); IgM typically starts to express 3 days after infection and lasts for ~ 3 weeks, while IgG starts to appear 7 days after infection and lasts for > 4 weeks. To develop a lateral flow ELISA which can assay these antibody targets in a single test, a paper-based 48-well sheet and coated SARS-CoV-2 S proteins at the odd rows and N proteins at the even rows was created (Figure 3B). For automation, an application- specific flow controller for the 48-well sheet into the final device to deliver Anti-IgM-HRP Docket No.10034-227WO1 to the first two rows and Anti-IgG-HRP to the last two rows was designed and integrated (Figure 8B and Figure 8C). A row of the multiplexed ELISA sheet was designed to analyze a different antibody type in the sample: 1st row for S-IgM, 2nd row for S-IgG, 3rd row for N-IgM, and 4th row for N-IgG. Next, the assay was tested with control samples prepared to simulate specimens from different stages of coronavirus infection. Blood plasma samples collected from healthy donors according to Institutional Review Board (IRB)-approved protocols were spiked with either of the IgM and IgG antibodies or with both in known quantities. Furthermore, the concentrations of the antibodies against SARS-CoV-2 spike and nucleocapsid proteins in a sample were specifically set to be different, the higher concentration type at 1:2187 titer and the less concentration type at 1:81 titer. When the samples spiked with IgM (Sample 1) or IgG (Sample 2) were assayed, the associated rows of the well sheet were observed to produce color changes: the top two rows for Sample 1 (Figure 3C) and the bottom two rows for Sample 2 (Figure 3D) demonstrating the specificity of the assay. Moreover, the relative concentrations of different antibody types (i.e., S versus N) were correctly reported with different numbers of colored wells. For the tested samples, the high- titer antibody produced 8 colored wells while the low-titer antibody consistently resulted in 5 colored wells. On the other hand, the samples spiked with both IgM and IgG antibodies (Sample 3) were observed to produce color changes in rows of the well sheet (Figure 3E). The sample containing both IgM and IgG at a high concentration generated 8 colored wells in rows while 5 colored wells were observed for the sample spiked at a lower concentration. Taken together, these results demonstrated the feasibility of this technology for performing multi-well multiplexed ELISA of different antibody types including those against different pathogens on the same assay. Calibrated depletion ELISA for concentration measurements Next, the concentration of a biomarker in a sample from a calibrated depletion ELISA process was estimated. As a medically relevant target, a depletion ELISA assay for troponin I, a specific cardiac biomarker, which is released into the bloodstream when a cardiac injury occurs with disruption of normal cardiac myocyte membrane integrity, was developed (Figure 4A). To capture troponin I, the wells were coated with a capture antibody at a concentration of 1 μg/ml and the ELISA reaction was automatically performed by an imprinted flow controller. To establish a calibration curve, first processed samples were prepared with varying concentrations of troponin I (5.3 pg/ml to 60 pg/ml) and recorded the number of colored wells (Figure 4B). The number of colored wells increased with higher troponin I concentration with ≥ 9 colored wells indicating abnormal levels of troponin I (> 30 pg/ml) Docket No.10034-227WO1 (Figure 4C). Next, the calibrated assay was applied to process samples with troponin I concentrations ranging from 0 to 60 pg/ml in a blinded fashion. Comparing results from eight different samples in a single assay (Figure 4D), three samples with abnormally high levels of troponin I were identified (Samples #5, #6, and #7) based on the observation that they produced ≥ 9 colored wells. Based on the calibration curve, troponin I concentrations in these samples were estimated to be 60, 40, and 50 pg/ml, which were later confirmed to be in close agreement with the spiked troponin I concentrations of 55, 38, and 48 pg/ml, respectively. Repeated blinded experiments showed that the troponin I concentration based on the number of colored wells with an accuracy limited by a quantization error can be consistently predicted (Figure 4E). These results demonstrated the feasibility of performing digital quantification of biomarkers using the lateral flow depletion ELISA test visually with no aid of an external imaging apparatus. Discussion A method to effortlessly perform this well-established yet intrinsically complex assay as a dipstick test was developed. This assay integrates serial sample dilution and automated manipulation of different reagents, on a disposable paper substrate, and provides results in a well plate format that can be used not only to detect but also to quantify target analytes in a multiplexed fashion. The depletion ELISA titer assay was purposely created in an inexpensive format on a paper substrate to ensure its practicality in different scenarios. While this assay can be operated in a laboratory equipped with 96-well plate readers, an advantage of the developed assay over conventional ELISA is that it offers the potential to perform multi-well ELISA outside of laboratories because of built-in paper-based automation. Furthermore, this device reduces reagent consumption and can lower assay cost given reagents