Devices and methods for performing lateral flow tests

Abstract

The present disclosure relates to a lateral flow testing device for performing a lateral flow test on a liquid sample, the device comprising: a test strip comprising a nitrocellulose membrane having a first capture reagent disposed on a first surface along a test line, the first capture reagent configured to capture a first analyte of the liquid sample; a sample pad disposed at a first end of the nitrocellulose membrane configured to receive a liquid sample; a test strip comprising a plurality of labeling molecules disposed on the first surface at a position between the sample pad and the test line, the labeling reagent configured to bind to the first analyte; and an electrode array disposed over the nitrocellulose membrane and configured to apply a potential across the first surface.

Description

[Technical field]

[0001] The present disclosure relates to lateral flow tests. [Background technology]

[0002] Lateral flow tests (LFTs), also known as lateral flow assays (LFAs) and lateral flow devices (LFDs), have become widespread in the field of medical diagnostics and are widely used in other contexts such as environmental monitoring. These were introduced in the 1980s and remain one of the most cost-effective platforms available for implementing disposable immunosensors (see, e.g., Campbell, RL, Wagner, DB and O'Connel, JP (1987) Solid-phase assay with visual readout, U.S. Pat. No. 4,703,017; Rosenstein, RW and Bloomster, TG (1989) Solid-phase assay employing capillary flow, U.S. Pat. No. 4,855,240; O'Farrell (2009) "Evolution in Lateral Flow-Based Immunoassay Systems", RC Wong, HY Tse (eds.), Lateral Flow Immunoassay, 1 DOI 10.1007 / 978-1-59745-240-3_1). In 2006, over 200 companies worldwide manufactured LFTs in a market that was then estimated at $2.1 billion (O'Farrell, op. cit.).

[0003] LFTs generally include a strip made of nitrocellulose that connects with a sample pad at one end and a wicking pad at the other end. This configuration ensures uniform capillary flow as the liquid sample is applied to the sample pad from the deposition point through the nitrocellulose towards the wicking pad. The configuration is assembled on a backing card for stability. Detection and visualization of the analyte is mediated by a pair of affinity reagents that bind to different surfaces on the analyte. Such affinity reagent pairs may be naturally occurring as polyclonal or monoclonal antibodies, or other affinity reagents selected for the purpose. The first reagent of each pair is labeled with an optically detectable reagent, such as microscopic latex beads or gold nanoparticles, and used in solution. Typically, optically detectable labels are so small that individual particles cannot be detected by eye, becoming detectable by eye only when a large number of particles accumulate to a very high concentration or amount at a particular location. To achieve this effect, the second (unlabeled) reagent of each pair is immobilized and used to capture the analyte and any labeled reagents that are bound to the analyte from the solution. The capture reagent is unlabeled and is printed in a visible line perpendicular to the direction of flow in what is known as a capture strip: accumulation of label in this line indicates that the capture reagent has added analyte from solution bound to the labeled reagent.

[0004] Typically, two visible lines are printed on the nitrocellulose membrane. As mentioned above, the one closest to the sample pad and the first the sample encounters is the capture reagent. This is the so-called test line. The second printed line contains an antibody or other capture reagent that binds directly to the labeled reagent and thus adds the labeled particle directly from solution: this is the so-called control line. It adds the labeled component added to the test, so if the test is performed correctly, the test line should always be visible. Soluble reagents, including binding affinity reagents labeled with latex beads or gold nanoparticles, can be included in the reagent pad during manufacture, in a separate reagent pad between the sample pad and the wicking pad, or added to the sample with the sample buffer before application to the membrane.

[0005] Although the capacity of the assay is tunable, several problems can exist with conventional LFT platforms.

[0006] · subjective visual readout of the sensor response leading to ambiguous interpretation of the results, moderate clinical competence, and the potential to exclude poorly visually impaired users from using the test; · For example, when the analyte is a trace toxic metal in a drinking water sample, or when blood, plasma or serum samples are required from patients who are neonatal or elderly, or who have hypotension and / or vascular collapse, the requirement that the amount or concentration of the analyte be above a detectable threshold commensurate with visual detection can be problematic when it is impractical to obtain large samples; The limited possibility of quantification, which means that LFTs cannot be used in many situations where other forms of follow-up testing are frequently required and where cheap disposable tests may be preferable, for example in glucose monitoring in diabetic patients.

[0007] Limited possibilities for multiplexing, especially when the test is used by someone who is not a trained practitioner, meaning that multiple LFTs must be used when more than one analyte or biomarker needs to be detected in the test.

[0008] Miniaturization of sample volume requirements below the microliter level has not been achieved; ·Can be difficult to integrate with on-board electronics and built-in QC functions; Reproducibility between studies can be difficult.

[0009] In view of the above, the present technology provides improved devices and methods for performing lateral flow tests. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Pat. No. 4,703,017 [Patent Document 2] U.S. Pat. No. 4,855,240 [Non-patent literature]

[0011] [Non-Patent Document 1] O'Farrell (2009) “Evolution in Lateral Flow-Based Immunoassay Systems”, RCWong, HYTse (eds.), Lateral Flow Immunoassay, 1 DOI 10.1007 / 978-1-59745-240-3_1 Summary of the Invention

[0012] Thus, the present technology provides devices and systems for performing electrochemical lateral flow tests that are non-optical based and can be implemented on off-the-shelf LFTs. The present technology also allows for the manufacture of new LFTs in cases where the introduction of LFTs was not previously practical, for example when the test requires high sensitivity, quantification, or multiplexing. The present technology further provides devices and methods for generating and capturing electrochemical signals from the results of conventional LFTs. The captured data can be transmitted, for example, to a computer, display, smartphone, other smart device, data storage, or transmission device, for analysis, storage, or onward transmission, for example. The present technology further provides a computer-implemented method for interpreting the results of a user's LFT.

[0013] One aspect of the present technology provides a lateral flow testing device for performing a lateral flow test on a liquid sample, the device comprising: a test strip comprising a nitrocellulose membrane having a first capture reagent disposed on a first surface along a test line, the first capture reagent configured to capture a first analyte of the liquid sample; a sample pad disposed at a first end of the nitrocellulose membrane configured to receive a liquid sample; a test strip comprising a plurality of labeling molecules disposed on the first surface at a position between the sample pad and the test line, the labeling reagent configured to bind to the first analyte; an electrode array disposed on the nitrocellulose membrane and configured to apply a potential across the first surface; and a housing enclosing the test strip and the electrode array, the housing comprising a support element configured to support the electrode array and the test strip in an initial position where the electrode array is spaced apart from the test strip.

[0014] In some embodiments, the housing may include a compressible portion configured to displace the electrode array from an initial position when the compressible portion is compressed.

[0015] In some embodiments, the support element can include a pivot coupled to the electrode array to allow the electrode array to rotate.

[0016] In some embodiments, the support element may further include a pin coupled to a rear end of the electrode array, the pin extending through a linear notch in the housing, such that movement of the pin rotates the electrode array about the pivot.

[0017] In some embodiments, the housing may further comprise a wedge element that, upon compression, compresses the compressible portion to cause the electrode array to rotate about the pivot.

[0018] In some embodiments, the electrode array can include a core formed of an insulating material and a plurality of electrodes disposed on the core.

[0019] In some embodiments, the core and / or the electrode array may be formed of a flexible material.

[0020] In some embodiments, the electrode array can have openings that allow the electrode array to flex.

[0021] In some embodiments, the housing can include an angled portion configured to support the test strip in an angled position.

[0022] In some embodiments, the device may further comprise a wicking pad having high liquid absorption capacity positioned at the second end of the nitrocellulose membrane and configured to provide a capillary force to drive the liquid sample from the sample pad along the nitrocellulose membrane.

[0023] In some embodiments, the device may further comprise a conjugate pad disposed on a first surface of the nitrocellulose membrane impregnated with the labeling reagent.

[0024] In some embodiments, the nitrocellulose membrane can further include a second capture reagent disposed on the first surface along the control line, the second capture reagent configured to capture the labeled molecule.

[0025] In some embodiments, the multiple electrodes can include one or more working electrodes positioned to overlap the test line and a background portion of the nitrocellulose membrane, the one or more working electrodes configured to apply a potential to a first surface of the nitrocellulose membrane.

[0026] In some embodiments, the multiple electrodes can include one or more counter electrodes, each positioned opposite a corresponding working electrode to complete an electrical circuit.

