Device for detecting molecules

Abstract

A lateral flow test device is disclosed in which a conjugation pad comprises a movable conjugation analyte comprising one or more analyte molecules conjugated to a detectable label. The first test zone comprising an immobilized analyte binding molecule defining a first binding site; or the conjugate pad comprises a movable analyte binding molecule and the first test zone comprises an immobilized capture molecule for immobilizing the analyte binding molecule, wherein the immobilized capture molecule defines a first binding site; or the conjugation pad comprises a movable analyte binding molecule and the first test region comprises an immobilized analyte binding molecule defining a first binding site. The second test region comprises an immobilized conjugated analyte binding molecule that binds the conjugated analyte and does not bind the unconjugated analyte molecule, and defines a second binding site. The number of molecules of the conjugated analyte is less than or equal to the number of second binding sites. Also disclosed are methods of detecting the presence of an analyte molecule in a test sample and diagnosing a disease or condition, a computer-implemented method of detecting the presence of an analyte molecule in a test sample, and a kit.

Description

[0001] Invention Field

[0002] This invention relates to a method and apparatus for performing lateral flow testing, and a kit comprising said apparatus. Specifically, the apparatus and method are used for performing quantitative lateral flow testing. Technical Background

[0004] Lateral flow testing (LFT) can be used to detect a variety of molecules and aids in the rapid diagnosis of diseases. LFTs are typically constructed from nitrocellulose membranes, with different zones of the membrane immobilizing biomolecules. Figure 1 a) When a sample is applied to a sample pad, it dissolves the conjugates deposited on the pad and migrates along the membrane via capillary action. As the sample migrates along the membrane, the conjugates or target analytes are captured by binding molecules (typically proteins) in the test area (e.g., a test line), forming a visible colored band indicating the presence of the target analyte in the sample.

[0005] Lateral flow assays (LFTs) come in several types, the two most common being sandwich assays and competitive assays. In a sandwich assay, the conjugation pad contains mobile, labeled conjugated molecules containing antibodies specific to the target analyte conjugated to the detectable label; the test line contains immobilized antibodies specific to the target analyte; and the control line contains immobilized secondary antibodies that bind to the detectable conjugation (…). Figure 1 a). The test sample is applied to the sample pad of the test device, where the target analyte in the test sample forms an analyte-conjugate complex with a movable detectable conjugate on the conjugate pad. This analyte-conjugate complex moves from the conjugate pad to the test line, where it is captured by an anti-analyte antibody immobilized on the test line, thus producing a visible positive reaction indicator line. Figure 1 b). Any detectable conjugate that does not bind to the target analyte will bind to the control line. Figure 1 (b) However, sandwich assays are only suitable for large molecular analytes with two or more binding sites, which allow both the conjugate and the anti-analyte antibody to bind to the first test region. Sandwich assays are not suitable for small molecules because small molecules cannot bind to two other molecules simultaneously.

[0006] Competitive assays are designed for small molecules, which typically have only one binding site. In a competitive assay, the conjugation pad contains a labeled conjugate that specifically binds to the target analyte in the test sample; and the test line contains immobilized trap molecules that specifically bind to the labeled conjugate. When a test sample without the target analyte is applied to the sample pad of the assay device, the labeled conjugate flows upward along the membrane and binds to the test line, thereby generating a detectable signal on the test line. When the target analyte is present in the test sample, the labeled conjugate binds to the target analyte, thereby inhibiting the binding of the labeled conjugate to the immobilized trap molecules on the test line. Therefore, if no detectable signal is present on the test line, it indicates the presence of the target analyte in the sample.

[0007] Both the sandwich method and the competitive method of flow finite element transfer (LFT) include a control line following the test line in the sample flow direction to indicate whether the flow of the marked conjugate is correct. Therefore, in a functionally feasible LFT, the control line will always produce a detectable signal regardless of the test results. In other words, the signal strength of the control line is independent of the signal strength of the test line; its only function is to indicate that the LFT is feasible.

[0008] Both types of LFT can be used to provide quantitative results to quantify the concentration of an analyte in a sample. For example, Insudex® uses a sandwich assay, quantifying by the intensity of the visible line in the first test area. Higher intensity indicates a higher concentration of the analyte in the sample. Once the test line develops color, the user must analyze the test line using a dedicated reader.

[0009] US 8,137,984 discloses a competitive LFT device for detecting the presence of caffeine. The device uses a conjugate comprising an anti-caffeine antibody and a secondary antibody conjugated to the particle; the test line comprises a molecule that binds to the anti-caffeine antibody. Therefore, the molecule on the first test line competes with caffeine in the test sample for binding to the anti-caffeine antibody. The device includes a control line comprising an antibody that binds to the secondary antibody of the conjugate.

[0010] US 2022 / 0146507 discloses a semi-quantitative barcode-type sandwich LFT device having two test lines for detecting β-trace protein (βTP). Each test line includes a binding moiety that binds to a complex formed by βTP and a detectable conjugate, wherein the detectable conjugate comprises a detectable label and a βTP-binding molecule (e.g., an antibody).

[0011] One drawback of competitive LFT is that the observed signal intensity is inversely proportional to the concentration of the target analyte present (Delmulle et al., J. Agric. Food Chem., 2005, 53, 3364–3368). This means that a signal can be detected in all samples except those with the highest concentration. This leads to inconsistent interpretation of results, requires extensive calibration to obtain accurate quantification, and results in low sensitivity. Furthermore, format-dependent negative readings indicate that the presence of the target analyte is detected in the region of lowest sensitivity in the dose-response curve (i.e., near the test line saturation point (i.e., maximum signal intensity)). This makes it difficult to improve the sensitivity of competitive LFT.

[0012] Both types of LFT (sandwich assay and competitive assay) have a drawback: the first test zone may become saturated when used for quantitative assays. In the sandwich assay, when the analyte in the test sample is present at a concentration higher than that of the immobilized antibody, the immobilized antibody in the first test zone becomes saturated with the conjugated analyte, resulting in saturation. This means that when a certain amount of analyte in the test sample is exceeded, the signal intensity (indicating the presence of the analyte) in the first test zone will not increase further. In the competitive assay, saturation occurs when the amount of target analyte in the sample is equal to or greater than the amount of analyte immobilized on the test line.

[0013] One way to prevent saturation is to increase the amount of antibody immobilized in the first test region of the sandwich LFT, thereby increasing the concentration range measurable by the lateral flow assay. However, this negatively impacts the quantitative resolution of the LFT.

[0014] Specifically, in sandwich and competitive LFT assays, quantification is achieved by inferring the concentration of the analyte in the test sample from the visual reaction generated by the conjugate (i.e., secondary inference). However, the increased detection range of LFT (i.e., the increased amount of antibody immobilized in the first test region of the sandwich assay) means that any change in the visual reaction represents a smaller percentage of the visual reaction. For example, in a sandwich assay, the binding of an analyte-conjugate complex to a first test region with 100 antibodies results in a 1% change in the intensity of the visible band in the first test region. However, the binding of an analyte-conjugate complex to a first test region with 1000 antibodies results in a 0.1% change in the intensity of the visible band. Therefore, the increased detection range of quantitative lateral flow assays is accompanied by a decrease in resolution, thereby reducing the accuracy of lateral flow assays.

