WO2022220754A1 - Methods, devices and systems for rapid detection of biomolecules - Google Patents

Abstract

Disclosed are devices, systems and methods for detecting biomarkers from samples. A device generally includes a sample collection and preparation unit, a vertical flow device for capturing and labelling of a desired biomarker, a chemical dispensing unit, a processing unit for performing a transformation of the biomarker, one or more light sources, light guiding means, and one or more detectors. The transformation of the bio marker can be of spectrum, colour or fluorescence. The transformation of the biomarker comprises mixing the sample with the one or more reagents selected from the group consisting of cellulose binding domain-tagged (CBD-tagged) capture reagent, reporter reagent, CBD- tagged capture reagent and reporter reagent that bind to different epitopes of the target biomarkers from desired pathogens or host organisms and CBD-tagged capture reagent and reporter reagent whereby the CBD-tagged capture reagent offers the same binding epitope to the desired biomarker in the sample and reporting reagent.

Description

METHODS, DEVICES AND SYSTEMS FOR RAPID DETECTION OF BIOMOLECULES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to US Provisional Patent Application No. 63/174,711 filed April 14, 2021. The disclosure of the application is incorporated herein for all purposes by reference in its entirety.

FIELD OF THE INVENTION

[0002 ] The presen tinvention generally relates to methods, devices and systems for detection of biomolecules. More particularly, the present invention relates to methods, devices and systems for low-cost and rapid detection of biomolecules from samples.

BACKGROUND

[0003] Rapid diagnostic tests (RDTs) are common tests employed for detection of desired antigens and biomarker molecules at Point-of-Care (POC) and in laboratory settings. Lateral flow assay (LFA) is a standard format ofRDTs. LFAs are constrained by several limitations, for instance (i) un-controllable orientation of immobilized captured reagents (ii) stationary phase of the immobilized captured reagents, which limits interaction time between the capture reagents and the target biomarkers, thus limiting sensitivity (iii) use of capillary' action through porous material with small pore sizes to relay fluid samples to the reporter and capture reagents, necessitating long assay times (15-30 minutes) or (iv) lack of quantification due to instrument - free approach or inaccurate quantification due to limitation of a reader unit's image analysis capability. Ail these limitations lead to low test sensitivity, low test specificity and unnecessarily long processing time.

[0004] In contrast, usage of cellulose matrix w ithin a vertical flow device and engineered proteins have been described to overcome some of these limitations. Cellulose binding domains (CBD) are known to bind strongly and rapidly to cellulose matrix^1,2. Engineering of capture reagents to carry CBD allows the capture reagent to be orientated on cellulose fibers with its binding region exposed to the aqueous solution phase^1-3. Alternatively, CBD tagged engineered protein capture reagents can be allowed to form a complex with the target biomarkers and reporter reagents in a solution phase prior to applying the mixture to the cellulose-based device³. Vertical flow formats of the cellulose-based device can be designed to offer short fluidic paths³, therefore allowing liquid to flow through rapidly, while ensuring effective capture of target biomarkers through CBD-cellulose interaction³. These prior technologies allow RDTs to be performed rapidly within 5-10 min while maintaining high test sensitivity and specificity.

[0005] The described technologies can be applied to a wide array of applications for detection of proteins and biomolecules. For instance, combination of these technologies with engineered proteins that bind specifically to SARS-CoV -2 antigen⁴ or antibodies⁵, allowed generation of RDTs that can diagnose CQVID-19 and determine immune status against SARS-CoV-2.

SUMMARY OF THE INVENTION

[0006] The present invention provides methods, devices and systems for low-cost surd rapid detection of biomolecules from samples. Generally, the methods, devices and systems of the present invention use vertical flow assays, combined with material dispensing procedures, real- time spectroscopy, and/or other additional, optional or alternative elements. The methods, devices and systems of the present invention are advantageous particularly in low-cost, fast, spectroscopic, colorimetric, and fluorescence sensing applications.

[0007] In various exemplary embodiments, the present invention provides a vertical flow device comprising one or more test strips, one or more absorbent pads, and a cassette for housing the one or more test strips and absorbent pads. In some exemplary embodiments, a cassette includes one or more chamber structures to host the one or more test strips and absorbent pads. A cassette also includes one or more openings to the one or more test strips. The one or more openings and chamber structures are generally aligned with each other. In some exemplary embodiments, a cassette includes additional, optional or alternative features such as protrusion structures disclosed herein. In some exemplary embodiments, a test strip is treated using one or more paper preparation processes disclosed herein.

[0008] In various exemplary embodiments, the present invention provides a vertical flow assay workflow' comprising a sample processing step, and a chemistry reaction step. In some exemplary embodiments, the sample processing step includes filtration of a sample such as saliva. The chemistry reaction step includes interaction of Cellulose Binding Domain (CBD) to cellulose matrix, engineered capture reagents, engineered reporter reagent and reporting molecules that produce optical signals. In some exemplary embodiments, the chemistry reaction step includes incubation that allows effective capture of desired target biomarkers.

[0009] In various exemplary embodiments, the present invention provides optical readout systems for at least two-point absorbance/fluorescence sensor platforms that includes lateral or vertical flow assays such as immuno-assays, and for real-time and/or end point detection, process control, continuous or triggered sensing and/or detection of reaction kinetics. A readout system generally includes an illumination unit and a reading unit. For instance, in some exemplary embodiments, a read-out system includes a light source (such as one or more light emitting diodes) illuminating the sample surfaces, a means (such as fibers and lenses) capturing the back- scatered light and guiding it to at least a single detector (e.g. photodiode), one or more additional, optional or alternative optics (such as a collimating lenses, filters, dispersive elements such as gratings or prisms, focusing lenses), one or more additional, optional or alternative detectors (such as one or more than one photodiodes or pixel array detectors), and electronics converting the photon flux received into an electric signal for further signal processing.

[0010] In various exemplary embodiments, the present invention provides methods for identifying biomarker(s) in the sample of interest. For instance, in some exemplary embodiments, following the read-out, subsequent data processing translates the electronic signal into one or more predefined or user-defined metrics such as transmittance, absorbance, reflectance, or emittance data. Further statistical analysis allows to predict one or more parameters such as concentration.

[0011] The present invention provides several advantages, including but not limited to:

• Rapid detection of biomolecules

• Ability to measure one or more points from a given space/surface in parallel;

• Ability to measure at least one spectral channel;

• Flexibility' to arrange measuring points/probes spatially,

• Real-time measurements to follow the time evolution or kinetics of the sample under rest;

• Flexibility to use in-field for rapid detection of pathogens;

• Small size; and

• Low system cost [0012] The devices, systems and methods of the present disclosure have other features and advantages that will be apparent from, or are set forth in more detail in, the accompanying drawings, which are incorporated herein, and the following Detailed Description, which together serve to explain certain principles of exemplary embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more exemplary embodiments of the present disclosure and, together w ith the Detailed Description, serve to explain the principles and implementations of exemplary embodiments of the invention.

[0014] Figure 1 illustrates an exemplary vertical flow device comprising treated cellulose paper and absorbent pad(s) in accordance with exemplary' embodiments of the present disclosure.

[0015] Figure 2 illustrates an exemplary system and method for rapid in-field detection of biological or chemical samples such as pathogens in accordance with exemplary embodiments of the present disclosure.

[0016] Figure 3A, 3B and 3C illustrate exemplary test cassettes of vertical flow devices in accordance with exemplary embodiments of the present disclosure.

[0017] Figure 4A, 4B, and 4C illustrate exemplary assays for rapid detection of biological or chemical samples in accordance with exemplary embodiments of the present disclosure.

[0018] Figure 5A illustrates an exemplary sample (e.g., saliva) collection unit in accordance with exemplary embodiments of the present disclosure.

[0019] Figure SB illustrates an exemplary pipette in accordance with exemplary_' embodiments of the present disclosure.

[0020] Figures 6A, 6B, 6C and 6D illustrate exemplary' methods for detecting antigens, antibodies or desired biomarkers in accordance with exemplary embodiments of the present disclosure.

[0021] Figure 7 illustrates an exemplary_' manual, high-throughput screening station in accordance with exemplary embodiments of the present disclosure. Such a screening station can be used at any suitable locations including but not limited to customs and airports. [0022] Figure 8 illustrates an exemplary rail system of a semi-automated work bench in accordance with exemplary embodiments of the present disclosure.

[0023] Figure 9 illustrates an exemplary semi -automated work bench in accordance with exemplary embodiments of the present disclosure.

[0024] Figure 10 illustrates an exemplary low volume pump in accordance with exemplary embodiments of the present disclosure.

[0025] Figure 11 schematically illustrates LED illumination and single collection fiber positioning with respect to a sample surface in accordance with exemplary embodiments of the present disclosure. Light emitted from the LEDs illuminates each sample surface and diffusely reflected light is captured by a collection fiber a distance ‘d’ from the sample surface. NA denotes the numerical aperture of fiber.

[0026] Figure 12 schematically illustrates an exemplary 2-input fiber spectrograph in accordance with exemplary embodiments of the present disclosure. Two input fibers displaced in the x-y plane are imaged through a dispersion grating onto an array detector on the x’-y plane, whereby x and x’ plane denote the dispersion direction of the grating, z and z’ denote the optical axis and the tilted optical axis directions, respectively. Each of the fibers may come from a different sample location.

[0027] Figure 13 illustrates captured spectral absorption coefficient estimates of HRP/TMB reaction of non -spiked (0 nMol) solution over measurement time with a spectral reader in accordance with exemplary' embodiments of the present disclosure.

[0028] Figure 14 illustrates an exemplary illumination unit in accordance with exemplary embodiments of the present disclosure. The illumination unit features two light sources (e,g., LEDs, LDs) on either side of the light collection / light guiding means (shown here are fibers) in respect to the sample surface. The two light sources may be selected to span different spectral bands. For instance, one light source may be selected to span the visible spectral band ranging from 400-750 ran, whereas the second light source may be selected to excite the spectral emission of a fluorophore reporter reagent such as Alexa Fluor dye 594.

[0029] Figure 15 illustrates an exemplary arrangement of two fibers per sample surface captaring reflectance and fluorescent emission triggered by two independent light sources in accordance with exemplary embodiments of the present disclosure. [0030] Figure 16 illustrates four channel spectrograph including two long-pass filtered fibers displaced along the y-direetion at the entrance of the device in accordance with exemplary embodiments of the present disclosure.

[0031] Figure 17 illustrates four channel spectrograph with two fibers displaced along x and y- direction at the entrance of the device in accordance with exemplary' embodiments of the present disclosure.

[0032] Figure 18 illustrates an exemplary single point/fiber. single detector element, narrow band reader unit in accordance with exemplary embodiments of the present disclosure.