[0027] In some embodiments, the plurality of electrodes can include one or more reference electrodes configured to act as reference points for the potential applied by the working electrode.

[0028] In some embodiments, the or each reference electrode may be positioned adjacent to the counter electrode.

[0029] In some embodiments, at least one working electrode-counter electrode pair can be disposed in a recess in the electrode array such that when contacted with a test strip, the recess forms a sampling well.

[0030] In some embodiments, the electrode array may be disposed on a printed circuit board PCB.

[0031] In some embodiments, the PCB may include multiple conductive layers.

[0032] In some embodiments, one or more of the conductive layers may be covered with a solder mask layer.

[0033] In some embodiments, the labeling molecule may include a molecule that exhibits catalytic activity, such as a gold nanoparticle.

[0034] In some embodiments, the device may further comprise a signal enhancer disposed on the first surface or sample pad of the nitrocellulose membrane configured to deposit on the surface of the labeled molecule for electrochemical detection of the labeled molecule.

[0035] In some embodiments, the device may further comprise an additional conjugate pad disposed on the first surface of the signal enhancer-impregnated nitrocellulose membrane.

[0036] In some embodiments, the signal enhancer may comprise multiple metal ions, and the multiple metal ions may be derived from a silver salt.

[0037] In some embodiments, the electrode array can include a plurality of gold-plated electrodes disposed on a nitrocellulose membrane that initiates the reduction of silver ions derived from the silver salt of the gold-plated electrodes.

[0038] In some embodiments, multiple metal ions may be provided in combination with a reducing agent.

[0039] In some embodiments, the reducing agent can be a hydroquinone solution.

[0040] In some embodiments, the device may be provided with a unique identifier, which may optionally include a barcode, a QR code, or a combination thereof.

[0041] In some embodiments, the housing may be provided with a window above the test line for visual evaluation of the test line.

[0042] In some embodiments, the device may further comprise a plurality of test strips incorporated within the housing.

[0043] In some embodiments, the device may further comprise an integrated electronic reader configured to generate electrical signals via the electrode array to drive electrochemical reactions in the test strip and receive the resulting electrochemical signals from the test strip via the electrode array; and a power source configured to provide power to the electrode array via the electronic reader.

[0044] In some embodiments, the electrode array may comprise at least one pair of electrodes, and the resulting electrochemical signal comprises a current across the at least one pair of electrodes, or a change in conductance, resistance, capacitance or impedance of an electrical circuit between the at least one pair of electrodes, or a combination thereof.

[0045] In some embodiments, the device may further comprise a communication interface configured to communicate with an external electronic device to transmit the received electrochemical signals.

[0046] In some embodiments, a first capture reagent can be further disposed on the first surface of the nitrocellulose membrane along a second test line in a different concentration than the first test line.

[0047] In some embodiments, the nitrocellulose membrane may further include a third capture reagent disposed on the first surface along a third test line, the third capture reagent configured to capture a third analyte of the liquid sample different from the first analyte.

[0048] In some embodiments, the electrode array may comprise a plurality of corresponding electrode pairs arranged such that a plurality of corresponding electrode pairs cover the nitrocellulose membrane background, a corresponding electrode pair covers a first test line, and a corresponding electrode pair of the plurality may cover each of the control line and / or the second test line and / or the third test line.

[0049] Another aspect of the present technology provides an electronic reader for reading results from a lateral flow test device, the lateral flow test device comprising a compressible portion, a receiving portion for receiving the lateral flow test device comprising a release mechanism configured to compress the compressible portion of the lateral flow test device; and a communication port configured to electrically couple to the lateral flow test device to generate an electrical signal to drive an electrochemical reaction in the lateral flow test device and to receive a resultant electrochemical signal indicative of the electrochemical reaction.

[0050] In some embodiments, the electronic reader may further comprise a power connection for receiving power from an external source.

[0051] In some embodiments, the electronic reader may further include an integral power source.

[0052] In some embodiments, the electronic reader may further comprise an optical reader for reading the unique identifier on the lateral flow test device.

[0053] In some embodiments, the release mechanism may comprise a linear actuator configured to compress a compressible portion of the lateral flow test device upon insertion of the lateral flow test device into the receptacle.

[0054] In some embodiments, the electronic reader may further comprise a timer configured to count down a measurement time defined by the length of time required to measure the result.

[0055] Another aspect of the present technology is a system for performing a lateral flow test on a liquid sample, the lateral flow test device comprising: a test strip, a nitrocellulose membrane having a first capture reagent disposed on a first surface along a test line, the first capture reagent configured to capture a first analyte of the liquid sample; a sample pad disposed at a first end of the nitrocellulose membrane configured to receive the liquid sample; a labeling reagent comprising a plurality of labeling molecules disposed on the first surface at a location between the sample pad and the test line, the labeling molecules configured to bind to the first analyte; an electrode array disposed on the nitrocellulose membrane and configured to apply a potential across the first surface; and a housing enclosing the test strip and the electrode array, the electrode array configured to bind to the first analyte of the liquid sample. A system is provided that includes a lateral flow testing device comprising: a test strip comprising a housing comprising a support element configured to support the electrode array and the test strip in an initial position spaced from the test strip; and an electronic reader comprising a receiving portion for receiving the lateral flow testing device comprising a release mechanism configured to compress the housing of the lateral flow testing device to bring the electrode array into contact with the test strip in a read-ready position; and an electronic reader comprising a communication port configured to electrically couple to the electrode array of the lateral flow testing device to generate electrical signals in the electrode array to drive an electrochemical reaction of the test strip and to measure resultant electrochemical signals from the electrode array indicative of the electrochemical reaction of the test strip.

[0056] In some embodiments, the housing may include a compressible portion configured to displace the electrode array from an initial position when the compressible portion is compressed.

[0057] In some embodiments, the release mechanism may be activated by the act of inserting the lateral flow test device into the receptacle.

[0058] In some embodiments, the support element may include a pivot coupled to the electrode array, and the release mechanism may include a protruding element configured to protrude into the compressible portion to rotate the electrode array upon insertion of the lateral flow test device into the receptacle.

[0059] In some embodiments, the protruding elements may be biased towards the centre line of the receptacle by an elastic element, and the protruding elements may be released into the compressible portion by the elastic element upon insertion of the lateral flow test device into the receptacle.

[0060] In some embodiments, the support element may include a pivot coupled to the electrode array to allow the electrode array to rotate and a pin coupled to a rear end of the electrode array, the pin extending through a linear cutout in the housing; and the release mechanism may include a guide groove on an inner surface of the receiver, the guide groove may be inclined toward a centerline of the receiver and configured to receive the pin to guide the pin toward the centerline upon insertion of the lateral flow test device into the receiver to rotate the electrode array.

[0061] In some embodiments, the support element may include a pivot coupled to the electrode array, and the housing further includes a wedge element, and the release mechanism comprises a surface against which the wedge element presses when the lateral flow test device is inserted into the receptacle such that the wedge element compresses the compressible portion and rotates the electrode array.

[0062] In some embodiments, the release mechanism may comprise a linear actuator configured to extend towards a centerline of the receiver upon actuation.

[0063] In some embodiments, the release mechanism may be configured to compress the compressible portion with a pressure in the range of 1N to 50N.

[0064] In some embodiments, the lateral flow test device may include a port having an exposed portion of the electrode array, the port positioned to mate with a communication port of an electronic reader.

[0065] A further aspect of the present technology provides a method of performing a lateral flow test on a liquid sample using a lateral flow test strip, the lateral flow test strip comprising: a nitrocellulose membrane having a capture reagent disposed on a first surface along a test line, the capture reagent configured to capture a predetermined analyte of the liquid sample; a sample pad disposed at one end of the nitrocellulose membrane configured to receive a liquid sample; and a plurality of labeling molecules disposed on the first surface between the sample pad and the test line, the labeling reagent configured to bind to the predetermined analyte, applying a potential across the first surface via an electrode array disposed on the nitrocellulose membrane to drive an electrochemical reaction of the lateral flow test strip; and measuring a resultant electrochemical signal from the lateral flow test strip through the electrode array.

[0066] In some embodiments, the method may further include depositing the liquid sample on the sample pad.

[0067] In some embodiments, the electrode array may comprise a plurality of corresponding electrode pairs, and the method may further include overlaying the corresponding electrode pairs on a nitrocellulose membrane background and on a corresponding electrode pair on a test line, and determining a difference in the resulting electrochemical signal between the electrode pairs overlaying the background and the electrode pairs overlaying the test line.