[0015] Quantitative LFT is also prone to errors in reading the intensity of the visible bands generated by the binding (or non-binding) of the conjugate in the first test zone. Specifically, the deterioration of the antibody used in the assay can lead to errors, causing sandwich LFT to be biased towards negative results and competitive LFT to be biased towards positive results, because the antibody bound to the detectable marker may not bind to the test line. Furthermore, the LFT reader itself may have systematic errors, or errors may occur due to ambient light affecting the signal intensity in the test zone. For example, Insudex® detects C-peptide (used for diabetes diagnosis) in the range of 0.17 ng / ml to 12 ng / ml. When the C-peptide concentration is approximately 80 ng / ml, a hook effect occurs, meaning that false low results are caused when the analyte concentration is too high. When measuring a C-peptide concentration of approximately 0.17 ng / ml, the variability of Insudex® is approximately 0.02 ng / ml, equivalent to a coefficient of variation of approximately 11%, meaning that quantitative readings may lead to misdiagnosis and that the product's application in large-scale screening is limited.

[0016] Another drawback of the competitive assay LFT is that its quantitative capability is severely limited by the presence of a control line. The presence of a control line means that an excess of the labeled conjugate must be added to the sample to allow sufficient labeled conjugate to flow towards the control line. However, this also means the loss of information about the amount of conjugate not bound to the test line, thus hindering the assay's ability to accurately quantify outlying concentrations of the target analyte.

[0017] Although ELISA tests can be used to quantify target analytes in samples, they are time-consuming, costly, and require specialized equipment and personnel.

[0018] Le et al. (“Dual recognition element lateral flow assay toward multiplexstrain specific influenza virus detection”, Anal. Chem, 2017, 89, 6781-6786) disclosed a lateral flow assay for detecting specific influenza virus strains, comprising a nucleic acid aptamer with an antibody. The lateral flow assay device has a first test line containing streptavidin and a control line containing an anti-mouse antibody. The antiviral antibody is conjugated to gold nanoparticles, while the aptamer is conjugated to biotin. The aptamer can be selected to be highly strain-specific. When the test sample contains the target virus, both the aptamer and the conjugated antibody bind to the virus, and the biotin conjugated to the aptamer enables the complex to bind to the streptavidin on the test line. Subsequently, the gold nanoparticles are detected on the test line. Regardless of the presence of the target virus, the antibody conjugated to the gold nanoparticles binds to the anti-mouse antibody on the control line. However, this lateral flow assay is not quantitative.

[0019] Therefore, there is a need for quantitative lateral flow assays to more accurately quantify target analytes in samples. In particular, there is a need for quantitative LFTs that can address saturation and resolution issues and reduce signal intensity readout errors. Furthermore, such LFTs can reduce the demand for ELISA assay kits, which, as mentioned above, are expensive and time-consuming. Summary of the Invention

[0020] This invention stems from a surprising discovery: by preloading a known amount of a detectable conjugated analyte into a lateral flow assay, the concentration of the target analyte can be calculated based on the signal generated by the detectable conjugated analyte; and by having a first test region where both conjugated and target analyte molecules can bind, and a second test region where only conjugated analyte molecules can bind, more precise quantitative or semi-quantitative lateral flow assays can be achieved. The detectable conjugated analyte comprises analyte molecules conjugated to a detectable label. The number of binding sites in the second test region is equal to or greater than the number of conjugated analyte molecules. When target analyte molecules are absent from the test sample, the conjugated analyte binds to the first test region and optionally to the second test region, thereby generating a detectable signal in the test region bound to the conjugated analyte. When target analyte molecules are present in the test sample, competition between the target analyte molecules and the conjugated analyte reduces the amount of conjugated analyte bound to the first test region, thereby reducing the detectable signal in the first test region. Unbound conjugated analytes (i.e., not bound to the first test region) can then bind to the second test region, thereby increasing the detectable signal intensity in the second test region (from no signal to a signal, or to a signal intensity higher than that without the target analyte). Therefore, the competition between the target analyte molecules in the sample and the conjugated analytes pre-loaded into the device means that the signal intensity in the first test region is inversely correlated with the signal intensity in the second test region (i.e., the signal intensities in the first and second test regions are interdependent), thus grading the response of the test regions relative to the amount of conjugated analyte. Therefore, the signal intensity in the second test region helps to validate the signal intensity in the first test region.

[0021] This invention also stems from a surprising discovery: more accurate quantitative lateral flow testing can be achieved when the signal intensity of the first test zone is inversely correlated with that of the second test zone. Specifically, the signal intensities of the first and second test zones can be compared to obtain a signal intensity ratio, which can be used to more accurately calculate the concentration of the target analyte in the sample. This ratio also means a reduction in systematic errors caused by the device used to read the signal intensity of the test zones, as well as errors caused by ambient light. In contrast, conventional quantitative LFT only considers the presence of a detectable signal in the first test zone when calculating the concentration of the target analyte in the sample, without considering the presence of a detectable signal in the second test zone. In conventional LFT, the second test zone is used only as a control to indicate that its conjugate is functional and has migrated along the test strip. However, in the lateral flow testing device of this invention, changes in signal intensity are measured in both the first and second test zones, thus providing two parameters for calculating the concentration of the target analyte in the sample.

[0022] This invention also stems from a surprising discovery: by providing the ratio of the signal intensity of the first test region to that of the second test region, more accurate quantitative lateral flow testing can be achieved. Specifically, by calculating the signal intensity ratio of the first and second test regions and using this ratio to calculate the concentration of the target analyte in the sample (especially when the sum of the signal intensities of the first and second test regions is considered 100%), errors caused by the background environment can be reduced. Furthermore, if the signal intensity ratio is calculated multiple times and an average is calculated using these multiple ratios, the error can be further reduced.

[0023] Quantitative errors caused by ambient light can be reduced by comparing the average ratio of signal intensities in the first and second test areas, calculated based on predicted signal intensities, with the measured ratio of signal intensities in the first and second test areas. Specifically, the signal intensity in the first test area is first measured, and then the signal intensity in the second test area is predicted based on the sum of the signal intensities being 100% (e.g., the maximum signal intensity), thus determining a first signal intensity ratio between the first and second test areas. Next, the signal intensity in the second test area is measured, and the signal intensity in the first test area is predicted based on the same basis, thus determining a second signal intensity ratio between the first and second test areas. Then, the average ratio of signal intensities is determined based on the first and second ratios. Finally, the ratio of the measured signal intensities is determined. This measured ratio of signal intensities can be used for ratiometric calibration of the average ratio of signal intensities. If the average ratio of signal intensities does not match the measured ratio, the average ratio of signal intensities can be re-determined using new measurements of the signal intensities recalibrated based on an adjusted scaling (or calibration) factor.

[0024] Therefore, in a first aspect of the invention, a lateral flow testing apparatus is provided, comprising a solid support structure including a sample receiving area, a ferrule pad, a first testing area, and a second testing area, wherein the solid support structure is configured to allow liquid to flow sequentially from the sample receiving area through the ferrule pad to the first testing area, and then to the second testing area, wherein...