[0033] Figure 19 illustrates an exemplary arrangement of single-LED-single-element-detector per sample surface for optically sensing the reaction kinetics, for instance, the reaction kinetics by HRP/TMB reporter molecules, in accordance with exemplary embodiments of the present disclosure,

[0034] Figure 20 illustrates an exemplary arrangement of single-LED, filtered single-element- detector per sample surface in accordance with exemplary embodiments of the present disclosure.

[0035] Figure 21 illustrates an exemplary timing diagram of read-out unit with LEDs as illumination in accordance with exemplary embodiments of the present disclosure.

[0036] Figure 22 illustrates time-series trace (average reflectance <R(t)>) of HRP/TM P reaction over measurement time t with a spectral reader in accordance with exemplary embodiments of the present disclosure.

[0037] Figure 23 illustrates time-series trace (average absorbance <k/s(t)>) of HRP/TMP reaction over measurement time 't with a spectral reader in accordance with exemplary embodiments of the present disclosure.

[0038] Figure 24 illustrates reflectance and absorbance traces for 0, 1, 2, and 4 nano-mol/L concentrations of the N-protein in the saliva sample in accordance with exemplary- embodiments of the present disclosure.

[0039] Figures 25A and 25B illustrate schematically an exterior of an exemplary- system in accordance with exemplary embodiments of the present disclosure.

[0040] Figures 26A and 26B illustrate schematically an interior of an exemplary system in accordance with exemplary embodiments of the present disclosure. [0041] Figure 27 is a flowchart schematically illustrating an exemplary process in accordance with exemplary embodiments of the present disclosure.

[0042] Figures 28A and 28B illustrate schematically a spectral reader unit with two-channel spectrometer engine measuring control and test spots simultaneously in accordance with exemplary embodiments of the present disclosure, wherein D, BB, L2, L1, G denote detector, beam blocker, lens 1 and lens 2 and grating respectively,

[0043] Figure 29 illustrates reflectance spectra of blood and plasma samples in accordance with exemplary embodiments of the present disclosure,

[0044] Figure 30 illustrates schematically an exemplary fluorescent reader unit in accordance with exemplary' embodiments of the present disclosure, where L, F, BS, and D denote lenses, filters, beam spliter and detectors, beam paths are indicated with circled numbers, arrows are drawn to indicate beam directions and serve as mere indicator lines and shall not describe or limit beam acceptance angles.

[0045] Figure 31 illustrates an exemplary_' pulse train of sample emission recorded by avalanche photo detector or diode (APD) over 512 data points in accordance w ith exemplary_'· embodiments of the present disclosure.

[0046] Figure 32 illustrates an exemplary' pulse train of LED excitation recorded by photodiode over 512 data points in accordance with exemplary embodiments of the present disclosure.

[0047] Figure 33 illustrates an exemplary_' temperature response of APD when measuring the emission intensity of a calibration cassette over device temperature in accordance with exemplary embodiments of the present disclosure.

[0048] Figure 34 illustrates an exemplary temperature response of LED and photodiode detector (PD) over device temperature in accordance with exemplary embodiments of the present disclosure.

[0049] Figure 35 illustrates exemplary temperature response curves of APD (blue) and PD (red) normalized to device operation temperature at e.g., 33 C and detector ratio D_(T) (black) in accordance with exemplary_' embodiments of the present disclosure.

[0050] Figure 36 illustrates exemplary_' D(T) and 4th order polynomial fit TC(T) (red) in accordance with exemplary embodiments of the present disclosure. [0051] Figure 37 illustrates exemplary temperature corrected responses in accordance with exemplary embodiments of the present disclosure.

[0052] Figure 38 illustrates schematically an exemplary read positions fluorescent reader unit in accordance with exemplary embodiments of the present disclosure.

[0053] Figure 39 illustrates schematically an exemplary read positions spectral reader unit in accordance with exemplary embodiments of the present disclosure,

[0054] As will be apparent to those of skill in the art, the components illustrated in the figures or disclosed herein are combinable in any useful number and combination. The figures are intended to be illustrative in nature and are not limiting.

DETAILED DESCRIPTION

[0055] Reference will now be made in detail to implementation of exemplary embodiments of the present disclosure as illustrated in the accompanying drawings. The same reference indicators will be used throughout the drawings and the following detailed description to refer to the same or like parts. Those of ordinary skill in the art will understand that the following detailed description is illustrative only and is not intended to be in any way limiting. Other embodiments of the present disclosure will readily suggest themselves to such skilled persons having benefit of this disclosure.

[0056] In the interest of clari ty, not all of the routine features of the implementations described herein are shown and described. It will be appreciated that, in the development of any such actual implementation, numerous implementation-specific decisions are made in order to achieve the developer's specific goals, such as compliance with application- and business-related constraints, and that these specific goals will vary from one implementation to another and from one developer to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, hut would nevertheless he a routine undertaking of engineering for those of ordinary skill in the art having the benefit of this disclosure.

[0057] Many modifications and variations of the exemplary embodiments set forth in this disclosure can be made without departing from the spirit and scope of the exemplary embodiments, as will be apparent to those skilled in the art. The specific exemplary embodiments described herein are offered by way of example only, and the disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

OVERVIEW

[0058] Embodiments of the present disclosure are described in the context of devices, systems and methods for rapid detection of biological or chemical samples. The devices, systems and methods of the present disclosure use spectroscopic reading platforms and define workflows leading to rapid, quantitative, and effective method for detection of desired target biomarkers, such as SARS-CoV-2 nucleocapsid proteins antibodies, from various types of biological examples. As disclosed herein, the devices, systems and methods of the present disclosure enable a rapid quantitative detection of biological or chemical samples such as virus antigen or antibodies within a short period of time, for instance, within 2, 3, 4 or 5 minutes from the time of sample collection.

[0059] In some exemplary embodiments, the devices and systems of the present disclosure include or are used with a vertical flow device/platform, such as the exemplary vertical flow device/platform illustrated in Figure 1, for sample collection, loading, detection, and/or other processes.

[0060] The process of the present disclosure enabling the rapid, in-field detection of pathogens such as SARS-CoV-2 within a short period of time (e.g,, within 2, 3, 4 or 5 minutes from sample collection) includes one or more steps. For instance, as illustrated in Figure 2, in some exemplary embodiments, the process includes: (1) the sample collection, (2) wet chemical mixing steps and incubation, (3) the dispensing of the sample onto the vertical flow device, (4) reading the sample output with the reading unit, and/or (5) the analytics and display of results from the reading.

[0061 ] While exemplary embodiments of the present invention are described with specific values regarding dimensions, time, volumes, temperatures, angles or other parameters, the specific values are given by way of example and are not intended to be in any way limiting. Values for dimensions, time, volumes, temperatures, angles or other parameters are readily adjustable to meet the needs of different applications. While some embodiments are described with saliva as a sample and SARS-CoV-2 as a target pathogen, it should also be noted that saliva and SARS- CoV-2 are given by way of example and are not intended to be in any way limiting. Samples can come from a wide range of application areas such as clinical, veterinary, agricultural, food, bio- defense and environmental industries, and any biological or chemical molecules can be the target. While some embodiments are described with LEDs as the illumination light sources, it should also be noted that LEDs are given by way of example and are not intended to be in any way limiting. Any suitable light sources can be used.

THE VERTICAL FLOW DEVICE - CASSETTE DESIGN

[0062] An exemplary vertical flow device generally includes one or more test strips, one or more absorbent pads, and a cassete for housing the test strip(s) and absorbent pad (s). A cassete of a vertical flow device can have various shapes, sizes and/or other configuration features. For instance, by way of example, Figure 3A illustrates a test cassette of a vertical flow device in various shapes (211). A cassette can contain one or more openings to the cellulose-based test strips for single or multiple test spots (212), single or multiple negative control spots (213) and/or single or multiple positive control spots (214). Additionally or optionally, a cassette can provide spaee(s) for QR and/or bar codes (215), or other identification means. A cassette can be a unitary piece or composed of multiple pieces. For instance, by way of example, Figure 3B illustrates a cassette including two pieces, atop piece (221) and a bottom piece (222). The two pieces can be assembled to form an enclosed cassette containing one or more chamber structures (223) inside the enclosing case and aligned with the cassette openings for hosting one or more test strips and one or more absorbent pads. The openings may be located at the top piece of the cassete. The opening shapes (224) may be, but are not limited to, a cylinder shape with perpendicular angle between the opening wall and the outside surface of the cassette or may comprise a slope to allow a wider opening. The inside surface around the perimeters of the openings may contain one or more protrusion structures. The protrusion structure may be a sharp peak (225) or have some width. In exemplary embodiments, the protrusion structure is not wider than a predetermined width such as about 2 nun (225). The cellulose-based test strips (226) may comprise single or several layers. Absorbent pads (227) cars be of any material that absorbs liquid, examples of the material including but not limited to cellulose, sponge, or cotton. It can comprise single or several layers. Absorbent pads can be placed underneath the test strips (226). The test strips (226) and absorbent pads (227) may be placed inside the chamber structure (223) and aligned to the openings (212 or 213 or 214) located at the top piece (221) of the cassette.

[0063] In some exemplary' embodiments, for example assays for rapid detection of virus antigen or antibodies such as SARS-CoV-2’s nucleocapsid (N) protein and neutralizing antibodies (NAbs), a cassette size of about 30 mm x 48 mm x 6 mm (231 and 232) or the like can be used. The cassette may contain two openings: One for a test spot and one for a control (e.g., positive control) spot. It may contain space(s) for QR/barcode codes that encode information, e.g., the cassette identification, batch, and reference numbers. Each of the openings may be square or circular, or the like, in shape and may have a chamfered edge. An exemplary circular cut-out may have a diameter of about 3,8 ± 0.5 mm (233) or the like. An exemplary protrusion structure at the inner face of an opening may be about 0.5 mm or the like in width (233). Chamber (223) sizes of about 9 mm x 16 mm or the like with the wall height of about 2.5 -2.9 mm or the like can be used

(234). Cellulose paper with an average thickness of about 150-180 μm or the like and pore size of about 11 μm or the like cars be used as test and control strips. Each strip may have, but not limited to, about 8.5 mm x 15.5 mm or the like in size or different sizes that can fit in the chamber. One, two, three or more layers of cellulose strip can be used for each test and control spots. Multiple layered strip (e.g,, the three-layered strip illustrated in Figure 3C) can be connected as one piece

(235). The paper can be folded alternately backward and toward (in a zig-zag motion) to stack the paper into three (or more) layers (235 and 2.36). The paper can be folded or corrugated horizontally, e g,, by means of a special tool akin to a waffle-iron , With the folds having about 0.5 - 1 mm height or the like, the surface of cellulose directly exposed to a dispensed liquid is significantly larger, thereby contributing to further speeding up of the process. This kind of “corrugated” paper is prc-fabricatcd on a special press. Absorbent pads can be cellulose paper having a similar width and length to the single layered test strip. The average thickness of the absorbent pad can be about 1.5 mm or the like. One, two or more absorbent pads can be used. In an exemplary embodiment, cassette assembly (237) is performed by placing the test strips such as the three-layered test strips (235) on top of the absorbent pads e.g., the two-layered absorbent pads (238). In an exemplary format, the test strips and absorbent pads stack are placed in the chamber (234 and 223). The top piece of the cassette can be assembled on top, with openings aligned above the test and control strips, holding test strips and absorbent pads together.