[0068] In some embodiments, the method may further include washing the nitrocellulose membrane with a buffer through the sample pad prior to applying the electric potential.

[0069] In some embodiments, the method may further include depositing a signal enhancer solution containing a plurality of metal ions onto the nitrocellulose membrane through the sample pad prior to applying the electrical potential.

[0070] In some embodiments, the multiple metal ions may be derived from a silver salt.

[0071] In some embodiments, the method may further comprise an activation step in which the signal enhancer solution is combined with a developing agent.

[0072] In some embodiments, the developing agent may include a reducing agent to enable spontaneous reduction of multiple metal ions on the surface of the labeled molecule.

[0073] In some embodiments, the method may further include measuring a distribution of metal resulting from at least a portion of the plurality of metal ions precipitated on the surface of the labeled molecule along the first surface of the nitrocellulose membrane to determine a concentration of the labeled molecule along the first surface.

[0074] In some embodiments, the method may further include measuring the distribution of reagents (eg, metal ions) along the nitrocellulose membrane that did not react with the labeled molecule (eg, did not precipitate on the surface).

[0075] In some embodiments, the method may further include performing the potential sweep at least two times, where a first cycle of the potential sweep resets the electrochemical reaction and a second cycle of the potential sweep measures the electrochemical signal.

[0076] In some embodiments, measuring a resultant electrochemical signal from the lateral flow test strip may include performing a potential sweep across the nitrocellulose membrane via the electrode array.

[0077] In some embodiments, detection of the current peak can indicate the local concentration of the metal ion.

[0078] In some embodiments, detection of the absence of a current peak at or near the test line can indicate the presence of a predetermined analyte in the liquid sample.

[0079] In some embodiments, measuring the resulting electrochemical signal from the lateral flow test strip may be based on voltammetric, amperometric, potentiometric, or impedance-based methods, or combinations thereof.

[0080] In some embodiments, measuring the resulting electrochemical signal from the lateral flow test strip may be performed using a linear sweep voltammetry method.

[0081] In some embodiments, the linear sweep voltammetry method can include sweeping a potential from a minimum potential to a maximum potential at a predetermined rate relative to a reference potential, where the predetermined rate can be in the range of 10 mV per second to 1000 mV per second.

[0082] In some embodiments, the minimum potential relative to the reference potential may be within the range of -1V to -0.1V, preferably -0.5V.

[0083] In some embodiments, the maximum potential relative to the reference potential may be in the range of +0.1V to +1V, preferably +0.5V.

[0084] In some embodiments, the linear sweep voltammetry method may include repeating a potential sweep a predetermined number of times.

[0085] In some embodiments, the linear sweep voltammetry method can include a deposition step of applying a low potential for a predetermined period of time before performing a potential sweep and / or applying a high potential for a predetermined period of time after performing a potential sweep.

[0086] In some embodiments, the low potential may be in the range of −1 V to −0.1 V relative to the reference potential, and / or the high potential is in the range of 0.1 V to 1 V relative to the reference potential.

[0087] In some embodiments, the predetermined period of time may be one minute or less.

[0088] In some embodiments, measuring the resulting electrochemical signal from the lateral flow test strip can be performed using pulse-based electrochemical techniques.

[0089] In some embodiments, the pulse-based electrochemical technique can include differential pulse voltammetry or square wave voltammetry.

[0090] Embodiments are described below with reference to the accompanying drawings. [Brief description of the drawings]

[0091] [Figure 1A] 1 shows an exemplary lateral flow assay strip. [Figure 1B] 1 shows an exemplary lateral flow assay with signal amplification using silver enhancement. [Diagram 2] 1 shows an exemplary printed circuit board (PCB) carrying an electrode array having multiple working, counter and reference electrodes. [Figure 3A] The bottom side of the PCB carrying the electrode array is shown with a schematic diagram of the PCB covering the LFT strip. [Figure 3B] 3B shows a conceptual diagram of overlapping the PCB and LFT strip of FIG. 3A in contact with the LFT strip. [Figure 4A] 1 shows an exploded view of an exemplary cassette containing an LFT PCB electrode array with top and bottom covers. [Figure 4B] 4B shows the exemplary cassette of FIG. 4A with exposed connections for electronic readout. [Figure 4C] 4C illustrates an exemplary electronic reader configured to read the cassette of FIG. 4B. [Diagram 5] 1 shows an exemplary electrode array layout. [Figure 6]FIG. 1 shows an exploded view of an exemplary lateral flow test device. [Figure 7A] 1 illustrates an exemplary system of a lateral flow test device and reader, according to various embodiments. [Figure 7B] 1 illustrates an exemplary system of a lateral flow test device and reader, according to various embodiments. [Figure 7C] 1 illustrates an exemplary system of a lateral flow test device and reader, according to various embodiments. [Figure 7D] 1 illustrates an exemplary system of a lateral flow test device and reader, according to various embodiments. [Figure 7E] 1 illustrates an exemplary system of a lateral flow test device and reader, according to various embodiments. [Figure 7F] 1 illustrates an exemplary system of a lateral flow test device and reader, according to various embodiments. [Figure 8A] A separate working electrode is shown overlaying the background, test and control lines of the LFA during an exemplary signal readout. [Figure 8B] FIG. 5B shows the results of the reaction of silver on the nitrocellulose membrane of the LFA during the exemplary signal readout of FIG. 5A. [Figure 8C] We show the reduction of Ag+ on AuNPs due to the formation of immune complexes, which increases the concentration of metallic silver locally at the locations where AuNPs accumulate. [Figure 8D] The concentration of Ag+ is shown decreasing in the corresponding regions of the film. [Figure 8E] In the example where a gold PCB is placed on the membrane, the potential sweep reveals peaks of metallic silver oxidation deposited on the gold PCB electrode, which are suppressed, indicating the accumulation of AuPN therein. [Figure 9] An exemplary linear sweep voltammetry is shown demonstrating the presence of a silver oxidation peak in the membrane background and the disappearance of the peak at the test / control line where AuNPs accumulate. [Figure 10]Shown is the result of sequential scanning of four PCB electrodes, where the first and third electrodes cover the background, the second electrode covers the test line, and the fourth electrode covers the control line, with both the test and control lines being positive, i.e., with accumulated AuNPs. [Figure 11] A schematic diagram of repeated LSV measurements is shown. [Figure 12] 1 shows exemplary differential pulse voltammetry. [Figure 13A] 1 shows a representation of visual testing of commercially available hCG LFTs with increasing concentrations of hCG. [Figure 13B] A visual assessment of the brightness of the test / control line of a commercial hCG LFT compared to background is shown, with brightness along the Y-axis being the ratio of the test / control line divided by the background. [Figure 13C] Electrochemical evaluation of the same test strips as in FIG. 13B is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] Turning first to FIG. 1A, an exemplary lateral flow test (LFT) includes a sample pad (where the sample is introduced into the test) overlaid with a conjugate pad. When the sample is introduced, the analyte migrates from the sample pad to the conjugate pad by capillary forces. The conjugate pad is impregnated with labeling molecules, which can be gold or latex nanoparticles coated with antibodies / affimers / aptamers. When introduced, the analyte interacts with the given labeling reagent (e.g. AuNP gold nanoparticles) and continues to migrate into the test under capillary forces. The conjugate pad is in contact with a nitrocellulose membrane on which a line of capture reagent (capture antibody / affimer / aptamer, etc.) is printed. As the analyte of interest flows through the line of capture reagent (test line), an immune complex is formed that attracts the AuNP to the test line. Another line is printed downstream of the test line (control line) that contains a reagent that can capture the AuNP without the presence of the analyte. The formation of the immune complex in the control line acts as a confirmation that the reagents under test are intact and the results of the test line are valid. Downstream of the test / control line, the nitrocellulose membrane connects to a wicking pad that has high liquid absorption capacity and drives the capillary action necessary to migrate the sample and label through the nitrocellulose membrane.