[0025] i) The conjugation pad includes a movable conjugation analyte comprising one or more analyte molecules conjugated to a detectable marker; ii)a) The first test region contains an immobilized analyte-bound molecule defining a first binding site. b) The conjugation pad contains a movable analyte-binding molecule, and the first test region contains an immobilized trapping molecule for immobilizing the analyte-binding molecule, wherein the immobilized trapping molecule defines a first binding site, or c) The conjugation pad contains a movable analyte-binding molecule, and the first test region contains an immobilized analyte-binding molecule defining a first binding site. iii) The second test region contains an immobilized conjugated analyte-binding molecule that binds to the conjugated analyte but not to the unconjugated analyte molecule, and defines a second binding site; In this case, the number of molecules of the conjugated analyte is less than or equal to the number of the second binding sites.

[0026] Conveniently, the number of molecules in the conjugated analyte is less than the number of second binding sites on the second test region.

[0027] Advantageously, the number of first binding sites is equal to the number of molecules of the conjugated analyte.

[0028] Preferably, the ratio of i) conjugated analyte, ii)a) immobilized analyte-binding molecule and iii) conjugated analyte-binding molecule is 1:1:1; or the ratio of i) conjugated analyte, ii)b) mobile analyte-binding molecule, ii)b) trapping molecule and iii) conjugated analyte-binding molecule is 1:1:1:1.

[0029] Conveniently, the detectable markers are nanoparticles, preferably gold nanoparticles.

[0030] Preferably, each analyte molecule of the conjugated analyte is bound to a detectable label via a linker molecule, preferably wherein the linker molecule is biotin-BSA.

[0031] Advantageously, the conjugated analyte binding molecule is specific to and can bind to the linker molecule, preferably wherein the conjugated analyte binding molecule is avidin, streptavidin, or polystreptavidin.

[0032] Conveniently, one of the analytes and analyte-binding molecules is an antibody that is specific to another of the analytes and analyte-binding molecules.

[0033] Preferably, the analyte-binding molecule is an anti-analyte antibody.

[0034] In a second aspect of the present invention, a method for detecting the presence of analyte molecules in a test sample is provided, the method comprising: i) Provide a lateral flow testing apparatus according to the first aspect. ii) Apply the test sample to the sample receiving area of ​​the lateral flow test device, allowing the test sample to migrate to the conjugation pad and mix with the conjugated analyte, and optionally mix with mobile analyte-binding molecules.

[0035] iii) Allows the test sample and conjugated analytes, and optionally mobile analyte-bound molecules, to sequentially migrate to the first and second test regions and contact the immobilized molecules in each of the first and second test regions. iv) Detect the signals in the first test area and the second test area respectively, wherein changes in the signal intensity in both the first test area and the second test area indicate the presence of analyte molecules in the test sample.

[0036] Preferably, the signal is an optical signal.

[0037] Advantageously, the method also includes: v) quantifying the concentration of analyte molecules in the sample based on the ratio of the signal intensity of the first test region to the signal intensity of the second test region.

[0038] Preferably, in step iv), the decrease in signal intensity in the first test area and the increase in signal intensity in the second test area indicate the presence of analyte molecules in the test sample.

[0039] Conveniently, the signal strength is measured as a percentage of the maximum signal strength, wherein step iv) includes measuring the signal strength of one of the first test area and the second test area, and predicting the signal strength of the other of the first test area and the second test area based on the fact that the sum of the signal strengths of the first test area and the second test area is 100% of the maximum signal strength.

[0040] Preferably, step iv) includes: a) Measure the signal strength of the first test area, and predict the signal strength of the second test area based on the fact that the sum of the signal strengths of the first test area and the second test area is 100% of the maximum signal strength, thereby calculating the first measured signal strength: predicted signal strength ratio.

[0041] b) Measure the signal strength of the second test area, and predict the signal strength of the first test area based on the sum of the signal strengths of the first and second test areas being 100% of the maximum signal strength, thereby calculating the second measured signal strength: predicted signal strength ratio.

[0042] c) Based on the first and second measured signal strength: predicted signal strength ratio, calculate the average signal strength of the first test area and the second test area respectively to determine the average first test area signal strength: second test area signal strength ratio.

[0043] d) Calculate the ratio of the measured signal strength in the first test area to that in the second test area. e) Compare the ratio of the average signal strength in the first test area to the signal strength in the second test area, and the ratio of the measured signal strength. f) When the ratio of average signal intensity in the first test region to signal intensity in the second test region matches the ratio of measured signal intensity, the concentration of analyte molecules in the sample is quantitatively determined based on this ratio. Steps a) and b) can be performed in any order.

[0044] Advantageously, during measurement, the signal strength of the first test area and the signal strength of the second test area are scaled using a calibration factor; and step iv) further includes: g) When the ratio of average signal strength in the first test area to signal strength in the second test area does not match the ratio of the measured signal strength calculated in step d, adjust the calibration factor and repeat steps a to f using the adjusted calibration factor.

[0045] When scaling with a calibration factor, a default or initial calibration factor can be used for one or more initial intensity measurements before adjusting the calibration factor. This default or initial calibration factor can be equal to 1 (however, other values ​​can also be used). When scaling with a calibration factor equal to 1, the scaled intensity will be equal to the unscaled intensity.

[0046] Conveniently, adjusting the calibration factor includes: Determine whether the sum of the measured signal strengths in the first test area and the second test area is greater than the maximum signal strength; In response to the sum of the measured signal intensities in the first and second test areas being greater than the maximum signal intensity, the calibration factor is reduced; In response to the fact that the sum of the measured signal strengths in the first and second test areas does not exceed the maximum signal strength, the calibration factor is increased.

[0047] Preferably, at least step a) of step iv) is performed using an optical signal reader, preferably a smartphone.

[0048] Test samples can be obtained from human, animal or plant individuals, environmental samples, or food or beverage samples.

[0049] In a third aspect of the invention, a method for diagnosing a disease or condition is provided, the method comprising performing the method of the second aspect, wherein the test sample is obtained from a human, animal, or plant individual.

[0050] In a fourth aspect of the invention, a computer-implemented method for detecting the presence of analyte molecules in a test sample is provided, the method comprising: After the test sample is applied to the lateral flow test device, the signal strength measurement values ​​of the first test area and the second test area of ​​the lateral flow test device are obtained according to the first aspect. Determine whether the signal strength in both the first and second test areas changes; In response to the determination that the signal intensity in both the first and second test regions has changed, an indication of the presence of analyte molecules in the test sample is output.

[0051] Acquiring signal strength measurements may involve receiving signal strength measurements from an external system. For example, this method can be performed by a first computing system. Signal strength measurements can be measured by a second computing system (e.g., a mobile device, a light sensor, or a camera). Signal strength measurements can be acquired by the second computing system and sent to the first computing system for further analysis to determine whether the signal strength in both the first and second test areas has changed. Alternatively, signal strength measurements can be determined by the first computing system from one or more light measurements in each of the first and second test areas and / or by displaying one or more images of the first and second test areas of the lateral flow testing device. One or more light measurements and / or one or more images can be acquired by the first computing system (e.g., by using a light sensor and / or a camera). Alternatively, one or more light measurements and / or one or more images can be acquired by the second computing system (e.g., a mobile device, a light sensor, or a camera) and sent to the first computing system to determine whether the signal strength in both the first and second test areas has changed.