[0064] In some exemplary embodiments, it is advantageous to include one or more recess areas in the chamber below the absorbent pads to allow for swelling when liquids are absorbed. Furthermore, one or more through holes in the lower shell below each test spot may allow for passive drainage or vacuum suction when required.

THE VERTICAL FLOW DEVICE - PAPER PREPARATION PROCESS

[0065] In some exemplary embodiments, cellulose-based materials are used as a testing matrix for vertical flow devices. Test strips are designed to detect the desired target biomarkers from samples. Control strips, e.g., negative control strips, are designed to host and enable quantification of non-specific chemical reactions that take place in the absence of target biomarkers, serving to generate baseline signals. The positive control spots are designed to host the chemical reaction that produces the desired signals, serving to verify performance and integrity of the chemical reagents and fluid flow processes.

[0066] Referring to Figure 4A, test strips may be used without pre-treatment (e.g., as blank cellulose paper) (311) or with hydrophobic materials to define liquid flow path (312). Hydrophobic materials can he applied to the topmost layer or to all layers of the cellulose stops, if multiple layers are used.

[0067] The test strips may be treated with blocking solution or a combination of capture reagents and blocking solution (313). Blocking solution serves to prevent non-specific binding of non- target molecules in the samples. It may comprise, but not limited to, casein-based and/or bovine serum albumin (BSA)-based solution. The treatment of the test strips with capture reagents leads to immobilization of the reagents on to the test spots. Negative control spots may be treated with blocking solution (314). Positive control spots may be treated with a combination of reagents that interact with the reporter reagent (e.g. target biomarker, antibodies, engineered binding proteins, each fused to and anchored to cellulose by a CBD) and blocking solution (315). The treatment of the cellulose-based positive control strips leads to immobilization of the reagents that interact with the reporter reagent, on to the positive control spots. Treatment of the cellulose strips can be applied to the topmost layer or to all layers of the strips, if multiple layers are utilized.

[0068 ] In some exemplary embodiments, for example assays for rapid detection of virus antigen or antibodies such as SARS-CoV-2’s N protein and neutralizing antibodies (nAbs), only test strips and positive control strips may be used. A liquid flow path is defined. In an exemplary embodiment, wax ink can be printed on the cellulose paper to define liquid flow path (321). The non-printed area is defined as a hydrophilic region where solution can be applied and will travel. A printed pattern can be printed to one, more than one or all layers of the paper strip to define a continuous liquid flow path. The printed paper can be baked, e.g., for about 1 min at about 150 °C or the like, to allow the wax ink to diffuse through the paper (or papers) to the desired thickness. After baking, the hydrophilic region may shrink slightly. An exemplary diameter of the hydrophilic region is in a range of about 6,5 - 6,8 mm or the like,

[0069] When using vertical flow devices featuring a protrusion ring (225) in the upper shell, wax printing may be omitted. [0070] In some exemplary embodiments, in the example test for rapid detection of SARS-CoV- 2^'s N protein, one or more layer of the test strips can be blocked by dispensing a blocking solution, such as casein-based blocking solution or the like (e.g., about about 10 μL ), to the hydrophilic region (322). Excess solution can be blotted away from underneath by any absorbing materials. The positive control spots can be treated by dispensing about CBD tagged SARS-CoV- 2 N protein (e.g., about 4 μL of about 2.5 μM N-CBD) or the like to the topmost layer of the strip. Subsequently, a blocking solution, such as casein-based blocking solution or the like (e.g., about 10 μL), can be dispensed to each layer of the paper strip.

[0071 ] Excess solution can be blotted away from underneath by any absorbing materials (322). In another example, the rapid detection of SARS-CoV-2 neutralizing antibodies, each layer of the test strips can be blocked by dispensing a blocking solution, such as casein- or bovine serum albumin (BSA)-based blocking solution or the like (e.g., about 10 μL), to the hydrophilic region (323). Excess solution can be blotted away from underneath by any absorbing materials. The positive control spots can be treated by dispensing CBD tagged SARS-CoV-2 receptor binding domain (e.g., about 4 μL of 2.5 μM RBD-CBD) protein or the like to the topmost layer of the strip. Subsequently, a blocking solution, such as 10 μL of casein- or BSA-based blocking solution or the like, can be dispensed to each layer of the paper strip. Excess solution can be blotted away from underneath by any absorbing materials (323).

[0072] After preparation, strips may be stored, for instance, at about 4 °C or the like, until usage.

SAMPLE COLLECTION UNIT

[0073] Several types of samples may be applied on the cellulose-based vertical flow devices. Samples may be collected and processed prior to dispensing it onto the vertical flow device.

[0074 ] In some exemplary embodiments, in an exemplary case of testing for SARS-CoV-2’s N protein from saliva, the saliva samples may be collected using off-the-shelf saliva collection devices such as the SuperSAL^TM by Oasis Diagnostics Corporation,

[0075] Alternatively, saliva samples may be collected by a saliva collection unit such as the collecting unit illustrated in Figure 5A. In some exemplary_' embodiments, the sample (e.g., saliva) collection unit includes a protection cap sealing a plunger tube, a plunger tube and seal, a sample barrel, a saliva filter piece that can apply shear force to the break mucus bond e.g. cotton, cellulose, mesh with pore size smaller than or equal to about 5 μm or about 4 μm or about 3 μm, a protection cap, and a removable sample botle with graduation lines. The exemplary design lends itself for low-cost manufacturing by injection molding processes.

[0076] In an exemplary collection operation, a technician retrieves the saliva collection unit, removes the protection cap and pulls the plunger tube to its maximum length. Subsequently, the test person places the plunger tube inside the mouth (looking downwards) and pushes the saliva inside the tube. Once a minimum volume, such as a volume of about 1 mL , is collected in the barrel, the technician closes the protection cap and presses the plunger down until sufficient volume is pressed through the saliva filter into the sample bottle marked with an indication line. The sample bottle is removed and a saliva pipette is inserted as illustrated in Figure 5B. The sample botle is capped with the saliva pipete, finalizing the sample collection process. The saliva pipette sampling volume is given by the length of the tubular section (Pipette Tube) at the bottom of the pipette. A graduation line marks a designated sample volume.

[0077] In some exemplary_' embodiments, in the case of testing for SARS-CoV-2 nAhs, whole blood samples may be obtained from a subject. An exemplary method for collecting whole blood is by pricking a fingertip with a lancet. Drops of blood may be collected using a capillary for immediate usage or for further processing. Alternatively, whole blood may be obtained from venous blood sampling using commercially available evacuated tube system e.g. Vacutainer from Becton, Dickinson. Collected whole blood can be used without further processing or after processing with known methods including, but not limit to, centrifugation, filtration, etc., to obtain plasma or serum samples.

WET CHEMISTRY AND PROCESSES

[0078] Referring to Figures 6A-6C, in some exemplary embodiments, an antigen, antibody or a desired biomarker can be detected using various methods including, but not limited to, match- paired capture and reporter reagents that bind to different epitopes of the target analyte ^1-4 (sandwich assay) (501) or capture reagents that offer the same binding epitopes to both target analytes and reporter reagents⁵ (competitive assay) (520). Reporter reagents (505 and 523) are tagged to signal reporting molecules (506) forming a functional reporter reagent (507 and 524). The signal reporting molecules (506) can be, but not limited to, enzymes e g. horse-radish peroxidase, nanomaterials e.g. gold nano-particles, fluorophore e.g. alexa fluor 594, chemiluminescence, etc. Capture reagents (502 and 521) can by tagged to CBD (503) forming a cellulose compatible capture reagent (504 and 522). [0079] In the sandwich assay condition (501 and 509), sample can be mixed with capture and reporter reagents and incubated in the liquid phase format (510) for anywhere from about 1 min to about 5 min before applying the mixture (510) to the test and positive control spots on the vertical flow device. Negative control spots are not used in this embodiment because the CBD tagged capture reagent (504) will capture the target analytes, if present, onto the test strips. Alternatively, if the capture reagents are pre-immobihzed on the cellulose test strips (511), sample can be mixed with reporter reagents (507 + 508) and incubated in the liquid phase format for 1-5 min before applying the mixture (512) to the test, negative control, and positive control spots on the vertical flow device. Optionally, washing reagent can be applied to the vertical flow device following application of the sample (510 and 512). If enzyme (e.g. HRP) is used as a reporting molecule, substrate reagent (e.g. 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate), can be subsequentially applied to the vertical flow device for signal generation.

[0080] In an exemplary competitive assay (520 and 527), samples can be mixed with capture reagents (522) and reporter reagents (524) and incubated in the liquid phase (528) for anywhere from about 1 min to 5 min before applying the mixture (528) to the cellulose-based vertical flow device.

[0081] In some exemplary embodiments, in an example test for rapid detection of SARS-CoV-2^’s N protein, sandwich assay format (501) can be used. CBD tagged binder protein engineered from rcSso7d (rcSso7d.NP1 CBD) can be used as a capture reagent⁴ (504) and biotin (BA) and maltose binding protein (MBP) tagged rcSso7d (BA-MBP-rcSso7d.NP2) can be used as a reporter reagent

(505)⁴. Streptavidin (SA) conjugated horse radish peroxidase (HRP) (SA-HRP) can be used as an enzymatic reporting molecule (506). Combination of BA-MBP-rcSso7d.NP2 (505) and SAHRP

(506), forms a complete functional reporter reagent (507).

[0082] in some exemplary embodiments, rapid detection of SARS-CoV-2 N protein from saliva can be performed according to the exemplary_' workflow illustrated in Figure 6C. For instance, (i) saliva sample processed through filtration can be mixed with reagent A (e.g., lysis buffer) to break to the vims membrane. Total volume of saliva and lysis buffer, in an exemplary- embodiment, is about 360 μL or the like; (ii) about 40 μL or the like of reagent B (e.g., comprising of rcSso7d.NPl -CBD (504), BA~MBP~rcSso7d.NP2 + SA-HRP (507)) can be added to the sample/lysis mixture; (iii) the mixture can be incubated, e.g., for anywhere from about 1 mm at about 20-25 °C or the like; (iv) about 40 μL or the tike of the mixture can be added to the test and control spots, respectively, on the cassette; (v) about 40 μL or the like of reagent C (e.g., Tween-20 based solution) can be added to test and control spots, respectively, to wash off non- specific molecules; (vi) about 40 μL or the like of reagent D (e.g., TMB) can be added to test and control spots, respectively, for signal generation; and (vii) signal can be detected using a spectral reader device including, but not limited to, the spectral reader unit disclosed herein,

[0083] In some exemplary embodiments, in an example test for detection of SARS-CoV-2 nAbs, competitive assay format (520) can be used, CBD tagged SARS-CoV-2 receptor binding domain (RBD-CBD) can be used as capture reagent⁵ (522) and Alexa fluorophore 594 tagged human angiotensin converting enzyme 2 (ACE2) receptor can be used as a reporter reagent⁵ (524).