[0093] According to the present technique, the sensitivity of AuNP-based LFT can be increased by the utilization of signal enhancement strategies that utilize enzymatic or non-enzymatic approaches, such as silver enhancement, among others. In the embodiment shown in FIG. 1B, the silver enhancement strategy is based on the use of a silver salt (silver lactate / nitrate or silver acetate) in combination with a reducing agent such as hydroquinone in solution, where the gold acts as a catalyst to allow the transfer of electrons from the reducing agent to the silver ions, which precipitate on the surface of the gold to form metallic silver. The combined silver salt and reducing agent solution can be supplied, for example, as an additional conjugate pad impregnated with a signal enhancer, or as an integrated reservoir inside the cassette that can be broken manually or automatically (and the solution inside released). The precipitated silver continues to catalyze the reaction, resulting in the deposition of a layer of silver on the gold surface, thus enlarging the AuNPs. According to the embodiment, the increased size of the AuNPs contributes to the higher sensitivity of LFT utilizing silver enhancement by visual evaluation of the test / control lines.

[0094] In an embodiment, an electrode array can be constructed and overlaid on the nitrocellulose membrane to capture signals generated on the test and control lines of the LFT. The electrode array can be composed of an inert insulating core material and conductive "pads" or electrodes made of conductive metal / polymer / nanomaterial or organic materials capable of electrical signal transduction. Additive or subtractive manufacturing techniques such as, but not limited to, photolithography, screen printing, inkjet printing, 3D printing can be used to fabricate the electrode array. In the embodiment shown in FIG. 2, the electrode array can be placed on a printed circuit board (PCB). The PCB can include multiple conductive layers that may be covered with a thin solder mask layer that prevents undesired pad exposure. The PCB shown is a conductive connection with a connector part, carrying two sides without interference from the LFT. In an embodiment, the PCB includes multiple electrodes forming a standard three-electrode cell configuration, with individual working electrodes (WE) overlaid on the test lines, control lines, and background (no lines) of the nitrocellulose membrane. Multiple or single counter electrodes (CE) may also be present on the PCB or a separate reference electrode (RE).

[0095] As shown in FIG. 3, the electrode array is placed on top of the LFT to align the background, test and control lines of the strip with each pair of working and counter electrodes. The bottom side of the PCB contacts the nitrocellulose membrane during the measurement, while other parts of the test (sample pad, conjugate pad, wicking pad) are avoided. In this embodiment, the electrodes of the electrode array are not functionalized, allowing for a long shelf life and enabling the method to be performed with any colloidal gold-based LFT, since immune complex formation occurs within the membrane matrix and not on the surface of the electrodes. In other embodiments, the electrodes of the electrode array may be alternatively functionalized as needed.

[0096] An electronic lateral flow test (eLF) device according to an embodiment is shown in FIG. 4A, in which the LFT and electrode array are sandwiched between an upper cover and a lower cover forming a housing. The device may be, for example, a disposable cassette, and may be made of renewable materials, such as, for example, compostable, e.g., corn-based plastic or other natural composites, bamboo, wood, etc. The cassette may be provided with a window for visually reading the test and control lines, but this is not required, as the results are read electronically.

[0097] In an embodiment, the cassette has an exposed connection configured to be inserted into an electronic reader for the results to be measured by the reader chip of the electronic reader, as shown in FIG. 4B. In an embodiment, the cassette is provided with a connection piece that can include, for example, a unique printed barcode or QR code. The connection piece can be configured to be inserted into a port of the electronic reader to perform the measurement, as shown in FIG. 4C. In an alternative embodiment, the reader chip may be integrated into the cassette along with a communication interface, for example a Bluetooth chip and a power source such as a Li-polymer battery. In either configuration, the integrated electronic LFT (eLF) may be single-use and disposable, for example for potentially infectious samples, or in an alternative embodiment, the cassette may be opened, the test strip replaced, and the cassette reused. Because the electronic reader does not come into contact with the sample, potential contamination is negligible and thus the reader is reusable.

[0098] Once the results are read, the test results may be instantly transmitted from the electronic reader, or from the cassette in embodiments where the electronics are integrated within the cassette, wirelessly, e.g. via Bluetooth / WiFi, or via a physical connection such as a USB port / AUX port based cable. The test results may be transmitted, e.g., to a computer for analysis, a communication device for onward transmission, a smartphone or other smart device with a corresponding app, or to a storage device. The data may be stored, analyzed, and / or transmitted in real time to relevant parties such as health authorities, medical services, caregivers, employers, or in the case of environmental monitoring to farmers, utilities, environmental agencies, public health agencies, etc.

[0099] In an embodiment, the reader may include a custom chip in the body with a power supply for a portable reader or with a power connection for a home or laboratory reader. The custom chip and firmware generate a signal to drive the electrochemical reaction and capture the resulting electrochemical signal, which may be a current or a change in the characteristics of the electrical circuit between the working and counter electrodes, such as its conductance, resistance, capacitance or impedance. A useful signal is obtained if there is a difference between the counter electrode aligned with the bare membrane (background) and the electrode aligned with the test line (expected to be positive if the test is functioning correctly) and the capture line (measures the presence of analyte). It will be clear to those skilled in the art that the characteristics of the electrical circuit will change depending on the number of label particles that accumulate on each line, and the strength of the signal is a proximity readout of this number and can be used to quantify the analyte. It is expected that multiple electrode pairs can be used to monitor multiple lines of a single strip, and that using an electronic reader to perform the result measurement so that the results are interpreted digitally means that multiple simultaneous readout data can be obtained in a single test by an untrained user without the risk of confusion.

[0100] In another embodiment, multiple test lines ranging in concentration of the reagent used to capture the label can be used to calibrate the assay and / or aid in quantitation of the analyte. Additionally, this embodiment allows for the incorporation of multiple test strips side-by-side or stacked on top of each other (e.g., separated by a PCB) into a single cassette, which is not possible when a visual / optical readout is performed.

[0101] The reader may have a single port so that one test is read at a time, or multiple ports may be provided to allow multiple simultaneous reads for higher throughput. In one embodiment, the reader incorporates a barcode reader, camera or other optical device used to read a unique barcode or QR code, etc. printed on the connecting part of a disposable or reusable cassette. The reader may incorporate a mechanical button that opens the sample port when the cassette is inserted into the reader. The reader may incorporate a countdown timer to ensure that the measurement is taken at the appropriate time. This also allows the use of kinetic measurements that may extend the measurement range of the test, for example allowing early readout of high levels of analyte that exceed the maximum limits of the assay if the measurement is carried out for the standard time required to measure low levels of analyte in other samples.

[0102] An embodiment of the reader designed for home use is provided. For example, on a single-use battery, each reader can perform 200 tests, allowing, for example, a family of four to perform one test per day for 50 days each. The reader may then be returned to the manufacturer to be reused to increase the sustainability of the reader. In another embodiment, the reader may be connected to an external power source for use with a higher number of samples per day or for extended periods of time.

[0103] According to this embodiment, the electrode array includes one or more working electrodes (WE), one or more counter electrodes (CE), and one or more reference electrodes (RE). The working electrodes are arranged to align on the test and control lines of the nitrocellulose membrane, and the counter electrodes are provided to complete a circuit with the corresponding working electrodes. According to this embodiment, the WE is smaller than the test and control lines of the nitrocellulose membrane to ensure proper alignment.

[0104] 5 illustrates an exemplary electrode array 500 according to an embodiment. The electrode array 500 comprises a plurality of working electrodes 501, 511, and 521, a plurality of counter electrodes 502, 512, 522, and a plurality of reference electrodes 503, 523 disposed on a dielectric core 510.

[0105] In this embodiment, the WE501 is configured to measure the background reagent of the nitrocellulose membrane. The CE502 is positioned opposite and in close proximity to the WE501. However, other configurations are possible that would be apparent to one skilled in the art. The RE503 is positioned adjacent to the WE501 and CE502 to ensure a stable application of a potential to the WE501. The three electrodes WE501, CE502 and RE503 form components of the sampling well 504 (shown in dotted lines). The sampling well 504 has a reduced height compared to the dielectric core 510 to allow the fluid sample to accumulate in the sampling well 504 and around the electrodes WE501 and CE502. The height difference can be achieved using any suitable and desired technique of the dielectric material, such as a solder mask, any photoresist, or injection molding.

[0106] The WE511 and CE512 form components of a second sampling well 514 with RE503. The two sampling wells 504 and 514 share the same RE503. In this embodiment, most of the reference electrode trace 505 is covered by a dielectric 510 to reduce the possibility of reactions occurring at the electrode surface of the RE503 that may affect the reactions in the WE501 and WE511 when the RE is in close proximity to the WE. For the same reason, the RE503 is placed on the side of the CE502 and CE512 to reduce the possibility of reactions occurring at the RE503 interfering with the reactions occurring at the WE501 and WE511.