[0052] Preferably, determining whether the signal strength in both the first test area and the second test area changes includes: In response to a signal strength in one of the first and second test areas being less than the maximum signal strength, it is determined that a change exists in one of the first and second test areas; and In response to the signal strength of the other of the first test area and the second test area being greater than the minimum signal strength, it is determined that there is a change in the other of the first test area and the second test area.

[0053] Conveniently, the signal strength measurement is acquired from one or more light measurement values ​​from each of the first and second test areas, and preferably, the signal strength measurement is acquired from one or more images from the first and second test areas of the display lateral flow test device.

[0054] In a fifth aspect of the invention, a kit is provided that includes the lateral flow testing apparatus according to the first aspect.

[0055] In some implementations, the conjugated analyte comprises two analyte molecules or one analyte molecule conjugated to a detectable marker.

[0056] As used herein, the term "immobilization" refers to the retention or fixation of molecules on a relevant part of a device using any suitable method known to those skilled in the art. Immobilized molecules are not released or moved upon contact with a test sample or by the flow of the test sample. Specifically, "immobilized" analyte-binding or trapping molecules are those molecules that are typically retained in the test area in use and thus bind to relevant complementary molecules contained in the liquid in contact with the test area.

[0057] As used herein, the term "mobile" means that the molecules in question can be releasably retained or fixed to a relevant portion of the device before use and released from that portion upon contact with the test sample. The mobile molecules can be displaced from their original retention / fixation point by lateral flow of the test sample.

[0058] As used in this article, the term "binding site" refers to a region or location on which a molecule can be anchored, particularly by complementary binding to a binding or trapping molecule.

[0059] As used herein, the term "unconjugated" refers to an analyte molecule that is not linked to a conjugated molecule. For example, an unconjugated analyte molecule can be an analyte molecule from a test sample.

[0060] As used herein, the term "peptide" refers to a polymer of amino acid residues. This term also applies to amino acid polymers, where one or more amino acid residues are modified residues or non-naturally occurring residues, such as artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers.

[0061] As used in this article, the term "polynucleotide" refers to a polymer of nucleic acid residues, including DNA and RNA.

[0062] The term "antigen" as used in this article refers to a molecule that can elicit an immune response in an individual.

[0063] As used in this article, the term "environmental sample" refers to a sample collected from a natural or man-made environment. For example, an environmental sample may be a water sample taken from a natural river or man-made water source, a soil sample, or a sample swabbed from a natural or man-made surface. Attached Figure Description

[0064] Figure 1 This is a schematic diagram of a typical lateral flow test strip (a) and a schematic diagram of the results obtained using the standard sandwich method (b).

[0065] Figure 2 This is a schematic diagram illustrating a capture ELISA method used to test the binding of BSA-biotin-caffeine conjugate to both anti-caffeine antibody and avidin.

[0066] Figure 3 This is a schematic diagram of an improved LFT design, which has two test lines: an anti-caffeine antibody test line and an avidin test line.

[0067] Figure 4 This is a schematic diagram of the synthesis reaction of the BSA-biotin-caffeine conjugate used in Example 1.

[0068] Figure 5 This is a graph illustrating the inhibition curve of a competitive ELISA using the BSA-biotin-caffeine conjugate. Data points were obtained in triplicate, and the error bars represent the standard deviation of the mean.

[0069] Figure 7 This is a schematic diagram of the initial LFA design, which has two test lines: an anti-mouse antibody test line and an avidin test line.

[0070] Figure 8 A schematic diagram illustrating the working principle of the improved LFT design shows the operation of a caffeine-free sample (a) and a caffeine-containing sample (b), respectively. Figure 8 (c) shows the expected test results when caffeine is absent or present in the sample.

[0071] Figure 9 Photographs of an improved LFT test strip running in buffer with different concentrations of glycerol in the presence (100 µg / ml) or absence (0 µg / ml) of caffeine.

[0072] Figure 10 Photographs of the improved LFT test strips using different concentrations of caffeine. The run buffer was PBS buffer containing 5% (v / v) glycerol, 2% (w / v) BSa, and 0.05% (w / v) Tween-20.

[0073] Figure 11 This is a semi-logarithmic plot, showing... Figure 10 The signal intensities of test lines 1 and 2 of the LFT test strip shown were analyzed using ImageJ software. Three copies of the measurement results were produced.

[0074] Figure 12 Photographs of MFT test strips after one week of storage, run with different concentrations of caffeine solution. The run buffer was PBS buffer containing 5% glycerol, 2% BSA, and 0.05% Tween-20.

[0075] Figure 13 This is a schematic diagram of an LFT design for testing the performance of test strips flowing laterally in buffer solutions.

[0076] Figure 14Photographs of LFT test strips run using synthetic urine containing different concentrations of NHPA. Solution volume: 200 µl.

[0077] Figure 15 ImageJ analysis of the pixel grayscale of each test line in the test strip for 14(a) is shown, as is line intensity of each test line (upper line: streptavidin line; lower line: antibody line) at each NHPA concentration calculated by subtracting the peak minimum from the background (b).

[0078] Figure 16 Photographs of LFT test strips run using synthetic urine containing different concentrations of NHPA. For each concentration, three test strips prepared from the same batch of conjugates were assembled and tested.

[0079] Figure 17 for Figure 16 ImageJ analysis of the signal intensity of antibody lines using different concentrations of NHPA is presented. Each data point is the average of five LFT test strips.

[0080] Figure 18 Photographs of LFT test strips run in the absence of (a) and presence of (b) synthetic urine containing NHPA, and schematic diagrams showing the positions of the analyte binding line (A) and streptavidin line (S).

[0081] Figure 19 Photographs of LFT test strips run with different concentrations of creatinine.

[0082] Figure 20 This is a semi-logarithmic plot, showing... Figure 19 The signal intensities of test lines 1 and 2 of the LFT test strip shown were analyzed using ImageJ software. Three copies of the measurement results were produced.

[0083] Figure 21 A schematic diagram of the sandwich LFT saturation mechanism is shown. There is twice the amount of analyte and anti-analyte antibody on the test line, therefore some analyte cannot bind to the test line, resulting in unquantified analyte.

[0084] Figure 22 A schematic diagram of an exemplary LFT of the present invention is shown, wherein the amount of analyte molecules in the test sample is the same as the amount of the conjugated analyte and the binding site on the first test line.

[0085] Figure 23 A schematic diagram of an exemplary LFT of the present invention is shown, wherein the amount of analyte molecules in the test sample is greater than the amount of binding sites of the conjugated analyte and the first test line.

[0086] Figure 24A schematic diagram of a conventional sandwich LFT is shown, in which the number of analyte molecules in the test sample is increased from 400 (A) to 415 (B), and the corresponding results obtained therefrom (C and D).