are contributors to the cost of performing ELISA. As an example, in this work, ~25X less reagent were used in this assay (Figure 2B) compared to the conventional ELISA (Figure 2C) that achieved similar results processing matched samples. The developed platform technology can also be further engineered to meet the sensitivity and specificity demands of a given application. Covalent surface modification techniques to immobilize recognition elements on a paper surface along with optimized blocking methods could be used to tune immunocapture rate and specificity for a well. Likewise, multi-well sheets precision manufactured using microfabrication techniques to reduce the size of well area could help achieve a uniform flow profile between stacked layers and thereby produce more homogeneous color development in a well and reduce variations in the well-to-well dilution factors. Additionally, design choices such as the thickness of the Docket No.10034-227WO1 paper substrate or the compression force holding the folded well plate are some of the parameters affecting assay performance and therefore provide opportunities for further optimization for a specific application. Based on the results on the analysis of SARS-CoV-2 antibody titers and troponin I, this technique can potentially be expanded to sensitive and specific detection of pathogens, hormones, drugs, or metabolites and has the potential to transform a variety of ELISA-based tests mostly performed at clinical laboratories into single-use, disposable dipstick tests to be used at the point of care or home. Given ELISA’s current popularity in its laborious form, the ability to self-test using dipstick ELISA tests can be transformative in the decentralized delivery of healthcare. Materials and Methods Fabrication of the paper-based 96 and 48-well sheet To fabricate the paper-based 96 and 48-well sheets, the following procedures were employed (Figure 5): (1) cellulose filter paper (Whatman grade 1 filter paper) was patterned by drawing lines with water-insoluble ink containing a hydrophobic resin (Sharpie metallic permanent marker, manufacturer part number 2003899). The drawn multiple lines form the pattern of 96- or 48- wells where a liquid is impenetrable. The patterned well sheet was then perforated along the vertical lines to aid alignment when folding. (2) The well sheet was folded along perforated vertical lines effectively creating a column of 8 wells with a vertical stack of 12 wells. (3) To immobilize the corresponding capture elements on a well through physical adsorption, the controlled concentrations of 50 μl recombinant SARS-CoV-2 S, N proteins, or troponin I capture antibody in phosphate-buffered saline (PBS) were loaded to a stacked well and incubated at room temperature for 2 hours. (4) After incubation, the stacked well was washed 3 times by loading 50 μl of washing buffer (PBS with 0.05% Tween 20) followed by unfolding to dry at room temperature. (5) The well sheet was folded again and 50 μl of a blocking buffer [10% (w/v) bovine serum albumin (BSA) in PBS] was applied to the stack and incubated for another 2 hours to prevent nonspecific binding of proteins. (6) Lastly, the well sheet was washed 3 times again and unfolded for fully drying at room temperature. Fabrication of the flow controller To fabricate the flow controller, the tissue paper (Kimtech Science Kimwipes delicate task wipers, Kimberly-Clark, Irving, TX) was immersed in a blocking buffer for 1 hour to prevent nonspecific binding. After washing with PBS, the paper was fully dried at room temperature and one side of the paper was covered with clear tape (Scotch Heavy Duty Docket No.10034-227WO1 Shipping Packaging Tape, 3M Scotch, MN, USA). On the uncovered side, the flow paths assigned for the 96-well sheet (Figure 8A), or 48-well sheet (Figure 8B) were drawn using an automatic drawing machine (Silhouette CAMEO, Silhouette America, Lindon, Utah). The silver color of 5 Sharpie metallic permanent was used for drawing the channel boundaries. The green color of Sharpie ultrafine point marker was used for creating timer gates to stall the capillary flow for the desired time. After drawing all patterns, the uncovered side of the paper was covered by a tape with holes punched to be used as sample and reagent inlets. Fabrication of device case SolidWorks software (SolidWorks Corp., Waltham, MA) was used to design the device case (Figure 6) and the design was printed using a 3D printer (FormLabs Form 3B) with High Temp V2 Resin as a structural material. The printed device was soaked in isopropyl alcohol (IPA) for 15 min to wash away uncured resin. Supporting structures were cut and the surface was rendered smoothly with sandpaper. After washing with deionized water (DI) water and drying, white paint (Rust-Oleum, Painters Touch 2X Spray Paint Matte White) was sprayed and fully dried. Recombinant antigens/antibodies and ELISA reagents Recombinant SARS-CoV-2 spike, nucleocapsid protein (used as surface-coated antigens), and the conventional ELISA kit to measure IgG titer (Figure 2C) were obtained from Abcam (Cambridge, MA, USA). Recombinant antibodies to spike protein IgM/IgG (S- IgM/S-IgG) and nucleocapsid IgM/IgG (N-IgM/N-IgG) were purchased from Absolute Antibody (Boston, MA, USA). HRP-conjugated anti-IgM, anti-IgG secondary antibodies, and TMB solution were obtained from BioVision (Waltham, MA, USA). For the troponin I assay, ELISA reagents