[0107] The third WE 521 and corresponding CE 522 are disposed within a third sampling well 524 in which the second RE 523 is provided.

[0108] In this embodiment, CE502, CE512 and CE522 may be connected to the same output, and RE503 and RE523 may also be connected to the same pin of an analog front end (AFE).

[0109] According to this embodiment, various mechanisms can be used to contact the electrode array with the nitrocellulose membrane to create isolated sample wells to determine the concentration of the reagent. Note that the concentration of the reagent is proportional to the concentration of the analyte due to localized immune complexes formed on the nitrocellulose membrane. In some embodiments, compression can be applied to the lateral flow test device to improve performance in the sampling wells, as described below.

[0110] In some embodiments, a 0.8 mm FR-4 core can be used as a suitable dielectric core, which can be filled with copper traces and pads and ENIG plated. Alternative plating techniques can include electrolytic or electroless plating, which can apply materials ranging from gold, silver, platinum, etc. In general, the thicker the plating, the more reliable the sensor. Plating thickness is conventionally described in microinches, with 1, 2, and 3 microinch plating being of particular interest. Various surface finishes will be apparent to those skilled in the art.

[0111] In this embodiment, the dielectric material is a fiberglass-based core that provides some flexibility. Alternatively or additionally, flexible electrode arrays may be used based on polyamide films known as flex PCBs. Screen spitting or inkjet printing, among other techniques, can also be used to deposit conductive material onto a rigid or flexible dielectric to form part of the lateral flow test device assembly or to print directly onto the base housing of the lateral flow test device assembly (top or bottom). The electrode array inside the LFT device allows electrical connection to the electronic reader without the electronic reader coming into contact with the sample inside the LFT device, thus allowing the electronic reader to be safely reused.

[0112] In an embodiment of the present technology, the electrode array of the LFT device is supported in its initial position spaced apart from the nitrocellulose membrane of the LFT device such that the electrode array does not initially contact the nitrocellulose membrane. After a liquid sample is deposited in the sample port of the LFT device, the electrode array contacts the nitrocellulose in a ready-to-read position. Figure 6 shows the structure of an exemplary lateral flow test device 600 according to an embodiment in an exploded view.

[0113] The device 600 comprises a housing including a top 601 and a bottom 606. The top 601 of the housing is provided with a vent 602, a QR code 603 that uniquely identifies the device 600 to allow a test to be associated with a particular person, a sample port 604 for receiving a liquid sample, and a compressible portion 605. The top 601 is configured to mechanically couple to the bottom 606, which forms a bed 607 for a lateral flow array strip (nitrocellulose membrane) 608 to ensure proper alignment of the LFA strip 608.

[0114] The electrode array 609 covers the LFA strip 608 and includes alignment features 611 that are positioned within opposing alignment features 610 in the base 606 to ensure minimal movement of the electrode array in the horizontal direction (parallel to the plane of the base 606). The electrode array 609 is provided with openings 612 that allow the absorbent pads 613 of the LFA strip 608 to expand as liquid is absorbed. The openings 612 further allow the electrode array 609 to flex. When the compressible portion 605 is compressed, it displaces the electrode array 609 from its initial position towards the LFA strip 608, bringing the electrode array 609 into contact with the LFA strip 608 in the read-ready position. The flexibility provided by the openings 612 in the electrode array 609 facilitates such displacement.

[0115] The transition from the initial position to the read-ready position results in a non-horizontal positioning of the electrode array 609. To ensure uniform contact between the electrode array 609 and the LFA strip 608, the bed 607 is configured with an inclined portion 614 to counter the non-horizontal positioning of the electrode array 609 in the read-ready position. A support feature / element 615 is provided on the bottom 606 configured to support the electrode array 609 in the initial position spaced apart from the LFA strip 608 and hold the electrode array 609 in the correct position when the electrode array 609 contacts the LFA strip 608 via the compressible portion 605, thus allowing a controlled transition of the electrode array 609 between the two positions.

[0116] An important consideration is the pressure exerted by the LFA strip 608 when it contacts the electrode array 609 by mechanical compression of the compressible portion 605. Applicant recognizes that changes in pressure have different effects on the electrochemical readout of the LFA strip 608. When the LFA strip 608 is compressed at low pressure (e.g. 1 N), electrical contact between the LFA strip 608 and the electrode array 609 is made and measurements can be made. When higher pressures are applied (e.g. 5 N), the electrochemical measurements become more reproducible due to a more uniform pressure distribution along the LFA strip 608 and the electrode array 609. When a particularly high pressure is used (e.g. 30 N), the membrane is compressed more and the flow of sample liquid through the nitrocellulose membrane during the measurement is more effectively stopped, resulting in easier separation of the local sampling pools created by the recesses / depressions of the electrode array 609, leading to a higher order of sampling resolution. Furthermore, increasing the pressure reduces the volume of the nitrocellulose membrane in each sampling pool, which in turn increases the number of labeled molecules per unit volume of the sampling pool, resulting in a higher conversion rate and a higher sensitivity of the assay. At very high pressures (e.g., 50 N), the compression becomes too high and most of the sample liquid is forced out of the sampling pool, increasing the resistance between the electrodes and impairing the flow of current.

[0117] Compression of the compressible portion 605 of the housing may, in some embodiments, be effected via one or more corresponding features (release mechanisms) of the electronic reader such that upon insertion of the lateral flow test device 600 into the electronic reader, the compressible portion 605 is compressed by one or more corresponding features of the electronic reader to displace the electrode array 609 from its initial position. Below are described various non-limiting examples of LFT devices and electronic readers having corresponding features. Note that in this specification, compressible portion simply refers to the section or portion of the housing that is to be compressed to displace the electrode array from its initial position (or displace the LFA strip, if desired). Although the compressible portion 605 is shown in FIG. 6 as being partially cut to allow compression, this is not considered to be necessary, and uncut compressible portions formed, for example, of flexible material or completely unmodified compressible portions are also possible.

[0118] 7A shows an LFT device with an electrode array supported inside a housing at an angle away from the LFA strip and a protruding rail on the upper inner surface of a corresponding electronic reader receptacle. When the LFT device is inserted into the electronic reader, the LFT device moves to a position where the protruding rail is above a compressible portion of the LFT device and engages the compressible portion to displace the electrode array, rotating the electrode array to allow contact between the electrode array and the LFA strip.

[0119] FIG. 7B again shows an LFT device with the electrode array supported inside the housing at an angle away from the LFA strip. A spring-compressed ball detent is provided in the corresponding electronic reader receiver, with a spring (or other elastic element) provided in the recess and a ball bearing protruding into the spring-biased receiver. When the LFT device is inserted into the electronic reader, the ball bearing is forced into the recess and the spring is compressed. Then, when the LFT device is further inserted, the compressible portion of the LFT device moves toward the recess and the ball bearing is released into the compressible portion under the force of the compression spring, compressing the compressible portion and displacing the electrode array.

[0120] 7C shows an LFT device in which the electrode array is supported inside the housing at an angle away from the LFA strip, and the electrode array is held in place by a pin at one end that extends through a cutout in the housing. The corresponding electronic reader is provided with a guide groove (or a pair of guide grooves, if a pair of pins are provided on one side of the electrode array) that descends from the entrance of the receptacle of the electronic reader toward the centerline of the receptacle. When the LFT device is inserted into the electronic reader, the pin of the electrode array engages the guide groove, which guides the pin along the cutout to rotate the electrode array and bring it into contact with the LFA strip.

[0121] 7D shows an LFT device with an electrode array supported within a housing at an angle away from the LFA strip. The housing of the LFT device includes a wedge-shaped portion that is pivoted at the front end of the LFT device and extends to a compressible portion of the housing. When the LFT device is inserted into the electronic reader, the receiver of the electronic reader pushes against the wedge-shaped portion, eventually forcing the thick end of the wedge-shaped portion into the compressible portion of the housing, displacing the electrode array and bringing it into contact with the LFA strip.

[0122] FIG. 7E shows an LFT device in which a flexible or partially flexible electrode array is used. In this example, the electrode array is supported at a split between the flexible and rigid parts. The inner surface of the corresponding electronic reader receptacle is provided with a protruding rail. When the LFT device is inserted, the housing of the LFT device slides on the protruding rail until the compressible part reaches the protruding rail, at which point compression of the compressible part pushes the rigid part of the electrode array, causing it to rotate towards and contact the LFA strip. The flexible part of the electrode array allows the front rigid part to be positioned in alignment with the connector (communication port) of the electronic reader while allowing the rear rigid part to be lifted away from the LFA strip.