[0087] Figure 25 A schematic diagram of an exemplary LFT of the present invention is shown, wherein the number of analyte molecules in the test sample is increased from 400 (A) to 415 (B), and the corresponding results obtained therefrom (C and D).

[0088] Figure 26 A schematic diagram of a calculation system for quantitatively measuring the concentration of analyte molecules in a sample based on the signal intensity of a first and second test region using an LFT, according to one embodiment, is shown.

[0089] Figure 27 This is a graph showing the relationship between the absorbance and concentration of BSA-biotin-creatinine. Data points were collected in triplicate, and the error bars represent the standard deviation.

[0090] Figure 28 This is a graph showing the relationship between the absorbance and concentration of free creatinine. Data points were obtained in triplicate, and the error bars represent the standard deviation. Detailed Implementation

[0091] This invention generally relates to a lateral flow testing device comprising a detectable conjugated analyte and two test regions. The conjugated analyte comprises analyte molecules conjugated to a detectable label. The first test region has one or more first binding sites that bind the conjugated analyte and analyte molecules from a test sample. The second test region has one or more second binding sites that bind the conjugated analyte but not the target analyte. The number of conjugated analyte molecules in the device is less than or equal to the number of second binding sites in the second test region. The signal intensity of the second test region never reaches 100% in the absence of analyte in the test sample, ensuring that the signal intensity of the second test region changes when analyte is present in the test sample.

[0092] solid support structure

[0093] The lateral flow testing apparatus includes a solid support structure on which other components and reagents for the lateral flow test are placed. For example, the solid support structure may be a backing card on which the other components and reagents for the lateral flow test are arranged. The backing card may be housed within a housing. In some cases, the solid support structure itself may comprise a housing, within which the other components and reagents for the lateral flow test are arranged. The solid support structure may be made of any suitable material known to those skilled in the art, such as plastic.

[0094] The solid support structure is configured to allow liquids (e.g., the test sample and any other liquid reagents or components added to the device) to flow sequentially from the sample receiving area to the conjugation pad, then to the first test area, and finally to the second test area. The device is configured to allow the liquid to contact and mix with the conjugated analytes and analyte-bound molecules (when present) on the conjugation pad, and to contact with a variety of molecules in the first and second test areas.

[0095] Sample receiving area

[0096] The sample receiving area is configured to receive a test sample, preferably in liquid form. The sample receiving area is also configured to receive any other liquid, such as a liquid reagent that can be added to the apparatus of the present invention.

[0097] The sample receiving area may contain any other components required or beneficial to LFT, which may be removable or fixed thereon. In some embodiments, the sample receiving area contains a buffer component for lateral flow testing before use of the apparatus. These buffer components are removable after the addition of the test sample and can be movably fixed to the sample receiving area by any suitable method known to those skilled in the art. For example, the buffer component may be dried onto the sample receiving area such that the addition of a liquid (e.g., a liquid test sample) causes the buffer component to be released from the sample receiving area and mixed with the test sample. Any suitable buffer component known to those skilled in the art can be used, such as sucrose and glycerol.

[0098] The sample receiving area can be made of any suitable material known to those skilled in the art. For example, the sample receiving area can be paper-based, woven mesh, or made of cellulose, nitrocellulose, or glass fiber. Preferably, the sample receiving area is made of nitrocellulose or glass fiber. The sample receiving area can be part of a single structure that includes a suture pad and optionally also includes a first test area and a second test area; or, the sample receiving area can be a structure separate from the suture pad and each of the first and second test areas. In some embodiments, the sample receiving area can be a region of the suture pad such that there is no distinction between the sample receiving area and the suture pad (i.e., the test sample is added to the suture pad).

[0099] Adhesive pad

[0100] The conjugation pad includes a movable conjugated analyte disposed on or inside the conjugation pad such that the conjugated analyte can be released and mixed with the test sample when the test sample comes into contact with the conjugation pad. For example, the conjugated analyte can be dried on the conjugation pad so that it is released or moved and mixed with the test sample upon contact with it.

[0101] In some embodiments, the conjugation pad further comprises a movable analyte-binding molecule, and the first test region comprises immobilized trapping molecules for immobilizing the analyte-binding molecule, wherein each immobilized trapping molecule defines a first binding site. The advantage of these embodiments is that the effect of any flow rate difference between the conjugated analyte and the analyte molecules in the test sample is reduced, thereby improving the accuracy of LFT. Specifically, the flow rate of a molecule is (at least partially) determined by its size. When there is a large size difference between the conjugated and unconjugated analytes (i.e., the analyte molecules in the test sample), the flow rates of the conjugated and unconjugated analytes differ, causing one of the conjugated or unconjugated analytes to arrive at the first test region before the other (typically the unconjugated analyte flows faster). This means that the conjugated analyte may not be able to compete sufficiently with the analyte molecules in the test sample (i.e., the unconjugated analytes) to bind to the analyte-binding molecule, potentially leading to inaccurate quantification of the analyte molecules in the test sample. By mixing analyte-bound molecules with the test sample and the conjugated analyte on the conjugation pad, the analyte-bound molecules can be simultaneously exposed to analyte molecules in both the conjugated analyte and the test sample, thereby reducing the impact of flow rate differences between the conjugated and unconjugated analytes (from the test sample).

[0102] In some implementations, the conjugation pad comprises a mobile analyte-binding molecule and a conjugated analyte, and the first test region comprises an immobilized analyte-binding molecule. The advantage of these implementations is that the mobile analyte-binding molecule acts as a concentration buffer, thereby introducing a minimum threshold concentration before a positive LFT result is displayed.

[0103] In some embodiments, the suture pad is part of the same structure including the first test region and the second test region, or it can be a structure separate from the first and second test regions. When the suture pad is a structure separate from the test regions, it can be attached to the structure including the first and second test regions by any suitable means known to those skilled in the art.

[0104] The fusion pad can be made of any suitable material known to those skilled in the art. For example, the fusion pad can be paper-based, woven mesh, or made of cellulose, nitrocellulose, or glass fiber. In some embodiments, the fusion pad is made of nitrocellulose or glass fiber.

[0105] First Test Area and Second Test Area

[0106] The first and second test areas can be different regions of a single structure, or they can each be separate structures or regions of separate structures. When the first and second test areas are separate structures or regions of separate structures, any suitable method known to those skilled in the art can be used to secure the structures together to allow liquid to flow from the structure containing the first test area to the structure containing the second test area. In all embodiments, the first and second test areas are arranged such that during LFT testing, liquid flowing through / along the lateral flow testing device reaches the first test area first, and then the second test area.

[0107] Preferably, the first test zone and the second test zone are separate regions of a single structure, which can be a permeable or semi-permeable membrane that allows liquid to flow on its surface or inside. In some embodiments, the first test zone and the second test zone are separate regions of a single nitrocellulose membrane.

[0108] In some implementations, the first test region comprises immobilized analyte-binding molecules. Each immobilized analyte-binding molecule defines a first binding site on the first test region.