[0084] In some exemplary embodiments, rapid detection of SARS-CoV-2 nAbs from whole blood, serum, or plasma can be performed following the exemplary workflow illustrated in Figure 6D. For instance, (i) whole blood, serum, or plasma sample can be mixed with reagent A (522) (for example, about 40 μL of whole blood, serum, or plasma sample can be mixed with about 40 μL of reagent A, resulting in the total volume of sample and reagent A about 80 μL, or the like; (ii) reagent B (524) can be added to the sample mixture (for example, about 40 μL or the like of reagent B, winch composes recombinantly expressed receptor binding domain (RED) of SARS-CoV-2 tagged to cellulose binding domain (CBD), can be added to the sample mixture); (iii) the mixture can be incubated, e.g., for about 5 min at about 20-25 °C or the like; (iv) the mixture (for example, about 40 μL or the like of the mixture) can be added to the test and control spots, respectively, on the cassette; (v) reagent C (e.g., phosphate buffer saline (PBS) based solution) can be added to test and controls spots, respectively, to wash off nonspecific molecules (for example, about 40 μL, or the like of reagent C can be added); and/or (vi) signal can be detected using a detector including, but not limited to, the spectral reader unit described thereafter.

[0085] Both presented w orkflows may be performed by an operator by hand, or in a semi- automated or fully automated fashion as disclosed herein.

[0086] It should be noted that sample volumes disclosed herein are exemplary and should not be interpreted to limit the workflow disclosed. Sample or reagents volumes may be adjusted for further process or throughput optimizations. CHEMICAL DISPENSING / WORK BENCHES FOR HIGH THROUGHPUT

SCREENING

[0087] Disclosed herein are manual and a semi-automated processing lines and work benches enabling high-patient-throughput testing as perceived for mass-screening.

[0088] Figure 7 illustrates an exemplary manual, high-throughput screening station in accordance with exemplary embodiments of the present disclosure. In some exemplary embodiments, examinees such as travelers register with a first operator and are handed a sample collection unit. Upon filling the sample collection unit, the examinee removes the sample bottle and places it onto a designated area of the 2^nd operators work bench. Saliva collection tube is discharged by the examinee. The examinee moves into a designated waiting area. The 2^nd operator withdraws sample volume, adds solution A and B and sets the mixture for incubation of 1 min. Subsequently, he/she passes the sample bottle to a designated area on the 3^rd operators^' work bench. The 3^rd operator withdraws a sample volume from the sample bottle and dispenses the volume on test and control spots of the vertical flow device referred here as cassette. The sample collection bottle is closed and discarded. 3^rd operator then manually dispenses solution C followed by D onto both spots of the cassette. Once the solution subsided, the operator places the cassette into the reading unit. The reading unit reports amount and concentration estimates and flags test results in either red (stop) or green (go) colors. In case of a confirmed infection (flagged by red), the examinee is re-routed to a medical station for further validation. In case of a detected non-infection (flagged by green), the examinee is allowed to proceed.

[0089] Figure 8 illustrates an exemplary rail system of a semi -automated work bench in accordance with exemplary embodiments of the present disclosure. In some exemplary embodiments, the cassette sits in the rail system. Below the rail system, push butons allow signaling when the cassette is to be moved forward to the next work station.

[0090] Figure 9 illustrates an exemplary semi-automated work bench in accordance with exemplary embodiments of the present disclosure. In some exemplary embodiments, in its operation, the examinee places the cassette and the sample bottle after sample collection into a rail and discards the sample collection device. Once placed the push button is pressed and the cassette and sample bottle are moved forward. At the second step, solution A is automatically dispensed into the sample bottle and moved forward. Location 3 automatically adds solution B. At station point 4, an operator adds a drop cap onto the sample bottle . The solution is manually shaken and placed back. The push of the button below the station moves the sample forward to position 5 at which a timer sets an incubation time of 1 minute. Upon completion, the operator then manually dispenses drops of the solution onto test and control spots of the cassette and discards the sample bottle. A push of the rail activation button moves the cassette to station 6 at which solution C is automatically dispensed onto the cassette and moved to station 7, Station 7 automatically dispenses Solution D and moves the cassette to the optical read-out unit. The read- out measures and displays the result on a screen. Upon completion, the cassette is automatically discarded at station 9 and the process is completed.

[0091] Figure 10 illustrates an exemplary small volume, automated reagent dispensing system (e.g., low volume pump) in accordance with exemplary embodiments of the present disclosure. In some exemplary embodiments, the system includes inlet and outlet tubes allowing the solution to enter or leave the system. In the filling position or passive position, the barrel is forced up by a spring to the upper position. Barrel entrance bore hole and fill tube align and solution can stream into the barrel body of designed fill volume. In the dispense position, an electric trigger signal signals a valve to compress/retract a plunger and the barrel moves to its lower position. In the lower position, barrel exit bore hole and exit tube position align and the volume can flow out of the barrel. Once the barrel is emptied, the trigger signal is removed, the valve opens and a loading spring pushes the barrel back into the upper filling position for the process to repeat.

SPECTRAL READERS

[0092] Disclosed herein are optical reader devices and systems for rapid, in-field detection of pathogens. Optical readers disclosed herein may be used in conjunction with the chemistry described herein or variants thereof. By way of example, two reader platforms are tailormade but not limited to the presented cassette design. Exemplary combinations of reporter reagents, illumination and reader configuration are listed in Table 1 below. They are non-limiting examples. Any suitable combination of reporter reagents, illumination and readers (or detectors) is encompassed by the present disclosure. In addition, other additional, optional or alternative reporter reagents, illumination and readers can be used. For instance, detectors, including but not limited to silicon photodiodes, silicon avalanche photo detectors (Si-APD), photo multiplier tubes (PMT), can be used.

Table 1 : Combinations of reporter reagents, illumination and reader configuration

[0093] Both systems may be differentiated into a spectroscopic, all-purpose or single-wavelength band, fixed-purpose optical read-out units. Each of the reader units includes an illumination unit and a reading unit. It is evident that combinations of either disclosed are conceivable and shall not be interpreted as limiting the use of the described reader units.

[0094] Examples of the spectroscopic, all-purpose optical read-out has been disclosed previously in patent application US Provisional Patent Application No. 63/136,777 “Multi-Point Spectral Devices, Systems and Methods for Real-Time Colorimetric and Fluorescence Sensing Applications,” the disclosure of which is incorporated herein for all purposes by reference in its entirety.

[0095] In some exemplary embodiments, a reading unit includes colorimetric reporter molecules such as Horseradish Peroxidase (HRP) and TMB. In some exemplary embodiments, the reader or variations of the reader detect the fluorescent reporter agent Alexa 594 or gold nanoparticle solutions.

[0096] In some exemplary embodiments, in the case of colorimetric reporter molecules such as HRP/TMB, it is advantageous for the reading unit to be equipped with abroad-band white light source such as high chromatic rating index (HRI) LEDs emitting a continuous spectral band ranging from about 390-750 nm or broader. The LEDs may be arranged to emit light and illuminate each sample surface under an angle, for instance an angle of 45°, from the samples’ surface normal and the diffuse reflected beam may be collected/captured by an arrangement of optical components including lenses, mirrors and fibers at 0°, parallel to the surface normal. It is obvious that any other arrangement of light illumination and collection angles are possible. Figure 11 depicts a single sample area, an ilium ination LED, and collection fiber schematically. Per sample area, a fiber is placed parallel to the samples surface normal at a distance ‘d’ from its surface satisfying the equation

where D denotes the sample surface diameter and α the half acceptance angle of the fiber given by its Numerical Aperture (NA) according to NA= nsinα, n denotes the refractive index of the medium between fiber and sample surface.

[0097] The spectral reading unit is designed to cover the spectral working band of the LEDs emission band, namely 390 to 700 run at least. In some exemplary embodiments, the spectral reading unit includes a collimating optics, a dispersive element such as a grating, a filter unit, or alternatively phase-sensitive interferometer, a focusing optics and an array detector, in the said spectral band such detectors include hut are not limited to CMOS and CCDs.

[0098] GRATING BASED - SPECTROGRAPH

[0099] The collimating optics of the spectral reading unit can be an achromatic doublet lens, a ruled / holographic (or replicas thereof) transmission grating, an achromatic doublet focusing lens and/or a monochrome CMOS detector. For instance, in some exemplary embodiments, the collimating optics may he an achromatic doublet lens of about 12.7 mm diameter and about 20 mm focal length, a ruled / holographic (or replicas thereof) transmission grating with about 600 hues per millimeter (i/mm) or similar, an achromatic doublet focusing lens of about 12.7 mm diameter and about 20 mm focal length and a monochrome CMOS detector such as the On Semiconductor (AR0144 or AR0134).

[00100] By transmission/reflection from the grating, light is split into spectral elements of a given bandwidth and leaves the grating under wavelength dependent angles according to the well- known diffraction formula:

whereby Qm denotes the diffraction angle, ns the diffraction order in integer values (1, 2, 3,

... ), A the wavelength and g the grating period.

[00101] In some exemplary embodiments, sn the case of the grating-based device, the focusing optics may be arranged to capture the 1^st order beam diffracted through/from a 600 l/mm grating at an diffraction (tilt) angle, such as an angle of about 19.3° from the surface normal of the grating, resulting in a dispersion on the detector of approximately 80 nm/mm over a wavelength band from 400-700 nm. Other arrangements of grating ruling number, tilt angles and focusing lens are possible covering the spectral band of 400-700 nm on the detectors surface. To name an alternative combination, a 830 line/mm grating with about 12.7 mm diameter collimating and focusing lenses of about 12 mm focal lengths, atilt single, e.g., an single of about 27.2°, covers the spectral width of approximately 400-700 nm at a dispersion of 90 nm/mm. Figure 12 depicts an exemplary 2 input fiber/channel spectrograph read-out unit schematically.

[00102] In an exemplary arrangement of 2 input fibers, an array detector of about 3.6 mm x 4.8 mm in size receives an image of tw o spectral streaks spanning approximately 400-750 nm in x’-y plane, each of which carrying the spectral composition of the fibers input spectrum. Each detector element {e.g,, pixel) along the x' axis in the array detector thereby receiving a differen t spectral wavelength component.

[00103] Referring to Figure 13, in some exemplary embodiments, a spectral reader is designed capturing of the spectral absorbance band of the HRP/TMB ranging from approximately 400-750 nm.