[0123] FIG. 7F shows an LFT device in which the electrode array is supported in a raised position spaced apart from the LFA strip. The rear of the electrode array covers the front of the LFA strip at the location of the compressible portion of the housing. The corresponding electronic reader is provided with a linear actuator, such as a button, lever, or any other suitable and desired mechanical activation mechanism, configured to extend inward toward the centerline of the receptacle of the electronic reader upon actuation. When the LFT device is inserted into the electronic reader, the linear actuator is actuated to depress the compressible portion of the LFT device, thus displacing the electrode array and bringing it into contact with the LFA strip. When in contact with the LFA strip, the electrode array may be placed in an inclined position, or if the electrode array is flexible, the electrode array may be allowed to bend across the interval of the LFT device.

[0124] Signal readout is based on working and counter electrodes in contact with a nitrocellulose membrane, for example as shown in the embodiment of FIG. 8A where the electrodes are on the underside of the PCB.

[0125] If the technology is implemented into a commercially available LFT by a third-party manufacturer, the LFT will be performed without any modifications to the existing LFT procedure. In some cases, if the sample type is such that it interferes with the LFT chemistry, this can be mitigated, for example, by the addition of a wash step, rinsing the LFT device with water (e.g., in a tap water sink) before the addition of reagents. The wash step can be carried out for as long as necessary or desired, for example, for a few seconds to a few minutes. The wash step removes unwanted species (e.g., Cl) while introducing species that may be useful in the silver enhancement reaction (e.g., water). -) after following the LFT manufacturer's protocol, the nitrocellulose membrane can be washed with 50 μL of water or any other suitable buffer added through the sample port to remove excess electrolytes from the membrane and add a signal enhancer such as silver enhancer. Typical silver enhancers available commercially usually require activation immediately prior to use by combining a "developer" and an "enhancer" stock solution in a 1:1 ratio. The developer allows for the spontaneous reduction of silver ions to metallic silver in the presence of gold nanoparticles and / or gold-plated electrodes. The resulting activated silver enhancer solution is then deposited on the membrane (50 μL) through the sample port and the reaction occurs within a 1-25 minute time frame. Contact between the electrodes and the nitrocellulose strip may be maintained throughout the LFA manufacturer's test, or the electrode array can be placed on top of the LFT strip after the LFT has been performed and the silver has reacted with the AuNPs. This may be required in some situations to ensure that the flow of the test is not impeded. Contact between the membrane and the electrode array can be achieved by manual compression (squeezing the cassette to collapse the internal struts) or by inserting the cassette into a reader to initialize the electrical readout.

[0126] After introduction of the activating silver solution, the AuNPs become coated with metallic silver, as shown in Figure 8B. The distribution of the deposited metallic silver along the nitrocellulose membrane shows an enrichment of AuNPs due to AuNPs catalyzing the reduction of free silver ions, thus enabling the deposition of metallic silver, as shown in Figure 8C. The increase in the amount of silver precipitate leads to a corresponding decrease in free silver ions on the nitrocellulose membrane, locally where the AuNPs are immunocomplexed, as shown in Figure 8D. The reduction of silver ions on the gold PCB is initiated by placing a gold-plated PCB electrode on the nitrocellulose membrane. The amount of available silver ions can be quantified by a potential sweep, where the reduced silver ions are oxidized at the gold electrode, forming the oxidation peak observed in Figure 8E. The height of the peak thus correlates with the local enrichment of free silver ions on the nitrocellulose membrane. When the accumulated AuNPs on the nitrocellulose membrane compete with the reduction of silver ions on the electrode, the silver oxidation peak disappears on the test / control line for positive samples, as shown in Figure 8E.

[0127] Electrochemical readout can be performed using any electrochemical technique such as voltammetric-based, amperometric-based, potentiometric-based or impedance-based methods. Linear sweep voltammetry (LSV) has been successfully used at a scan rate of 50 mV / s from -0.5 to +0.5 V against a reference potential such as pseudo-Au PCB RE, as shown in Figure 9. The low starting potential of LSV ensures that the positive Ag+ ions in the vicinity of the electrode are first reduced at the electrode and deposited on the PCB surface before the potential is increased such that an oxidation peak can be observed between 0.1 and 0.2 V against the pseudo-Au PCB RE. The potential continues to increase to +0.5 V against the pseudo-Au PCB RE, ensuring complete oxidation of silver at the PCB electrode.

[0128] Figure 10 shows the results of sequential scans of four PCB electrodes, where the first and third electrodes cover the background, the second electrode covers the test line, and the fourth electrode covers the control line, and both the test and control lines are positive, i.e., have accumulated AuPN. The bars represent the average of three replicates, and the error bars are the standard deviation. When the PCB electrodes are first brought into contact with the LFT membrane, the initial LSV scans do not show the pattern shown in Figure 9. As can be seen in Figure 10, in the first scan of all four electrodes, the peak heights do not follow any pattern. Only when multiple LSV scans are performed can the current drop for the test and control lines be observed. Nevertheless, the peaks of silver oxidation from the background-covered electrodes remain stable throughout scans 2-12, and no drop in current can be observed.

[0129] Repeated exposure to low and high potentials (by LSV measurements) causes silver ions to be reduced / oxidized and repelled away from the electrode. This increases diffusion and encourages the migrating ions to interact with AuNPs if they are present near the electrode. The interaction of silver ions with AuNPs results in the reduction of silver at the AuNPs, which competes with the reduction of silver ions at the PCB electrode. This is particularly important to accurately assess the concentration of silver ions at the test / control line, since the AuNPs are not immobilized on the electrode surface and reside in the bulk of the membrane. Repeated LSV measurements, as shown diagrammatically in Figure 11, result in potential cycling from low to high potentials, resulting in repeated reduction and oxidation of silver, as well as attraction and repulsion of silver ions. This enhances the diffusion of silver ions, allowing silver to react more efficiently with the AuNPs.

[0130] To increase the sensitivity of the assay, the height of the background current peaks is important, as it is the deviation from the current of these peaks that demonstrates the presence of an electrochemical "signal". To obtain a high signal-to-noise ratio, a deposition step can be used in which a low potential, such as -0.4 vs. pseudo-Au PCB RE, can be used for a predefined time, such as 10 s to 1 min prior to the potential sweep. In this scenario, positive silver ions are attracted to the negatively charged electrode and reduced at the electrode surface. The deposition potential and time, among others, are two parameters that can be adjusted to obtain a higher background current. Furthermore, pulse-based electrochemical techniques, such as differential pulse voltammetry (DPV), can be used to detect low levels of silver ions. Electrochemical deposition and DVP allow a 10-fold increase in the obtained current levels, which again decreases with the number of scans in the AuNP line, as shown in Figure 12.

[0131] It should be noted that the methods described herein may involve multiple independent potential sweeps, as well as coordinated combinations of multiple potential sweeps.

[0132] When multiple potential sweeps are performed in succession, the time difference between electrode measurements can affect catalytic events at the electrode. For example, the high potential at the end of the potential sweep can oxidize one or more species, such as metallic silver, that precipitate on the surface of the catalytically active electrode. A possible mitigation is to perform a series of potential sweeps, where an early cycle of the potential sweep is used to establish a baseline time point.

[0133] For example, a potential sweep can be performed on a first electrode, followed by the same potential sweep on a second electrode, a third electrode, and a fourth electrode. This series of potential sweeps can be considered as one cycle of potential sweeps. After each sweep of the first cycle is completed, all silver species are oxidized in the vicinity of the electrodes. After completing the first cycle of potential sweeps, additional cycles can be repeated in which each electrode is incubated in the LFT device for the same controlled period of time.

[0134] This method has been tested using a commercially available hCG LFT. Figures 10A, 10B and 10C show a comparison of visual and electrochemical readouts on a commercially available hCG LFT, with Figure 13A representing the visual assessment of increasing hCG concentrations, Figure 13B representing the visual assessment of the brightness at the test / control line compared to the background, with the brightness shown on the Y-axis being the ratio of the test / control line divided by the background, and Figure 13C representing the electrochemical assessment for the same test strips of Figure 13B, with the current ratio on the Y-axis being the ratio of the test / control line current divided by the background. All data points were performed in duplicate (N=3), with symbols representing the mean and error bars showing the standard deviation. For data points where no error bar is present, the symbol is larger than the error bar.