[0109] In some embodiments, the conjugation pad comprises a movable analyte-binding molecule and a movable conjugated analyte, and the first test region comprises an immobilized trapping molecule for binding and immobilizing the analyte-binding molecule. Each immobilized trapping molecule defines a first binding site on the first test region. The advantage of these embodiments is that the effect of any flow rate difference between the conjugated analyte and the analyte molecules in the test sample is reduced, thereby improving the accuracy of LFT, as discussed above.

[0110] In some embodiments, the conjugation pad comprises a mobile conjugated analyte and a mobile analyte-binding molecule, and a first test region comprises an immobilized analyte-binding molecule. Each immobilized analyte-binding molecule defines a first binding site on the first test region. The advantage of these embodiments is that the mobile conjugated analyte acts as a concentration buffer, thereby introducing a minimum threshold concentration before a positive LFT result is displayed. Specifically, analyte molecules in the test sample compete with the conjugated analyte for mobile analyte-binding molecules on the conjugation pad. Once an analyte molecule has bound to an analyte-binding molecule, it can no longer bind to the immobilized analyte-binding molecule on the first test region. In the first test region, there is competition between the conjugated analyte and any analyte molecule that is not bound to a mobile analyte-binding molecule.

[0111] In each configuration of the conjugation pad and the first test region, the second test site contains an immobilized conjugated analyte-binding molecule that binds to conjugated analytes but not to unconjugated analytes (i.e., analyte molecules from the test sample). Each immobilized conjugated-analyte-binding molecule defines a second binding site. Therefore, any conjugated analyte not bound to the first test region will bind to the second test region. As the concentration of analyte molecules in the test sample increases, fewer conjugated analyte molecules are able to bind to the first test region, thus reducing the signal intensity generated by the first test region, while more conjugated analyte molecules bind to the second test region, thus increasing the signal intensity generated by the second test region. Therefore, the signal intensity of the second test region depends on and is inversely correlated with the signal intensity of the first test region. To increase the signal intensity in the presence of one or more analyte molecules in the test sample, the number of second binding sites in the second test region must be equal to or greater than the number of conjugated analyte molecules in the lateral flow test device. Specifically, since the first test region always has at least one first binding site, after lateral flow testing in the absence of analyte molecules in the test sample, the second test region always has at least one second binding site that is not bound to a conjugated analyte molecule. Therefore, the presence of analyte molecules in the test sample can increase the number of analyte molecules bound to the second test region by at least one molecule, thereby increasing the signal intensity of the second test region.

[0112] The first and second test regions can each be of any suitable shape, such as square, rectangle, circle, triangle, ellipse, or linear. Preferably, the first and second test regions are each linear, formed by fixing the associated analyte-binding or trapping molecules and conjugated analyte-binding molecules into a linear shape. For example, when the first and second test regions are regions on the membrane, they can each be linear on the membrane. Preferably, both the first and second test regions span the entire width of the path of liquid flow along / through the membrane (i.e., perpendicular to the direction of liquid flow). This has the advantage that the analyte molecules and conjugated analytes must contact the first test region first, then the second test region, thereby preventing false positives in the second test region due to conjugated analyte binding. Furthermore, this configuration ensures that the test sample and conjugated analyte molecules cannot bypass the test regions, which would otherwise distort the test results.

[0113] Conjugated analyte

[0114] Each molecule of the conjugated analyte used in the apparatus and method of the present invention comprises an analyte molecule conjugated to a detectable marker. One or more analyte molecules are conjugated to each detectable marker. In some embodiments, only one analyte molecule is conjugated to each detectable marker.

[0115] The analyte molecule conjugated to the detectable label must compete with the analyte molecules in the test sample for analyte binding in the conjugation pad and / or the first test region. Preferably, the analyte molecule conjugated to the detectable label is of the same type as the analyte molecule in the test sample. For example, if the analyte molecule to be detected in the test sample is caffeine, then the analyte molecule conjugated to the detectable label can also be caffeine.

[0116] By varying the number of analyte molecules conjugated to each detectable label, the minimum concentration threshold required for a positive result in the test sample can be altered. For example, increasing the number of analyte molecules conjugated to each detectable label results in stronger competition between the conjugated analyte and the analyte-binding molecule, as the ratio of molecules that the analyte-binding molecule can bind to (i.e., the test sample and the conjugated analyte molecules) is more heavily weighted to favor the conjugated analyte. Therefore, in some embodiments, each conjugated analyte comprises two or more analyte molecules conjugated to each detectable label, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 analyte molecules conjugated to each detectable label.

[0117] In some embodiments, each detectable label is conjugated with two analyte molecules, the conjugation pad contains a mobile analyte-binding molecule, and the first test region contains an immobilized analyte-binding molecule. Preferably, the mobile analyte-binding molecule and the immobilized analyte-binding molecule are the same. Embodiments with both mobile and immobilized analyte-binding molecules offer the advantage of reducing the impact of different analyte molecule flow rates in the test sample and the conjugated analyte, for the same reasons described above. Furthermore, the mobile analyte-binding molecule can also act as a concentration buffer, for the same reasons described above. Moreover, since each detectable label is conjugated with two analyte molecules, the conjugated analyte can bind to both the mobile and immobilized analyte-binding molecules simultaneously; whereas analyte molecules from the test sample can only bind to one of the mobile or immobilized analyte-binding molecules. This further enhances the concentration buffering effect of these embodiments.

[0118] In some implementations, each detectable marker is saturated with analyte molecules (i.e., the number of analyte molecules conjugated to each detectable marker is the maximum number that can conjugate to that detectable marker). Since target analyte molecules vary in size, the specific number of analyte molecules conjugated to a detectable marker will depend on the target analyte molecules in question.

[0119] The analyte molecule is conjugated to a detectable marker using any suitable method known to those skilled in the art. Specifically, the detectable marker remains detectable during the conjugation of the analyte molecule to the detectable marker. In some embodiments, the analyte molecule is conjugated to the detectable marker via a linker. The linker can be any suitable linker known to those skilled in the art. For example, the linker may contain biotin and / or BSA. In some embodiments, the linker is biotin-BSA.

[0120] The detectable marker can be any suitable molecule known to those skilled in the art that generates a detectable signal whose intensity can be detected or measured. For example, the detectable marker can be dye particles, carbon particles, fluorescent markers, latex particles, gold particles, magnetic particles, etc. In some embodiments, the detectable marker generates an optical signal, such as a color change, or the appearance and / or disappearance of a visible marker. Therefore, the signal intensity can be a change in light intensity (positive or negative) detected in a given test area. For example, the detectable marker may produce red when it binds to a test site. An increase in measured intensity may not actually refer to an increase in the intensity of the red light frequency, but rather to an increase in the intensity of the red light frequency relative to other light frequencies (e.g., increased absorption of other frequencies). Alternatively, the increase in signal intensity may also refer to a decrease in the intensity of reflected light. For example, in an example where a red dye binds to an otherwise white test area, the increase in signal intensity may be related to an overall decrease in the intensity of reflected light (e.g., all visible frequencies due to the dye's absorption of non-red light), or a decrease in the intensity of one or more non-red frequencies.

[0121] In a preferred embodiment, the detectable marker is a detectable nanoparticle, preferably a gold nanoparticle.