[00104] In some exemplary embodiments, in the case of fluorescent reporter molecules such as Alexa Fluor dye 594 or strongly scattering marker such as gold nanoparticles, it is advantageous for the reading unit to be equipped with a narrow-band light source such as single- color LEDs or laser diodes or lasers emitting a narrow spectral band tailored to the absorption baud of the reporter molecule,

[00105] In an exemplary_' arrangement for Alexa Fluor dye 594, a laser diode (LD) emitting light in a spectral band ranging from about 500-590 nm such as 532 nm diode would be advantageous. Alternatively, LEDs emitting narrow-spectral band radiation centered at about 590 nm such as AlGalnP LEDs or LEDs emitting a similar emission spectrum may be utilized. The optical arrangement of the sample illumination as described in Figure 11 may be adapted to replace the white LED disclosed herein with a single-color LED or a laser diode. Furthermore, excitation filters, commonly applied in fluorescent detection systems, may be utilized to limit the spectral emission band of a broad-band light source (e.g. Xenon flash lamps, white LEDs, or Tungsten Halogen light sources) to the absorbance peak of Alexa Fluor 594 (590 nm wavelength). These filters may include traditional transmission (interference or absorbance) filters, dichroic beamsplitters, or may include prism- or grating based spectral filter systems. Alternatively, the illumination unit may he further equipped with the aforementioned light sources as illustrated in Figure 14.

[00106] In some exemplary embodiments, in the case of a spectrograph disclosed herein, the optical arrangement may be further equipped with a long-pass (LP) filter upstream or downstream the fibers. Such LP filters may be permanently or manually inserted into the beam when the illumination is switched to the fluorescent emission measurement. LP filters are required to separate the fluorescent from the excitation radiation reflected from the sample surface. In the case of Alexa Fluor 594, LP filters with a cut-on wavelength of about 600 nm or similar may be used. Such filter may include hard coated and/or dichroic filters and/or beamsplitters.

[00107] In an alternative optical arrangement, each sample surface may be further equipped with additional light guiding means such as an additional fiber designed to transport the fluorescent emission to the spectral reading unit as illustrated in Figure 15.

[00108] In such a two-fiber-collection unit per sample surface, the spectrograph further includes additional, alternative or optional fibers in its input beam. The placement of the fibers at the entrance of the spectral reading unit may be varied for the additional, alternative or optional input fibers.

[00109] For instance, in the case of LP filtered fibers, the additional fiber may be displaced vertically from the original sample fibers described previously. All fibers leading into the spectrograph are displaced vertically on top of each other (along the y-axis). In this ease, the detector receives additional spectral streaks along i ts vertical y-axis by the number of fibers added. It should be noted, however, that each of the fibers designed for the fluorescent emission must be equipped with a suitable LP filter to cut off directly reflected radiation of the excitation light source. The LP filter may either be placed at the fibers^' entrance or exit. Figure 16 depicts an exemplary 4 fiber channel spectrograph in which two additional fibers are filtered at the exit of the fibers entering the spectrograph.

[00110] Referring to Figure 17, in a further arrangement, the fibers assigned to the fluorescent emission may be displaced in both directions in the x-y plane of the spectrographs entrance plane. In such an arrangement, the displacement of the fibers along the x and y axis results in a relocation of the spectral band imaged onto the detectors' x’-y plane. In an exemplary embodiment using a 600 l/mm grating, about 19.3° tilt, and about 20 mm focal length lenses, as disclosed herein, a displacement of the entrance fiber position of about 1 mm along the y-axis results in a spectral band shift of about 90 nm on the detector plane. Thus, by shifting the location of the entrance fibers transporting the fluorescent emission, the spectral bandwidth received by the array detector can be tailored to the specific spectral requirement.

[00111] In an exemplary arrangement for the fluorescent reagent Alexa Fluor dye 594, a shift of the fibers guiding the fluorescent emission by about 2.2 mm (200/90) along the x-axis, results in a spectral shift of about 200 nm at the detectors surface. In such an exemplary arrangement, the placement of LP filters may be omitted.

[00112] Similar concepts can be adopted for other fluorescent or nano-particle based reporter reagents.

[00113] READOUTS BASED ON SINGLE DETECTOR ELEMENT PER SAMPLE POINT

[00114] In the event low-cost read-outs are required, the spectrograph may be replaced by reading units using a single element detector (e.g., a single pixel detector, a single photodiode detector or the like) per sample surface. Con trary to the spectroscopic reading unit such read-outs must be tailor-made to the specific marker reagent in combination with the chemistry disclosed herein.

[00115] The present disclosure provides a number of spectral reading units for low cost, in- field readers on the basis of photodetectors and refers to the photodetectors as a ^"‘single-element- detector” in general. In some exemplary embodiments, these detectors include Si-based detectors such as photodiodes or silicon avalanche photodiodes. In some exemplary embodiments, single element detectors include photo multiplier tubes. Silicon-based photo-detectors allow' low cost read-outs with a spectral sensitivity typically covering a wide spectral range from about 300 - 1200 nm. In the operation of a single-element-detector, each photo-detector element, (e.g., pixel, photodiode or the like) is further equipped with an Analog -to-Digital converter (ADC) transferring the photo-generated voltage change of the photo-diode into a digital signature read by a computation unit for further processing.

[00116] GRATING BASED - SINGLE ELEMENT NARROW SPECTRAL BAND FILTER UNIT

[00117] Referring to Figure 18, in some exemplary embodiments, the array detector of the spectrograph is replaced by a single-element-detector in the x’-y plane. In this case, the grating acts as a filter unit capturing a bandwidth determined by the spatial extent of the individual detector element or by an upstream aperture. Per sample point, light from the sample surface is collected by means of optical fibers, lenses or mirrors and is transmitted/reflected of a grating structure having a ruled structure of about 200 lines to about 1200 lines per millimeter. A single detector element is placed a distance from the grating structure at an angle to receive electromagnetic radiation of a spectral bandwidth determined by the ruling number, the tilt angle, the spatial extent of the detector element and the distance from the grating structure. In some exemplary embodiments, focusing lenses may be omitted,

[00118] LED FILTERED - SINGLE ELEMENT DETECTOR

[00119] Alternatively, the spectral emission profile of the LED illuminating the sample surface may be used to tune the read-out to a specific marker molecule. Therefore, a LED in the illumination unit may be broad-band (white) or narrow-band, single colored.

[00120] For instance, referring to Figure 19, in some exemplary embodiments, each sample surface is illuminated with a single LED and the diffuse reflected beam is captured by a single element detector. A LED and a detector are held in a fixture or frame providing positional means for a 45/0° arrangement of the LED source and the detector. Light emitted from the light source under the angle of about 45° may be guided by the fixture and directed onto the sample surface, whereby the detector records the scattered beam at about 0° from the surface normal . Specular reflected light is guided away from the illumination unit by a 45° bore hole in the fixture and may be trapped in a light trap at its end.

[00121] In some exemplary embodiments, in the ease of HRP/TMB marker, the LED may be selected to emit light in the absorbance bands of the HRP/TMB, specifically in the violet (e.g., centered on about 390 nm wavelength) or dark red (e.g., about 660 nm wavelength) part of the electromagnetic spectrum (refer to Figure 13). Both wavelength bands are highly sensitive to the HRP/TMB reporter molecule in the chemistry_' described herein. Alternatively, a white LED illumination covering the spectral range from about 400-750 nm may be used. In either case, the reflected/scattered light is captured by a single detector element per sample surface. In terms of detector noise reduction, it may be advantageous to spectrally filter the single-element-detector as illustrated in Figure 20. In the ease of HRP/TMB markers, long- or band- pass spectral filters allowing the spectral bands of either from about 380-400 nm, or from about 600-700 nm to pass, allowing maximum sensitivity to the chemical reaction. Furthermore, it may be advantageous to include further single element detectors measuring the emission beam of each light source. These additional detector elements will allow to compensate for spectral or power drfts of the light source during operation.

[00122] LED BASED - FILTERED SINGLE ELEMENT DETECTOR

[00123] Similarly, for the use of reporter reagents such as the Alexa Fluor 594, a single LED illumination with a filtered single-element-detector provides a low-cost read-out system as illustrated in Figure 20. In the case of Alexa Fluor 594, the reader may be equipped with LEDs having a center emission wavelength of about 570 or about 590 nm. Alternatively, a laser diode with an emission wavelength of about 532 nm may be used as an excitation light source. Upon excitation of the fluorescent label in the sample surface, the emitted fluorescent beam may be captured parallel to the sample surface normal by an arrangement of filter and single-element- detector. A LP filter with a cut-on wavelength of about 600 nm or similar upstream the single - element-detector filter, however, is necessary_' to filter elastically scattered radiation. Such filter arrangements may include, dichroic beamspliter arrangements commonly used in fluorescent microscopes.

MEASUREMENT AND ANALYSIS

[00124] In various exemplary embodiments, after placing the cassette into the reading units, the reader produces spectral intensities “S”, at an interval of Δt, over a measurement time span t^", In the case of a two-test spot cassette featuring test and control spots, the spectral reading system produces two spectral intensity channels S1 and S2. Each spectral intensity channel spans N spectral values. N is an integer equal to or greater than 1. For instance, in some exemplary embodiments, in the case of spectrograph as presented earlier, N is a vector of multiple spectral points (e.g., 640 spectral points, N=640) whereas N=1 in the case of a single- element-detector.

[00125] Referring to Figure 21, there is depicted an exemplary tim ing diagram of the read- out. In some exemplaiy embodiments, the illumination of the sample (e.g. the cassette) is timed at an interval At. The CMOS electronics for data capture is set to the same interval and captures the spectral data after the illumination is switched on. Once the data is captured, the illumination is switched off until the next trigger signal. After data is recorded both spectral channels (S1 and S2 of length N) are sent to an external processor for computation.

[00126] In an alternative timing diagram, particularly important for thermally unstable detectors, the read-out frequency of S1 and S2 may be doubled to record two reading per measurement event: for instance, one bright (S1_b, S2_b.) with illumination on and one dark (S1_d, S2_d) field reading with illumination off. Thermal drifts are compensated by subtracting S1_b-S1_d and S2_b-S2_d.

[00127] In some exemplary embodiments of a measurement, e.g. for HRP/TMB marker molecules, the spectral channels are recorded in a time series with an interval over a time period, for instance, an interval of 1 second spanning a total time of from about 30 seconds to about 5 minutes. The interval of the time series can be identical, varied, or incremented in a predetermined way, e.g. to measure a decrease or increase of a signal. In some exemplary embodiments of the measurement of, e.g. a fluorescent marker molecule, the spectral channels may be recorded at a single instant, t.

[00128] In some exemplary embodiments, the signal S per channel is converted to a reflectance R by dividing it with the signal of a reference measurement taken by the same device either in parallel or In sequence.

[00129] The reference signal may be taken with a white surface of a known reflectance spectrum. Alternatively, the reference signal may be set as the first time instant, when the cassette is placed into the read-out unit or as a blank reading before the chemistry has been added to the cassette.