were obtained from Human Troponin I Type 3, Cardiac (TNNI3) ELISA Kit DIY Materials (MyBioSource, San Diego, CA, USA). Preparation of IgM/IgG and troponin I samples Whole blood samples were collected from healthy donors according to Georgia-Tech IRB approved protocol. The collected blood was centrifuged at 1500 x g for 10 mins to remove the cells and supernatant was used for the plasma samples. To prepare antibody samples with known antibody titers, 810 μg/ml of recombinant IgM and IgG samples were first analyzed using a conventional ELISA kit (BioVision, Waltham, MA, USA) to determine the antibody titers. Then, the samples were serially diluted with PBS (Figure 2A-Figure 2D) or plasma (Figure 3A-Figure 3E) to set the antibody titer from 1:19683 (810 μg/ml) to 1:27 (1.11 μg/ml). Diluted samples were analyzed again using conventional ELISA kits to ensure the estimated titers are correct. To prepare varying concentrations of troponin I sample Docket No.10034-227WO1 (Figure 4B) used for establishing the calibration curve, an initial troponin I sample (60 pg/ml) was serially diluted with the plasma to set the concentrations from 5.3 pg/ml to 60 pg/ml. The blinded troponin I samples (Figure 4D) of arbitrary concentrations were prepared by a third party. These concentration values were disclosed after the assay was completed. Calculation of the effective dilution factor in depletion ELISA To calculate the effective concentration of IgG based on the optical signal amplitude from a row of stacked wells, the Hill equation was employed as θ is the fraction of occupied binding sites and can be represented as the ratio of the measured to the maximum color intensity. Then, the Hill equation becomes as To estimate the remaining n in the equation, the color intensities for the first well in each row (i.e., all in the first column) were measured to get the values of I and [L]. The equation was then fitted to the experimental data using a curve fitting tool to obtain the values of the remaining parameters; Imax= 161.5, [LSO] = 0.4871, and n = 0.2819. With the equation built, IgG of the subsequent wells were estimated from the measured color intensities and the well-to-well dilution factors were calculated by comparing the concentration between two adjacent wells. Quantification of color intensity The images of the unfolded well sheet were captured after the depletion ELISA assay and individually analyzed using the ImageJ program. The raw images were first split into separate RGB channels, and the red channel image was used for quantification; the red-orange is a complementary color to the blue-green color of the reacted TMB solution. Measured gray intensities in wells from each row were subtracted by the intensity from the negative control row for normalization. Other advantages which are obvious, and which are inherent to the invention, will be evident to one skilled in the art. It will be understood that certain features and sub- Docket No.10034-227WO1 combinations are of utility and may be employed without reference to other features and sub- combinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

Docket No.10034-227WO1 Sequences SEQ ID NO.1: SARS-CoV-2 Spike protein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKT QSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEY VSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLV DLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGT ITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFN ATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS FVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYR LFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVV VLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGR DIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIH ADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPR RARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTM YICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDF GGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKF NGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGV TQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLS SNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTV YDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCG SCCKFDEDDSEPVLKGVKLHYT SEQ ID NO.2: SARS-CoV2 Nucleocapsid protein MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTAL TQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFY YLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTL PKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLL LDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRR GPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTY Docket No.10034-227WO1 TGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQ QTVTLLPAADLDDFSKQLQQSMSSADSTQA SEQ ID NO.3: Troponin I MADGSSDAAREPRPAPAPIRRRSSNYRAYATEPHAKKKSKISASRKLQLKTLLLQIA KQELEREAEERRGEKGRALSTRCQPLELAGLGFAELQDLCRQLHARVDKVDEERY DIEAKVTKNITEIADLTQKIFDLRGKFKRPTLRRVRISADAMMQALLGARAKESLDL RAHLKQVKKEDTEKENREVGDWRKNIDALSGMEGRKKKFES

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Claims