[0135] The test was performed and analyzed visually using ImageJ software by reading the brightness of the test / control line compared to the background shown in Figures 13A and 13B. Due to the non-zero brightness of the background, the signal is reduced to a minimum of 0.6 AU in the positive hCG samples. When the same test is evaluated electrochemically, a nearly complete drop in signal to zero is observed in samples with high hCG concentrations, as shown in Figure 13C. Similar intelligence can be observed in both cases, indicating that the two methods can be used interchangeably, with electrochemical detection avoiding the subjective decision-making of the LFT user.

[0136] The technology described herein allows lateral flow tests to be performed with improved accuracy and allows results to be measured objectively using signal enhancers that precipitate into labeling particles that allow the test line to be measured using an electrode array. Thus, the technology described herein improves the efficacy and usefulness of LFTs.

[0137] It will be apparent to those skilled in the art that many improvements and modifications can be made to the exemplary embodiments described above without departing from the scope of the present technology.

Claims

1. A lateral flow test device for performing a lateral flow test on a liquid sample, comprising: a test strip, the test strip comprising: a nitrocellulose membrane having a first capture reagent disposed on a first surface along a test line, the first capture reagent configured to capture a first analyte of the liquid sample; a sample pad disposed at a first end of the nitrocellulose membrane configured to receive the liquid sample; a labeling reagent comprising a plurality of labeling molecules disposed on the first surface at a location between the sample pad and the test line, the labeling molecules configured to bind to the first analyte; The lateral flow test device comprises: an electrode array disposed over the nitrocellulose membrane and configured to apply an electrical potential across the first surface; and a housing enclosing the test strip and the electrode array, the housing including a support element configured to support the electrode array and the test strip in an initial position in which the electrode array is spaced from the test strip; A device comprising:

2. The device of claim 1 , wherein the housing comprises a compressible portion configured to displace the electrode array from the initial position when the compressible portion is compressed.

3. The device of claim 2 , wherein the support element comprises a pivot coupled to the electrode array to allow the electrode array to rotate.

4. 4. The device of claim 3, wherein the support element further comprises a pin coupled to a rear end of the electrode array, the pin extending through a linear notch in the housing, and movement of the pin rotates the electrode array about the pivot.

5. The device of claim 3 , wherein the housing further comprises a wedge element, wherein upon compression, the wedge element compresses the compressible portion to cause the electrode array to rotate about the pivot.

6. The device of claim 1 , wherein the electrode array comprises a core formed of an insulating material and a plurality of electrodes disposed on the core.

7. The device of claim 6 , wherein the core and / or the electrode array are formed of a flexible material.

8. The device of claim 1 , wherein the electrode array has openings that allow the electrode array to flex.

9. The device of claim 1 , wherein the housing comprises an angled portion configured to support the test strip in an angled position.

10. 10. The device of claim 1, further comprising a wicking pad having high liquid absorption capacity disposed at a second end of the nitrocellulose membrane and configured to provide a capillary force to drive the liquid sample from the sample pad along the nitrocellulose membrane.

11. 11. The device of claim 1, further comprising a conjugate pad disposed on the first surface of the nitrocellulose membrane impregnated with the labeling reagent.

12. 12. The device of claim 1, wherein the nitrocellulose membrane further comprises a second capture reagent disposed on the first surface along a control line, the second capture reagent configured to capture the labeled molecule.

13. 13. The device of claim 12, wherein the plurality of electrodes comprises one or more working electrodes positioned to overlap the test line and a background portion of the nitrocellulose membrane, the one or more working electrodes configured to apply an electrical potential to the first surface of the nitrocellulose membrane.

14. 14. The device of claim 13, wherein the plurality of electrodes comprises one or more counter electrodes each positioned opposite a corresponding working electrode to complete an electrical circuit.

15. 15. The device of claim 14, wherein the plurality of electrodes comprises one or more reference electrodes configured to act as reference points for the potential applied by the working electrode.

16. 16. The device of claim 15, wherein the or each reference electrode is positioned adjacent to a counter electrode.

17. 17. The device of claim 14, 15 or 16, wherein at least one working electrode-counter electrode pair is disposed in a recess in the electrode array such that the recess forms a sampling well when contacted with the test strip.

18. 18. The device of claim 1 , wherein the electrode array is disposed on a printed circuit board (PCB).

19. 20. The device of claim 18, wherein the PCB comprises multiple conductive layers.

20. 20. The device of claim 19, wherein one or more of the conductive layers are covered with a solder mask layer.

21. 21. The device of claim 1, wherein the labeling molecules comprise molecules that exhibit catalytic activity, such as gold nanoparticles.

22. 22. The device of any of claims 1 to 21, further comprising a signal enhancer disposed on the first surface of the nitrocellulose membrane or on the sample pad, configured to precipitate on the surface of the labeled molecule for electrochemical detection of the labeled molecule.

23. 23. The device of claim 22, further comprising an additional conjugate pad disposed on the first surface of the nitrocellulose membrane impregnated with the signal enhancer.

24. 24. The device of claim 22 or 23, wherein the signal enhancer comprises a plurality of metal ions, optionally derived from a silver salt.

25. 25. The device of claim 24, wherein the electrode array comprises a plurality of gold-plated electrodes, the plurality of gold-plated electrodes disposed on the nitrocellulose membrane initiates reduction of silver ions derived from the silver salt of the plurality of gold-plated electrodes.

26. 26. The device of claim 25, wherein the plurality of metal ions is provided in combination with a reducing agent.

27. 27. The device of claim 26, wherein the reducing agent is a hydroquinone solution.

28. 28. A device as claimed in any preceding claim, wherein the device is provided with a unique identifier, which may comprise a barcode, a QR code or a combination thereof.

29. 29. The device of any of claims 1 to 28, wherein the housing includes a window above the test line for visual evaluation of the test line.

30. 30. The device of any of claims 1-29, further comprising a plurality of test strips incorporated within the housing.

31. an integrated electronic reader configured to generate electrical signals via the electrode array to drive an electrochemical reaction in the test strip and to receive resultant electrochemical signals from the test strip via the electrode array; and 31. The device of claim 1, further comprising: a power source configured to provide power to the electrode array via the electronic reader.

32. 32. The device of claim 31, further comprising a communication interface configured to communicate with an external electronic device for transmitting the received electrochemical signal.

33. 33. The device of claim 31 or 32, wherein the electrode array comprises at least one pair of electrodes and the resultant electrochemical signal comprises a current across the at least one pair of electrodes, or a change in conductance, resistance, capacitance or impedance of an electrical circuit between the at least one pair of electrodes, or a combination thereof.

34. 34. The device of any of claims 1-33, wherein the first capture reagent is further disposed on the first surface of the nitrocellulose membrane along a second test line at a different concentration than the first test line.

35. 35. The device of any of claims 1 to 34, wherein the nitrocellulose membrane further comprises a third capture reagent disposed on the first surface along a third test line, the third capture reagent configured to capture a third analyte of the liquid sample different from the first analyte.

36. 36. The device of any of claims 1 to 35, wherein the electrode array comprises a plurality of corresponding electrode pairs, the plurality of electrodes being arranged such that a corresponding electrode pair of the plurality covers the background of the nitrocellulose membrane, a corresponding electrode pair covers the first test line, and a corresponding electrode pair of the plurality may optionally cover each of the control line and / or the second test line and / or the third test line.

37. 1. An electronic reader for reading a result from a lateral flow test device, the lateral flow test device comprising a compressible portion, the electronic reader comprising: a receiving portion for receiving the lateral flow test device comprising a release mechanism configured to compress the compressible portion of the lateral flow test device; and an electronic reader comprising: a communication port configured to electrically couple to the lateral flow test device to generate electrical signals to drive electrochemical reactions within the lateral flow test device and to receive resultant electrochemical signals indicative of the electrochemical reactions;

38. 38. The electronic reader of claim 37, further comprising a power connection for receiving power from an external source.

39. 39. The electronic reader of claim 37 or 38, wherein the electronic reader further comprises an integral power supply.

40. 40. An electronic reader according to any of claims 37 to 39, wherein the electronic reader further comprises an optical reader for reading the unique identifier of the lateral flow test device.