[00130] In some exemplary embodiments, the reflectance R is further converted to absorption coefficient (k/s), for instance, by Kubelka-Munk’s formula^6,7:

Note, ‘k’ and ‘s' denote absorption and scattering coefficient, respectively,

[00131] Time series data may be visualized using any metric defined on the signal S in real time. For example, in some exemplary embodiments, a metric for a color change is defined by an average reflectance over the spectral sample points N.

[00132] For instance, in the case of HRP/TMB marker reagent, the color change is then captured as a decay curve with respect to time as illustrated in Figure 22. [00133] In some exemplary embodiments, the time-series data is visualized using the absorbance coefficients estimated from the reflectance data by Kubeika-Munk⁶·⁷ as illustrated in Figure 23. The ploted metric may be computed as an area under the absorbance curve or at a particular wavelength ,

[00134] In an exemplary embodiment, the color development rate of HRP/TMP marker molecules is useful to monitor reactions in time-series data. For instance, by way of example, Figure 24 illustrates the monitoring of color change rate when the chemistry is exposed to different concentrations of N -protein of the SARS-CoV-2 antigen. As seen in the figure, samples with higher N-protein concentrations show a higher rate of color change and likewise absorbance rates. Thus, the slope of the absorbance curve (as an example) can be used to estimate the rate of color change and therefore the concentration of the sample under investigation. In some exemplary embodiments, prior to estimating die rate of color change, the reaction rate versus target molecule concentration is determined in a device calibration step,

[00135] In some exemplary_'· embodiments, reaction changes such as the color development rate of HRP/TMP marker molecules are statistically analyzed using known methods such as Principal Component Analysis (PCA) or Partial Least Square Regression (PLS) to name a few. In this ease, the Principal Component loading functions may be used to estimate Principal Component scores as a metric for tracing the reaction rate over time.

METHODS OF CALIBRATION

[00136] In order to estimate the concentration of unknown samples, in some exemplary embodiments, time trace data is measured and stored in a calibration routine. In some exemplary embodiments, the calibration routine includes one or more of the following exemplary steps:

1) Record time-series data of reaction change versus e.g. protein concentration;

2) Process signal S to compute metric value;

3) Estimate a first curve fit for each time trace of a metric using fitting functions such as polynomials or exponentials;

4) Map curve fitting coefficien ts of first curve fit versus protein concentrations;

5) Estimate second curve fit for each of the coefficients determined in step 4 using fitting functions such as polynomials or exponentials; and

6) Formulate predictor function for unknown concentrations based on second curve fit. RESULTS

[00137] Time-trace data disclosed herein can be used to estimate an unknown concentration value for a marker molecule such as HRP/TMB. In some exemplary_' embodiments, the method includes one or more of the following exemplary_'· steps:

1. Dispense a sample and relevant chemistry;

2. Optically monitor, using a reader, the reaction, thereby providing signal S over time t;

3. Process, using a software, the signal S into a metric;

4. Fit th e time trace of the metric to yield fitting coefficients, which may change with every_' new point of the time-senes; and

5. Determine, using a predictor function, a concentration based on the fitting coefficients for ever}_' new point of the time-series.

[00138] In some exemplary_'· embodiments, as the reaction proceeds, more data points are collected to improve the accuracy of the predicted concentration.

EXEMPLARY SYSTEM

[00139] Disclosed hereinafter are exemplary system and processes for detection of proteins and biomolecules in accordance with some embodiments of the present disclosure. The system is generally referred to as the ‘Thrixen CoVIm”.

[00140] The Thrixen CoVlm neutralizing Antibody (nAbs) Test is an integrated system of instrument, vertical flow cassettes and reagents for the qualitative and quantitative detection of nAbs of biological samples, e.g., from finger prick and venous blood samples taken from human subjects. The system can be used for the testing (e.g., point-of-care testing) and verification of protection of an individual from vims infections. For instance, as a non-limiting example, the system can be used for the testing and verification of protection of an individual from SARS- CoV-2 infections. It serves a dual purpose of mass-screening for virus, and vaccination profiling within a population.

[00141] Results from the nAbs test report the identification and level of virus (e.g., SARS- CoV-2) nAbs. nAbs prevent the virus from infecting host ceils and thus from replicating. nAbs bind to the receptor binding domain (RDB) located on the spike (S) protein of the virus, and block the vims from binding to the host cell angiotensin converting enzyme 2 receptor (ACE2), an initial process required for viral infection. Thus, hosts with sufficient nAbs status (e.g. through vaccination) confer protective immunity against vims infections which prevent the vims from replicating, as such posing minimal infectious risk to the population.

[00142] In various exemplary_' embodiments, CoVIm test determines nAb status by detecting fluorescent intensity signals generated from RBD and fluorescent conjugated ACE2 (ACE2-F1) complex. In the absence of nAbs, RBD and ACE-F1 form maximal numbers of complex, generating high fluorescent signal intensity. In the presence of nAbs, the antibodies bind to RBD, disrupting the RBD/ACE2-F1 complex formation, and inhibiting fluorescent signal intensity. The test is sensitive all nAbs isotypes (e.g. IgG, IgM, IgA, IgD, IgD).

[00143] CoVIm test results are displayed as % inhibition whereby a cut-off of a certain percentage, such as 30% inhibition, distinguishes detectable from non-detectable nAbs status.

[00144] The Thrixen CoVIm test can be used by trained personnel specifically instructed and trained in in-vitro diagnostic procedures and proper infection control procedures, for instance, at point-of-care or other institutions such as hospitals or laboratories.

[00145] THE INSTRUMENT

[00146] Referring to Figures 25A-25C and 26A-26B, the Thrixen CoVIm neutralizing Antibody instrument is designed to read and quantify the presence of a fluorescent marker molecule in whole, human blood samples utilizing a proprietary chemistry'. In some exemplary embodiments, the instrument is referred to as Thrixen COVIM fluorescent reader platform. The instrument includes a hardware device, its electronics and onboard software necessary for full automation of biomarker detection from test cassettes. In some exemplary_' embodiments, the instrument (Thrixen COVIM fluorescent reader platform) includes a cassette loading bay, a fluorescent and an absorbance reader unit, a computation unit and an LCD display.

[00147] Table 2 below lists some specifications for the instrument in accordance with an exemplary embodiment. It should be noted that this is by way of example and is non-limiting.

One or more or all of the specifications can be readily varied without departing from the spirit and scope of the exemplary embodiments. For instance, the instrument can have other dimensions (e.g., smaller or larger size) or different shapes. Parts (e.g., shell or baseplate) of the instrument can be made of different materials. Other parameters (e.g., temperature or humidity) can be configured differently as well. Table 2: Instrument Specifications

[00148] In an exemplary embodiment, the instrument is about 185 mm by 185 mm footprint and is 200 mm tall. In another exemplary embodiment, the instrument has a size smaller or larger than 185 mm by 185 mm by 200 mm. In some exemplary embodiments, the instrument has a different shape. The instrument is powered by a conventional external AD/DC power adapter. In some exemplary embodiments, the instrument includes one or more or all of the following:

* Display/ Touchscreen Unit (1);

* QR Code Camera Unit (2);

* Power Supply Electronics (3);

* Computation Unit (4);

* Cassette Loading Bay (5);

* Housing Base (6);

* Position Stage (7):

* Spectral Reader Unit (8);

* Fluorescent Reader Unit (9); and/or

* Upper Housing Shell (10). [00149] Figure 27 is a flowchart schematically illustrating an exemplary process, including the instrument operation, in accordance with exemplary embodiments of the present disclosure. It should be noted that the processes disclosed herein and exemplified in the flowchart can be, but do not have to be, executed in full or in the order as they are presented.

[00150] To operate the instrument, a user connects the external power supply to the inlet on the back of the device. Subsequently, the user switches the device on, which is at the backside of the device in an embodiment, and waits for the software boot up. The instrument can he operated in two main program modes: Calibration or Test Mode. In some embodiments, at every start of the instrument, the software requests the user automatically to run through a calibration procedure.

[00151] In either operation the user places a test cassette into the cassette loading bay and starts the measurement via the touchscreen. Subsequently, the QR code camera captures the QR code of the cassete, and a position stage moves the cassette inside the housing into reading position 1 aligning the Control spot underneath the Fluorescent Reader Unit. In position 1, the Fluorescent Reader Unit flashes an illumination (e.g., a 0.5 second LED illumination) on the sample spot and records the fluorescent backscatered light, for instance, with a Silicon- Avalanche-Photodiode (SiAPD) or any suitable detectors.

[00152] In some exemplary embodiments, the LED flashes for a duration at a frequency and a duty cycle, such as a total duration of about 0.5 seconds, at a 300 Hz frequency and a 50/50 duty cycle, while the APD registers the signal over the same total duration. From the registered signal of the APD (intensity over sampling duration), an average peak fluorescent signal intensity is computed and stored with reference to sample position 1. The LED modulation allows to remove background drifts of the APD electronic circuitry.

[00153] Once completed, a stage moves the cassette to position 2. where the Test spot is measured in the same way. Subsequently, a stage moves the cassette to a third position which records the reflectance of both spots in parallel. In this position, an Absorbance or Spectral Reader Unit flashes white LEDs and records the backscattered white light information completing all spectral measurements. Finally, the software computes the nAbs concentration via proprietary algorithm. The touchscreen displays the result while the cassette is pushed back out to the cassette loading bay.

[00154] READER DESCRIPTIONS AND COMPUTATIONS [00155] The system is able to determine the neutralizing Antibody (n Abs) concentration from a number of sample types. For instance, in some exemplary embodiments, the system is able to determine nAbs concentration from two sample types, such as whole blood samples and plasma samples. Both sample types can be dispensed onto the cassette. Once the sample is dispensed and the cassette is placed into the reader, the system analyses the sample type using a spectroscopic sensor.

[00156] Spectral Reader Unit

[00157] Figures 28A and 28B illustrate schematically a spectral sensor with two-channel spectrometer engine measuring control and test spots simultaneously, wherein D, BB, L2, L1, G denote detector, beam blocker, lens 1 and lens 2 and grating respectively. In some exemplary- embodiments, the sensor utilizes two fiber inlets coming from the two sample points of the cassette, namely the control and test spots. When the cassette is placed underneath the spectral sensor unit, the system flashes two white LEDs (e.g, emission spectrum is covering 380 to 730 nm w avelength) and illuminates the cassette control and test spots under a certain angle (e.g., 45 degrees) at the same time while two fibers positioned above control and test spots collect the back-scattered light parallel to the sample surface normal of both sample points (test and control) at the same time. The fibers transport the back-scattered beam into the spectrometer unit which images the two fiber inlets via a diffraction grating onto an array detector. While the system is set up as a two-channel spectrograph, the concept can be applied to more than two channels.