[0122] By using a pre-set amount of conjugated analyte and a pre-set number of binding sites in the first test region, the minimum concentration of analyte molecules in the test sample required to generate a detectable signal in the second test region can be pre-set. For example, increasing the amount of conjugated analyte in the lateral flow test device will increase the minimum concentration of target analyte required to cause a change in signal intensity in the first and second test regions, because competition between the conjugated analyte and the analyte molecules in the test sample will favor a larger number of conjugated analytes.

[0123] Analyte molecule

[0124] Analyte molecules can be any target analyte molecule, especially those associated with disease or pathological conditions. Other target analyte molecules include hormones, antibiotics, impurities (such as food and beverage contaminants), and contaminants. Analyte molecules can be peptides, polynucleotides, or organic or inorganic compounds.

[0125] In some implementations, the analyte molecule is an antigen, such as a protein, peptide, or fragment thereof. Exemplary antigens include bacterial or viral proteins or fragments thereof, such as coronavirus proteins or fragments thereof. The target protein may be, for example, an enzyme or a protein hormone.

[0126] The analyte molecule can be a biomarker of a disease or condition, or an immunological biomarker for a disease; therefore, detecting and quantifying the biomarker helps in the diagnosis and / or monitoring of the associated disease or condition, or its immune response. For example, the analyte molecule can be a marker of pregnancy, renal function, bacterial infection, or viral infection. The biomarker can be a metabolite, lipid, steroid, protein, peptide, polynucleotide, or fragment thereof. In some embodiments, the analyte molecule is 3-nitrohydroxyphenylacetic acid (NHPA). In some embodiments, the analyte molecule is an antibody. In some embodiments, the analyte molecule is creatinine.

[0127] The analyte molecule can be a drug, a nutraceutical drug, or other organic or inorganic compound, which is useful for detection and quantification. In some embodiments, the analyte molecule is caffeine.

[0128] Analyte-binding molecule

[0129] Any molecule capable of binding to both the analyte molecule in the test sample and the analyte molecule in the conjugated analyte can be used as an analyte binding molecule. Therefore, an analyte binding molecule is suitable for binding to both the analyte molecule in the test sample and independently to the analyte molecule in the conjugated analyte (i.e., the analyte binding molecule is suitable for binding to both the analyte molecule in the test sample and the analyte molecule in the conjugated analyte, but only one at a time). The analyte binding molecule is preferably specific to the analyte molecule (both in the test sample and the conjugated analyte). In some embodiments, the analyte binding molecule is an antibody specific to the analyte molecule (i.e., an anti-analyte antibody). For example, if the analyte molecule is NHPA, the analyte binding molecule could be an antibody specific to 3-nitrotyrosine (3-NTyr), which is known to cross-react with NHPA (Wisastra et al., “Antibody-free detection of proteintyrosine nitration in tissue sections”, ChemBioChem, 2011, 12, 2016-2020). In another example, if the analyte molecule is caffeine, the analyte-binding molecule may be an anti-caffeine antibody known to those skilled in the art. In another example, if the analyte molecule is creatinine, the analyte-binding molecule may be an anti-creatinine antibody.

[0130] In some embodiments, the analyte molecule is an antibody, as described above, and the analyte-binding molecule is a molecule that the antibody specifically binds to. For example, the analyte-binding molecule may be an antigen, and the analyte molecule may be an antibody specific to that antigen. In other instances, the analyte molecule is an antibody, and the analyte-binding molecule is an antibody specific to that analyte molecule (i.e., an anti-antibody).

[0131] Capture molecules

[0132] In some embodiments, the first test region includes a capture molecule, which can be any suitable molecule capable of binding and immobilizing an analyte-binding molecule, thereby retaining the analyte-binding molecule in the first test region. In some embodiments, the capture molecule is capable of binding a complex formed by the analyte-binding molecule and a conjugated analyte or an analyte molecule from the test sample (i.e., the capture molecule is specific to the complex formed by the analyte-binding molecule and the conjugated analyte, or specific to the complex formed by the analyte-binding molecule and an analyte molecule from the test sample). Preferably, in these embodiments, the capture molecule does not bind to analyte-binding molecules that have not yet bound to a conjugated analyte or an analyte molecule from the test sample. In some embodiments, the immobilized capture molecule is an antibody specific to the analyte-binding molecule. In some embodiments, the analyte-binding molecule is an antibody, and the capture molecule can be an anti-antibody complex antibody that is specific to analyte-binding molecules that have already bound to a conjugated analyte or an analyte molecule from the test sample.

[0133] Conjugated-analyte-binding molecule

[0134] The conjugated analyte binding molecule in the second test region can bind to the conjugated analyte without binding to the unconjugated analyte molecule (i.e., the analyte molecule from the test sample). Any suitable conjugated analyte binding molecule can be used, for example, a molecule that specifically binds to a detectable label (or, if present, a linker of the conjugated analyte).

[0135] In some embodiments, the linker comprises biotin, preferably biotin-BSA, and the conjugated analyte is avidin, streptavidin, or polystreptavidin.

[0136] Signal detection

[0137] The signal generated by the detectable marker of the conjugated analyte can be detected in any manner suitable for the type of signal to be detected. In some embodiments, the detectable marker generates an optical signal, which is detected and its intensity measured using an optical signal sensor and / or reader. For example, a smartphone (e.g., using the smartphone's camera) can be used to detect the optical signal and measure its intensity. Alternatively, a reader including a lateral flow test holder configured to receive a lateral flow test sample for measurement can be used to detect the optical signal and measure its intensity. The holder may include one or more walls configured to enclose the lateral flow test (e.g., to block external light). The reader may include a light source for illuminating the lateral flow test during measurement. This allows control over the amount of light incident on the lateral flow test during measurement. Reference will be made below. Figure 26 Further discussion on signal detection methods.

[0138] Detection of analyte molecules in test samples

[0139] In the apparatus and method of the present invention, changes in signal intensity in both the first and second test regions indicate the presence of one or more analyte molecules in the test sample. Specifically, the presence of analyte molecules in the test sample means that there is competitive binding between the analyte molecules and conjugated analyte molecules to the first test region. When the competition reaches a point where the first binding site in the first test region is saturated with analyte molecules and / or conjugated analytes, any conjugated analytes that fail to bind to the first test region will bind to the second binding site in the second test region. The more analyte molecules that bind to the first binding site, the more conjugated analytes that fail to bind to the first binding site, and therefore the greater the decrease in signal intensity in the first test region. The more conjugated analytes that fail to bind to the first binding site, the more conjugated analytes will bind to the second binding site, and therefore the greater the increase in signal intensity in the second test region.

[0140] In some embodiments, the presence of one or more analyte molecules in the test sample can be indicated by a signal intensity reduction in the first test region to below 100%. Therefore, in these embodiments, the device is configured such that, when no analyte molecules are present in the test sample, either all first binding sites in the first test region bind to the conjugated analyte (either there is an excess of conjugated analyte, or the number of first binding sites is the same as the number of conjugated analyte molecules), or all conjugated analytes bind to the first binding sites (either there is an excess of first binding sites, or the number of conjugated analyte molecules is the same as the number of first binding sites).