41. 41. An electronic reader according to any of claims 37 to 40, wherein the release mechanism comprises a linear actuator configured to compress the compressible portion of the lateral flow test device upon insertion of the lateral flow test device into the receptacle.

42. 42. The electronic reader of any of claims 37 to 41, wherein the electronic reader further comprises a timer configured to count down a measurement time defined by the length of time required to measure the result.

43. 1. A system for performing a lateral flow test on a liquid sample, comprising: A lateral flow test device, comprising: a test strip, the test strip comprising: a nitrocellulose membrane having a first capture reagent disposed on a first surface along a test line, the first capture reagent configured to capture a first analyte of the liquid sample; a sample pad disposed at a first end of the nitrocellulose membrane configured to receive the liquid sample; a labeling reagent comprising a plurality of labeling molecules disposed on the first surface at a location between the sample pad and the test line, the labeling molecules configured to bind to the first analyte; an electrode array disposed over the nitrocellulose membrane and configured to apply an electrical potential across the first surface; and a housing enclosing the test strip and the electrode array, the housing including a support element configured to support the electrode array and the test strip in an initial position in which the electrode array is spaced from the test strip; Equipped with the lateral flow test device comprising an electronic reader, the electronic reader comprising: a receptacle for receiving the lateral flow test device comprising a release mechanism configured to compress the housing of the lateral flow test device to bring the electrode array into contact with the test strip in a ready-to-read position; and a communications port electrically coupled to the electrode array of the lateral flow test device and configured to generate electrical signals at the electrode array to drive an electrochemical reaction of the test strip and to measure resultant electrochemical signals from the electrode array indicative of the electrochemical reaction of the test strip; A system comprising:

44. 44. The system of claim 43, wherein the housing comprises a compressible portion configured to displace the electrode array from the initial position when the compressible portion is compressed.

45. 45. The system of claim 44, wherein the release mechanism is activated by the action of inserting the lateral flow test device into the receptacle.

46. 46. ​​The system of claim 45, wherein the support element comprises a pivot coupled to the electrode array, and the release mechanism comprises a protruding element configured to protrude into the compressible portion to rotate the electrode array when the lateral flow test device is inserted into the receptacle.

47. 47. The system of claim 46, wherein the protruding elements are biased toward a centerline of the receiving portion by an elastic element, and the protruding elements are released into the compressible portion by the elastic element when the lateral flow test device is inserted into the receiving portion.

48. the support element includes a pivot coupled to the electrode array to allow the electrode array to rotate and a pin coupled to a rear end of the electrode array, the pin extending through a linear cutout in the housing; 46. ​​The system of claim 45, wherein the release mechanism comprises a guide groove on an inner surface of the receptacle, the guide groove being inclined toward a centerline of the receptacle and configured to receive the pin to guide the pin toward the centerline when the lateral flow test device is inserted into the receptacle to rotate the electrode array.

49. 46. ​​The system of claim 45, wherein the support element comprises a pivot coupled to the electrode array, the housing further comprises a wedge element, and the release mechanism comprises a surface against which the wedge element presses when the lateral flow test device is inserted into the receptacle, such that the wedge element compresses the compressible portion and rotates the electrode array.

50. 46. ​​The system of claim 45, wherein the release mechanism comprises a linear actuator configured to extend toward a centerline of the receiver upon actuation.

51. 51. The system of any of claims 44 to 50, wherein the release mechanism is configured to compress the compressible portion with a pressure in the range of 1N to 50N.

52. 52. A system according to any of claims 43 to 51, wherein the lateral flow test device comprises a port having an exposed portion of the electrode array, the port being arranged to mate with the communications port of the electronic reader.

53. 1. A method of performing a lateral flow test on a liquid sample using a lateral flow test strip, the lateral flow test strip comprising: a nitrocellulose membrane having a capture reagent disposed on a first surface along a test line, the capture reagent configured to capture a predetermined analyte of the liquid sample; a sample pad disposed on one end of the nitrocellulose membrane configured to receive the liquid sample; and a plurality of labeling molecules disposed on the first surface between the sample pad and the test line, the labeling molecules comprising labeling reagents configured to bind to the predetermined analyte. The method is applying an electrical potential across the first surface via an electrode array disposed on the nitrocellulose membrane to drive an electrochemical reaction of the lateral flow test strip; and and measuring a resultant electrochemical signal from the lateral flow test strip passing through the electrode array.

54. 54. The method of claim 53, further comprising depositing the liquid sample on the sample pad. Law.

55. 55. The method of claim 53 or 54, wherein the electrode array comprises a plurality of corresponding electrode pairs, the method further comprising overlaying corresponding electrode pairs on the nitrocellulose membrane background and further overlaying corresponding electrode pairs on the test lines, and determining a difference in the resulting electrochemical signal between the electrode pairs overlaid on the background and the electrode pairs overlaid on the test lines.

56. 56. The method of any of claims 53-55, further comprising washing the nitrocellulose membrane with a buffer through the sample pad prior to applying a potential.

57. 56. The method of any of claims 53 to 55, further comprising depositing a signal enhancer solution containing a plurality of metal ions onto the nitrocellulose membrane through the sample pad prior to applying an electrical potential.

58. 58. The method of claim 57, wherein the plurality of metal ions is derived from a silver salt.

59. 59. The method of claim 57 or 58, further comprising an activation step of combining the signal enhancer solution with a developing agent.

60. 60. The method of claim 59, wherein the developing agent comprises a reducing agent to enable spontaneous reduction of the plurality of metal ions on the surface of the labeled molecule.

61. 61. The method of any of claims 57 to 60, further comprising measuring a distribution of metal resulting from at least a portion of the plurality of metal ions precipitated on the surface of the labeled molecule along the first surface of the nitrocellulose membrane to determine a concentration of the labeled molecule along the first surface.

62. 62. The method of claim 61, further comprising measuring the distribution of metal ions along the nitrocellulose membrane that have not reacted with the labeling molecule.

63. 63. The method of any of claims 53 to 62, wherein measuring a resulting electrochemical signal from the lateral flow test strip comprises performing a potential sweep across the nitrocellulose membrane via the electrode array.

64. 64. The method of claim 63, further comprising performing the potential sweep at least two times, a first cycle of the potential sweep resetting the electrochemical reaction and a second cycle of the potential sweep measuring the electrochemical signal.

65. 65. The method of claim 63 or 64, wherein detection of a current peak indicates a local concentration of the metal ion.

66. 66. The method of claim 63, 64 or 65, wherein detection of the absence of a current peak at or near the test line indicates the presence of the predetermined analyte in the liquid sample.

67. 67. The method of any of claims 53 to 66, wherein measuring the resulting electrochemical signal from the lateral flow test strip is based on a voltammetric, amperometric, potentiometric, or impedance-based method, or a combination thereof.

68. 68. The method of any of claims 53-67, wherein measuring the resulting electrochemical signal from the lateral flow test strip is performed using a linear sweep voltammetry method.

69. 69. The method of claim 68, wherein the linear sweep voltammetry method comprises sweeping a potential from a minimum potential to a maximum potential at a predetermined rate relative to a reference potential, the predetermined rate may be in the range of 10 mV per second to 1000 mV per second.

70. 70. The method of claim 69, wherein the minimum potential relative to the reference potential is in the range of -1V to -0.1V, preferably -0.5V.

71. 71. The method of claim 69 or 70, wherein the maximum potential relative to the reference potential is in the range of +0.1V to +1V, preferably +0.5V.

72. 72. The method of any of claims 69 to 71, wherein the linear sweep voltammetry method comprises repeating the potential sweep a predetermined number of times.

73. 73. A method according to any of claims 69 to 72, wherein the linear sweep voltammetry method comprises a depositing step of applying a low potential for a predetermined period of time before performing the potential sweep and / or applying a high potential for a predetermined period of time after performing the potential sweep.

74. 74. The method of claim 73, wherein the low potential is in the range of -1 V to -0.1 V relative to the reference potential and / or the high potential is in the range of 0.1 V to 1 V relative to the reference potential.

75. 75. The method of claim 73 or 74, wherein the predetermined period of time is one minute or less.

76. 76. The method of any of claims 53-75, wherein measuring the resulting electrochemical signal from the lateral flow test strip is performed using a pulse-based electrochemical technique.

77. 77. The method of claim 76, wherein the pulse-based electrochemical technique comprises differential pulse voltammetry or square wave voltammetry.

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