[00158] Spectral Reflectance R. The spectrophotometer determines the spectral reflectance R of each of its channels according to:

whereby I_s and I_s,Ref denote the spectral intensity (Is) received from a test cassette (position 4) in comparison to the reference surface (Ref) recorded on position 5,

[00159] As each spectrometer as well as each spectrometer^'s channel has its own, specific wavelength vector, the reflectance reads must be interpolated to a common wavelength vector in order to compare spectra across spectrometers and channels.

[00160] Sample type (e.g.. Blood versus Plasma). In order to determine the sample type, Atto S computes the reflectance spectrum of a test cassette R and estimates peak ratio (PR) between the closest wavelength channels. For instance, to determine blood and plasma types,

Ato S computes the reflectance spectrum of atest cassette R, as illustrated in Figure 29, and estimates peak ratio (PR) between the two closest wavelength channels, such as at λ₁ — 575 nm and λ₂ — 700 nm, according to:

whereby and

denotes the reflectance at the nearest wavelength point to wavelength 1 and wavelength 2, respectively.

[00161] The nearest neighbor wavelength values are used to avoid data interpolating to save computation time. It should be noted that this is a non-limiting example, and other neighbor wavelength values can be used in a similar fashion.

[00162] Then, sample types are determined in accordance with whether they are above or below a threshold. For instance, blood or plasma samples are determined by the condition: PR ≥ 1.4 = blood and PR < 1 .4 = plasma,

[00163] Fluorescence Reader Unit

[00164] Once the sample type is determined, the fluorescent emission of the test spot is measured by the fluorescence reader unit. The nAbs concentration is determined, for instance, using Eq. 8 and Eq. 9, as explained in more detailed below.

[00165] In some exemplary embodiments, the unit includes an amber LED configured to emit a light, such as a light of 590 nm, into the 1^st beam path as illustrated in Figure 30. The LED emission is triggered by the system once the stage positioned the sample underneath the fluorescent unit. The LED is emitting a pulse train (e.g., 300 Hz, 50/50 duty cycle). The emitted light pulse train is collimated by an aspheric lens (L1) and filtered by a short-pass excitation filter (F1) downstream the lens. Spectrally, the short pass filter allows shorter wavelength, such as wavelength smaller than 600 nm, to pass, whereas longer wavelengths, such as wavelength greater than 600 nm, are rejected. Additional optical elements such as linear polarizers may be used to condition the LED emission further. Further downstream tire filter and/or polarizer, the excitation beam is directed by a dichroic beam splitter (BS) towards the sample surface. On the path towards the sample, a second aspheric lens (L2) focuses the beam onto the sample surface below and completes the 1^st beam path.

[00166] For the 2^nd beam path, the same aspheric lens (L2) collects and collimates back- scattered light from the surface and directs the emitted light path towards the dichroic beam splitter (BS). Upon transmission through the dichroic beam spliter, the emission beam is further filtered by a long-pass emission filter (F3) before it is focused by another aspheric lens (L3) onto the APD detector (D1) on top completing the 2^nd beam path. Spectrally, the long-pass filter allows longer wavelengths, such as wavelength greater than 600 nm, to pass whereas shorter wavelengths are rejected. An example modulated pulse train received by the APD is shown in Figure 31.

[00167] The dichroic beam splitter is advantageous in the setup as it allows a high reflection of the shorter wavelength (<600 nm) excitation beam while is enables a high transmission of the emited beam flux (>600 nm) coming from the sample surface. The combination of the dichroic beam splitter (BS) with the emission filter (L3) enables high optical transmission for the emission path while enabling a high optical re_jection for the excitation beam on the way towards the APD detector (D1).

[00168] Furthermore, a 3^rd beam path transmitting from the LED through the dichroic beam splitter (BS) is focused by a fourth aspheric lens (1,4) onto a conventional photo diode (D2). The 3rd beam path is used to determine the output power of LED emission as illustrated in Figure 32. On the path toward the photo diode (PD) an absorbance filter and/or linear polarizer (F4) may be used to reduce the beam intensity emitted from the LED and prevent the detector (D2) from saturation. The polarizer is advantages to reduce/adjust direct light coming from the LED and to avoid sensitivity of back-scattered light coming from the sample surface. The absorbance filter is advantages to reduce the overall light intensity.

[00169] It should he noted that the purpose of the PD is to have a direct intensity read of the excitation beam without the influence of the sample reflectance. Thus, an arrangement of polarizer, absorbance (F4) and/or apertures at the focal point of the aspheric lens (L4) may be advantageous. Furthermore, the optical system allows for an additional but optional filter (F2).

[00170] From the pulse train data received by either the avalanche photo diode (D1) or the photo diode (D2) an average signal peak signal strength is computed by removing the dark photo current or background intensity signal when the LED is modulated and in its off position and estimating the average peak signal strength of the light emissions. Mathematical means such as FFT may be applied to filter and clean the signal and derive an average signal strength.

[00171] The signal output power is, how ever, dependent on LED output power and the device temperature. Thus, each Atto S unit applies a thermal correction factor. Furthermore, as the signal out of each fluorescent unit varies an additional inter-device correction factor is applied as described below. [00172] In some exemplary embodiments, one or two device correction algorithms are applied to data recorded from the fluorescent reader unit: Temperature drift compensation (Tc) and/or Inter-device correction (Ic).

[00173] Temperature Drift Compensation Tc. Temperature drifts of the fluorescent reader unit including temperature drifts of LED excitation and fluorescent emissions read with a APD detector were experimentally verified, for instance, were experimentally verified to change by up to 30% from 15 C to 38 C device temperature.

[00174] To compensate for reader temperature drifts, a calibration cassette is placed into the reader unit during a device calibration procedure. In the procedure, the device is switched on at room temperature and the calibration cassette fluorescent signal is recorded over a period of time (e.g., 1 hour) in an interval (e.g., an interval of 1 minute). Typical device temperature response curves for a calibration cassette are plotted in Figure 33 and Figure 34, As shown, the APD or PD output of a temperature uncompensated reader may vary_' up to 20% over a temperature increase from 20 C to 34 C.

[00175] In some exemplary embodiments, as illustrated in Figure 34, both reads are then normalized to its respective intensity read at operating temperature, e.g., 33 C, and divided by each other to yield a temperature variation of the APD (D_T) after power drift compensation of the LED measured by the PD:

[00176] Subsequently, (D_T) is fitted with a polynomial least-square estimate. For instance, in some embodiments, as illustrated in Figure 35, (D_T) is subsequently fitted with an 4^th degree polynomial least-square estimate TC<T;. according to:

[00177] Then, the fluorescent reader temperature drift is corrected, for instance, by applying:

whereby p1 to p5 denote the polynomial fitting parameters, PD _(Tref) the PD output at device reference temperature (Tref), PD the photodiode output, TC the temperature correction terms, and T the temperature reported by the Atto S reader unit, respectively. [00178] After compensation the linear response of the system remains constant within approximately 2% from a mean value as illustrated in Figure 37. The remaining 2% may he attributed to positional differences or fluorescent decay of the calibration cassette alone. After correction the APD signal varies by max 5%.

[00179] In some exemplary embodiments, APD_(TC) values are fed into the inter-device correction model.

[00180] Inter-device Correction . After thermal drift compensation each reader unit is calibrated to the same intensity output for a set of calibration samples referenced to a calibration device.

[00181] For instance, in some embodiments, a plurality of white reference cassettes (e.g.,

5) and a plurality of paper cassettes (e.g., 8) printed with an increasing density of black ink to vary the auto-fluorescent emission of the paper are first measured by a reference device and then measured by the calibration device.

[00182] Subsequently, the response APD_(TC) of both devices are plotted into a 2D scatter plot and fitted with a polynomial, e.g., a 1^st degree polynomial according to: (Eq . 6)

whereby p1 and p2 are polynomials and APD_(TC)Ref refers to the temperature compensated output of the reference calibration unit.

[00183] Then, the inter-device corrected APD_CCorr output is computed by:

[00184] Read Positions on Cassette Loading Tray

[00185] Figure 38 depicts the loading tray holding a test cassette and indicate the reader position for the fluorescent reader unit in accordance with exemplary embodiments of the present disclosure. In Figure 38, (a) indicates a cassette loading tray, (b) indicates a white reference surface (b) and (e) indicates a test cassette held by the cassette loading tray. Red circles indicate fluorescent reader measurement points I (reference), 2 (control spot), and 3 (test. spot).

[00186] Figure 39 depicts the loading tray holding a test cassette and indicate the reader position for the spectral reader unit in accordance with exemplary embodiments of the present disclosure. In Figure 39, (a) indicates a cassette loading tray, (b) indicates a white reference surface (b) and (c) indicates a test cassette held by the cassette loading tray. Red circles indicate 2 -parallel -read spectrophotometer channels and its measurement points on the loading tray: 4 (cassette read), 5 (reference read).

[00187] Data Analysis Modes

[00188] Calibration - Acquiring of Reference Data. In the calibration mode, a calibration cassette is placed in the reader and the calibration routine is initiated. Excitation intensity is recorded by the PD detector (PD_(T)) on a first tray position whereas the fluorescence intensity of the test spot ( APD_cal ) spot may be recorded by the APD on a different tray position and stored as reference values for test mode computations. In some exemplary embodiments, both values are stored in a device internal storage and used for device correction and computation purposes.

[00189] Test - Computation of %Inhibition for Blood and Plasma Samples. In the test mode, the system is setup to analyze samples, e.g., blood as well as plasma samples. For each sample type (determined by the spectral reader unit), the system reports % Inhibition indicating the effectiveness of nAb inhibiting ACE2 and RBD complex formation.

[00190] In some exemplary embodiments, a test with a %Inhibition of less than a percentage, e.g., <30%, is considered tested negative for nAbs.

[00191] In some exemplary embodiments, for blood test, % Inhibition is computed by:

%Inbibition = (Eq, 8)

whereby:

APD_CCorrTest is the fluorescent intensity recorded on the test spot of a cassette by the APD detector

AP D_CCorr,Cal is the latest fluorescent intensity recorded in the calibration routine are experimentally

determined the ratios of minimum and maximum fluorescent intensity of blood samples (B) with and without nAbs versus fluorescent intensity_' recorded from a calibration cassette, respectively.

[00192] In some exemplary embodiments, for plasma test, % Inhibition is computed by:

whereby: are experimentally

determined the ratios of minimum and maximum fluorescent intensity of plasma samples (P) with and without nAbs versus fluorescent intensity recorded from a calibration cassette, respectively.

[00193] Results. In some exemplary embodiments, the system reports the % inhibition value to the user according to the table below:

[00194] The terminology used herein is for the purpose of describing particular implementations only and is not intended to be limiting of the claims. As used in the description of the implementations and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[00195] As used herein, the term “and/or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. It will be further understood that the terms “composes” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[00196] As used herein, the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. “About” can mean a range of ± 20%, ± 10%, ± 5%, or ± 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” means within an acceptable error range for the particular value. The term “about” can have the meaning as commonly understood by one of ordinary skill in the art. The term “about” can refer to ± 10%. The term “about” can refer to ± 5%.