Docket No.10034-227WO1 CLAIMS What is claimed is: 1. A device comprising: a paper-based well sheet functionalized with a recognition element, the paper-based well sheet comprising hydrophobic lines demarcating at least two columns having a plurality of wells on the paper-based well sheet, wherein the plurality of wells form a grid comprising at least two rows and at least two columns, wherein the paper-based well sheet is folded such that the at least two rows of wells are disposed on one another to result in one folded row of wells; a flow controller; a blotting paper, wherein the paper-based well sheet is disposed between the blotting paper and the flow controller; and a case, wherein the paper-based well sheet, the blotting paper, and the flow controller are together contained within the case. 2. The device of claim 1, wherein the paper-based well sheet has a dimension of from 70 mm to 90 mm by from 120 mm to 140 mm. 3. The device of any one of claims 1-2, wherein the wells have a dimension of from 5 to 7 mm by from 5 to 7 mm. 4. The device of any one of claims 1-3, wherein the wells hold from 3 to 5 µL of a sample prior to saturation of the paper-based well sheet. 5. The device of any one of claims 1-4, wherein the recognition element is an antigen. 6. The device of claim 5, wherein the antigen is a viral antigen. 7. The device of claim 6, wherein the viral antigen comprises a SARS-CoV-2 antigen or an influenza antigen. 8. The device of any one of claims 1-7, wherein SARS-CoV-2 spike proteins are disposed in the columns with odd numbers and SARS-CoV-2 nucleocapsid proteins are disposed in the columns with even rows. 9. The device of claim 8, wherein SARS-CoV-2 spike proteins comprise spike protein IgM and spike protein IgG antibodies. 10. The device of any one of claims 8-9, wherein the SARS-CoV-2 nucleocapsid proteins Docket No.10034-227WO1 comprise nucleocapsid protein IgM antibodies and nucleocapsid protein IgG antibodies. 11. The device of claim 5, wherein the antigen is related to a disorder. 12. The device of claim 11, wherein the disorder is a cardiovascular disorder, disease, or cancer. 13. The device of any one of claims 1-12, wherein the device comprises 48 wells. 14. The device of claim 13, wherein the grid comprises 4 columns and 12 rows. 15. The device of any one of claims 1-12, wherein the device comprises 96 wells. 16. The device of claim 15, wherein the grid comprises 8 columns and 12 rows. 17. The device of any one of claims 10-16, wherein IgM is disposed in the first two columns and IgG in the last two columns. 18. The device of any one of claims 1-17, wherein the wells are coated with a capture antibody. 19. The device of any one of claims 1-18, wherein the flow controller comprises a sample input, wherein the number of sample inputs is equal to the number of columns. 20. The device of any one of claims 1-19, wherein the flow controller comprises at least one ELISA reagent inlet. 21. The device of claim 20, wherein the flow controller comprises four ELISA reagent inlets. 22. The device of any one of claims 20-21, wherein the ELISA reagent includes a washing buffer, 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate, Ab-HRP, Anti-IgM-HRP, Anti- IgG-HRP, or any combination thereof. 23. The device of any one of claims 1-22, wherein the flow controller comprises a first channel comprising a first delaminating timer, wherein the first channel comprises a first washing buffer, a second channel comprising a second delamination timer, wherein the second channel comprises a secondary Ab-HRP, a third channel comprising a third delamination timer and a fourth delamination timer, wherein the third channel comprises the washing buffer; and a fourth channel comprising a fifth delamination time, a sixth delamination timer, a seventh delamination timer, and an eighth delamination timer, wherein the fourth channel comprises a TMB substrate, wherein the washing buffer leaves the first channel first, the secondary Ab-HRP leaves the second channel second, the washing buffer leaves the third channel third, and the TMB substrate leaves the fourth Docket No.10034-227WO1 channel fourth. 24. The device of any one of claims 1-23, wherein the flow controller comprises a first channel and a second channel both comprising a washing buffer, a third channel comprising a first delamination timer, wherein the third channel comprises a secondary Anti-IgM-HRP, a fourth channel comprising a second delamination timer, wherein the fourth channel comprises a secondary Anti-IgG-HRP, a fifth channel comprising a third delamination timer and a fourth delamination timer, wherein the fifth channel comprises a TMB substrate, and a sixth channel comprising a fifth delamination timer, a sixth delamination timer, and a seventh delamination timer, wherein the sixth channel comprises a washing buffer, wherein the washing buffer leaves the first channel and the second channel first, the secondary Anti-IgM-HRP leaves the third channel second, the secondary Anti-IgG-HRP leaves the fourth channel third, the TMB substrate leaves the fifth channel fourth, and the washing buffer leaves the fourth channel fifth. 25. A method of detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising contacting a sample with the device of any one of claims 1-24. 26. A method of detecting IgG antibody, IgM antibody, or any combination thereof comprising contacting a sample with the device of any one of claims 1-24. 27. A method of detecting a cardiac injury comprising contacting a sample with the device of any one of claims 1-24. 28. A method of detecting troponin I in a sample comprising contacting the sample with the device of any one of claims 1-24. 29. The method of any one of claims 25-28, wherein the sample comprises blood, urine, semen, saliva, or any combination thereof.