REFERENCES

[00197] The present disclosure cites the following references, the disclosure of each article is incorporated herein for all purposes by reference in its entirety.

1. Sikes Johnson, H., Miller, E. A. & Sung, K.-J. PROTEIN FOR RAPID, EFFICIENT CAPTURE OF ANTIGENS. PCT/US2018/055582 (2019).

2. Sikes Johnson, H., Miller, E. A. & Sung, K.-J. PROTEIN FOR RAPID, EFFICIENT CAPTURE OF ANTIGENS, US Patent Application No. 16/158,506 (2019),

3. Kongsuphol, P, Huan, J. & Sikes Johnson, H. A METHOD OF DETECTING AN ANALYTE AND RELATED SYSTEMS. IPOS Patent Application No.

10202009197S (2020).

4. Sikes Johnson, H. & Miller, E. A. No TiteENGINEERED PROTEINS THAT BIND THE SARS-CQV-2 NUCLEOCAPSID PROTEIN. US Provisional Application No, 63/111,769 (2020),

5. Sikes Johnson, H. ENGINEERED PROTEIN FOR RAPID, EFFICIENT CAPTURE OF PATHOGEN -SPECIFIC ANTIBODIES. US Provisional Application No. 63/111,781 (2020).

6. Kubelka, P. and Munk, F. (1931), ‘Έin beitrag zur optik der farbanstriche” Z. Tech. Phys., 12:593-601.

7. Michael E. Myriek, Michael N. Simcock, Megan Baranowski, Heather Brooke,

Stephen L. Morgan and Jessica N. McCutcheon (2011), ‘^‘The Kubelka- Munk Diffuse

Reflectance Formula Revisited”, Applied Spectroscopy Review s, 46:2, 140-165, DOI: 10.1080/05704928.2010.537004.

Claims

WHAT IS CLAIMED IS:

1. A method for rapid detection of one or more molecules in a biological or chemical sample, the method comprising: collecting a sample; transforming, biochemically, the collected sample; detecting optical signals emitted from the transformed sample; and determining, based on the detected signals, one or more parameters associated with the one or more molecules in the sample.

2. The method according to claim 1, wherein the determining of one or more parameters comprises determining a concentration of a molecule in the one or more molecules.

3. The method according to any one of preceding claims, wherein the collecting of the sample comprises taking saliva from humans or animals.

4. The method according to any one of preceding claims, wherein the collecting of the sample comprises taking blood from humans or animals.

5. The method according to any one of preceding claims, wherein the method is performed using a system for manual, semi-automated, or fully-automated sample preparation.

6. The method according to claim 5, where the system for sample preparation is an essentially monolithic device.

7. The method according to claim 6, wherein the system for sample preparation is a lab-on- a-chip.

8. The method according to any one of preceding claims, wherein the transforming of the collected sample comprises: marking the one or more biomolecules with one or more reporter agents.

9. The method according to claim 8, wherein the marking of the one or more reporter agents are performed using one or more vertical flow devices.

10. The method according to any one of claims 8-9, wherein the marking of the one or more reporter agents comprises: a. collecting and filtrating saliva; b. treating the saliva with lysing agents for lysis of pathogen membrane; c. mixing the treated saliva with one or more reagents selected from the group consisting of CBD-tagged capture reagent, reporter reagent, CBD-tagged capture reagent and reporter reagent that bind to different epitopes of the target biomarkers from desired pathogens or host organisms and CBD-tagged capture reagent and reporter reagent whereby the CBD-tagged capture reagent offers the same binding epitope to the desired biomarkers in the processed saliva sample and reporting reagent; and d. applying the mixture sample onto a vertical flow device.

11. The method according to any one of preceding claims, wherein the sample comprises whole blood/serum/plasma.

12. The method according to any one of preceding claims, where the method is performed by a system comprising a vertical flow device, wherein the vertical flow device comprises a cassette having a top piece and a bottom piece, wherein one or more openings are formed at the top piece and one or more chamber structures are formed at the bottom piece to host one or more paper strips and one or more absorbent pads.

13. The method according to claim 12, wherein the one or more openings and chambers in the cassette are aligned with each other, wherein the cassette further comprises one or more protrusion structures, each formed at an inner surface of the cassette and around at least a portion of a perimeter of a corresponding opening in the one or more openings.

14. The method according to any one of claims 12-13, wherein a test strip in the one or more strips comprises one or more layers of cellulose material with a single or multiple layered absorbent pad assembled underneath die test strip.

15. The method according to any one of claims 12-14, wherein the test strip comprises a multiply folded or corrugated top layer of cellulose material.

16. The method according to any one of preceding claims, wherein the detecting of optical signals is performed by a system comprising: at least one light source for illuminating at least two sample points; a guiding means for directing light from the at least two sample points; and a detector for detecting light from the guiding means.

17. The method according to claim 16, where the light is incident on the sample and the detected light is scattered or emitted from the transformed sample.

18. The method according to claim 16, where the light is incident on the sample and the detected light is fluorescent light excited by the incident light.

19. The method according to any one of claims 16-18, wherein a spectrum of the light is measured over one or more ranges selected from the group consisting of a VIS/NIR range, a portion of the VIS/NIR range, a broadband range contained in the VIS/NIR range, a narrowband range contained in a VIS/NIR range, a spectral line contained in the VIS/NIR range, and multiple spectral features contained in the whole VIS/NIR range

20. The method according to tiny one of preceding claims, wherein the biological or chemical sample comprises a pathogen.

21. The method according to any one of preceding claims, wherein the determining of the one or more parameters comprises; determining an instantaneous concentration of a reporter agent,

22. The method according to claim 21, wherein one or more lower bounds to a concentration of a pathogen are derived from the instantaneous concentration of the reporter agent.

23. The method according to any one of preceding claims, wherein the detecting of optical signals is performed in real-time from an onset of chemical reaction through an incubation phase towards a saturation.

24. The method according to any one of preceding claims, wherein the determining of one or more parameters is performed in real-time from an onset of chemical reaction through an incubation phase towards a saturation,

25. The method according to any one of claims 21-24, wherein the determining of one or more parameters is based on a time-derivative of the detected signals from the onse t of the chemical reaction through the incubation phase towards the saturation, thereby shortening a time for obtaining a result.

26. A method for rapid detection of a biomolecule in a biological or chemical sample disclosed herein.

27. The method of claim 26, wherein the sample is saliva from humans or animals.

28. The method of any one of claims 26-27, wherein the biomolecule is a pathogen.

29. The method of claim 26-28, comprising: processing a sample; dispensing the processed sample onto a vertical flow de vice; detecting, using a spectral reader, optical signals emitted front the processed sample; and determining one or more parameters associated with the biomolecule based on the detected optical signals.

30. The method of claim 29, further comprising: collecting the sample.

31 . The method of claim 30, wherein the collecting of the sample is performed using a sample collection unit disclosed herein.

32. The method according to any one of claims 29-31 , further comprising: performing a calibration disclosed herein.

33. The method according to any one of claims 29-32, wherein the processing of the sample comprises: performing wet chemistry disclosed herein.

34. The method according to any one of claims 29-33, wherein the detecting of optical signals is performed over a time period at a time integral, wherein the time interval is a constant or varies over time.

35. The method according to any one of claims 29-34, wherein the determining of one or more parameters comprises: con verting the detected signals into a metric; obtaining fitting coefficients by fiting time trace of the metric; and determining, based on the fitting coefficients, a concentration associated with the biomolecule in the processed sample.

36. A method for high throughput screening of a biomolecule in a biological or chemical sample disclosed herein.

37. A method for preparing a test strip disclosed here.

38. A method for a vertical flow assay, the method comprising a sample processing step and a chemistry reaction step disclosed herein.

39. A system for performing the method of any one of preceding claims.

40. A test strip prepared by any one of paper preparation methods disclosed herein

41 . A cassette disclosed herein.

42. The cassette of claim 41, comprising: a first piece comprising one or more openings; and a second piece coupled to or monolithically formed with each other, the second piece comprising one or more chamber structures to host one or more paper strips and one or more absorbent pads, wherein the one or more chamber structures are aligned substantially with the one or more openings.

43. The cassette according to claim 42, wherein the cassette further comprises one or more protrusion structures, each formed at an inner surface of the cassette and around at least a portion of a perimeter of a corresponding opening in the one or more openings.

44. A vertical flow device disclosed herein.

45. The vertical flow device of claim 44, comprising: one or more test strips, one or more absorbent pads, and a cassete for housing the one or more strips and absorbent pads.

46. A spectral reader disclosed herein.

47. The spectral reader of claim 46, comprises: at least one light source for illuminating at least two sample points; a guiding means for directing light from the at least two sample points; and a detector for detecting light from the guiding means.

48. A system for rapid detection of a biomolecule in a biological or chemical sample disclosed herein.

49. The system of claim 48, comprising: a collection and preparation unit for collecting and preparing a sample; a vertical flow device for capturing and labelling of a desired biomarker in the sample; a chemical dispensing unit; a processing unit for performing a transformation of the biomarker; one or more light sources for illuminating the sample; a light guiding means for directing light emitted from the sample; and o one or more detectors for detecting the light emitted from the sample.

50. The system of claim 49, wherein the transformation of the biomarker is of spectrum, color or fluorescence.

51. A system of claim 48, comprising: a fluorescent reader unit disclosed herein; an absorbance or spectral reader unit disclosed herein, and a software configured to compute a nAbs concentration based on measurements obtained by the fluorescent reader unit and absorbance or spectral reader unit.

52. A method for rapid detection of a biomolecule in a biological or chemical sample, using a system disclosed herein.

53. The method of claim 52, comprising: measuring, using a fluorescent reader unit of the system of the system, a fluorescent backscattered light from a sample spot and a fluorescent backscattered light from a test spot; measuring, using an absorbance or spectral reader unit of the system , a backscattered white light from the sample spot and a backscattered white light from the test spot, determining a nAbs concentration based on measurements obtained by the fluorescent reader unit and absorbance or spectral reader unit.

54. The method of claim 53, wherein the fluorescent backscattered light from the sample spot and the fluorescent backscattered light from the test spot are measured sequentially.

55. The method of claim 53 or claim 54 wherein the backscattered white light from the sample spot and the backscattered white light from the test spot are measured concurrently.

56. The method of any one of claims 53-55, further comprising one or more of the following: placing a test cassette into a cassete loading bay of a system; capturing a QR code or a barcode of the test cassette; positioning the test cassette to a first position for measuring the fluorescent backscattered light from the sample spot; positioning the test cassette to a second position for measuring the fluorescent backscattered light from the test spot: positioning the test cassette to a third position for measuring the backscattered white light from the sample spot and the backscattered white light from the test spot.