[0141] In some embodiments, the presence of one or more analyte molecules in the test sample can be indicated by an increase in the signal intensity of the second test region from below 100%. In these embodiments, when no analyte molecules are present in the test sample, some or none (but not all) of the second binding sites bind to the conjugated analyte. Therefore, when one or more analyte molecules are present in the test sample, conjugated analytes that did not bind to the first test region due to competition with analyte molecules in the test sample will instead bind to the second test region, thereby increasing the signal intensity of the second test region. Therefore, the signal intensity of the second test region is inversely correlated with the signal intensity of the first test region.

[0142] In some embodiments, the presence of one or more analyte molecules in the test sample can be indicated by an increase in the signal intensity of the second test region from 0%. Therefore, in these embodiments, when no analyte molecules are present in the test sample, all conjugated analyte molecules bind to the first test region, while no (or very few, such that no detectable signal is generated) molecules bind to the second test region.

[0143] The signal strength of the first and second test areas is detected based on the signals present in the first and second test areas within the shortest possible time after the test sample is applied to the sample receiving area. It should be understood that signal strength detection can be performed directly using the first and second test areas themselves (i.e., directly from the physical first and second test areas) or using replicas of the first and second test areas (e.g., photographs). Specifically, sufficient time must be allowed before signal strength detection to allow the test sample to migrate to the suture pad, the first test area, and subsequently the second test area. The signal strength of the first and second test areas can be detected based on the signals present in the first and second test areas after any suitable minimum time, such as at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes. Preferably, the signal strength of the first and second test areas is detected based on the signals present in the first and second test areas at least 15 minutes after the test sample is applied to the sample receiving area.

[0144] The signal strength of the first and second test areas can be detected based on the signals present in the first and second test areas up to the longest possible time elapsed after the test sample is applied to the sample receiving area. For example, the signal strength can be detected based on the signals present in the first and second test areas no more than 1 hour, no more than 50 minutes, no more than 45 minutes, no more than 40 minutes, no more than 35 minutes, or no more than 30 minutes after the test sample is applied to the sample receiving area. In some embodiments, the signal strength of the first and second test areas is detected based on the signals present in the first and second test areas no more than one hour after the test sample is applied to the sample receiving area.

[0145] Quantitative

[0146] The apparatus and method of the present invention can provide semi-quantitative or quantitative results. Specifically, the lateral flow assay apparatus of the present invention is manufactured with known amounts of each component, namely the conjugated analyte, the first binding site and the second binding site, and optionally a mobile analyte-binding molecule, thereby creating an apparatus with an appropriate detection threshold for the target analyte in question. Specifically, each target analyte has a different threshold concentration that indicates the relevant condition (e.g., the presence of a disease or pathological condition, an acceptable threshold level of impurities, etc.). Those skilled in the art will know how to adjust the amounts of the conjugated analyte, the first binding site and the second binding site, and optionally the mobile analyte-binding molecule to provide a specific positive reaction threshold (i.e., detection of the target analyte in the test sample).

[0147] Therefore, changes in signal intensity in both the first and second test regions indicate that the concentration of analyte molecules in the test sample is higher than the threshold concentration. Specifically, the signal intensity change in the first test region is a decrease in signal intensity, due to increased competition between the conjugated analyte and the analyte molecules in the test sample, resulting in a reduction in the number of conjugated analyte molecules bound to the first test region. Conversely, the signal intensity in the second test region increases inversely, due to an increase in the number of conjugated analyte molecules not bound to the first test region, thus increasing the number of conjugated analyte molecules bound to the second test region.

[0148] Therefore, the method and apparatus of the present invention quantitatively test the concentration of analyte molecules in a sample by measuring the change in signal intensity in a first test region and a second test region. Since the threshold concentration of analyte molecules in the test sample required for a change in signal intensity is known, as discussed above, a further decrease in the signal intensity in the first test region and a further increase in the signal intensity in the second test region are correlated with an increase in the concentration of analyte molecules in the test sample.

[0149] Furthermore, the apparatus and method of the present invention can more accurately quantify analyte molecules in a sample because they do not lose information about the amount of conjugates not bound to the first test region and / or bound to the second test region. As mentioned above, conventional LFT requires an excess of labeled conjugates to ensure the control line is a valid control, but this means the loss of information related to the amount of labeled conjugates bound to the control line. In contrast, in the apparatus and method of the present invention, the amount of detectable conjugated analyte is equal to or less than the number of binding sites in the second test region, thus avoiding the corresponding information loss.

[0150] It should be noted that the apparatus and method of the present invention do not require a separate control line, because the presence of a detectable signal in the first test area or the second test area indicates that the test is valid. The absence of both lines indicates that the test is invalid.

[0151] In some embodiments, quantification of analyte molecules in a sample is performed by measuring the signal intensities of a first test region and a second test region and calculating the ratio of these two signal intensities. This ratio indicates the number of analyte molecules in the test sample. Specifically, as described above, the apparatus of the present invention is manufactured using known and predetermined amounts of each component, thereby allowing the amount of conjugated analyte bound to each of the first and second test regions to be known even in the absence of analyte molecules.

[0152] The signal strength of the first and second test regions can be measured. This can be achieved by acquiring one or more images of the first and second test regions. The locations of the first and second test regions in each of the one or more images can be obtained through user input or by using image recognition methods (such as edge detection and / or machine learning). The signal strength of each test region can be determined based on the intensity of one or more pixels representing that test region. This can be based on a single pixel or the sum of the intensity values ​​of multiple pixels (e.g., average intensity).

[0153] The pixel intensity values ​​can then be converted into signal strength representing the amount of intensity change (e.g., relative to a calibration area). The signal strength can be expressed as a percentage of one or both of the maximum and minimum signal strengths (e.g., it can be normalized). The maximum signal strength can be a predefined value (e.g., based on previous calibration measurements) or can be determined by measuring the signal strength of a lateral flow test calibration area (e.g., a calibration line with a predefined color (or color change) corresponding to a fully saturated test area (e.g., a signal strength of 100%)). The signal strength can be determined as a percentage of the maximum signal strength. This percentage can also be based on the minimum signal strength. This minimum signal strength can be predefined or measured (e.g., a calibration area with a predefined color corresponding to a fully unsaturated test area (e.g., a signal strength of 0%)). While in this embodiment 100% represents a fully saturated curve and 0% represents a fully unsaturated curve, it should be understood that the opposite may be true depending on the line's response to the analyte.

[0154] In some implementations, analyte molecules in the test sample are quantified by measuring the signal intensity of one of the first and second test regions, and then predicting the signal intensity of the other test region based on the sum of the signal intensities of the first and second test regions being 100%. This measurement and subsequent prediction yields the measured-to-predicted ratio of signal intensities in the first and second test regions.

[0155] In some implementations, the signal strength of a first test area is measured, and then the signal strength of a second test area is predicted based on the sum of the signal strengths of the first and second test areas being 100%. This yields a first measured signal strength: predicted signal strength ratio. The signal strength of the second test area is measured, and then the signal strength of the first test area is predicted based on the sum of the signal strengths of the first and second test areas being 100%. This yields a second measured signal strength: predicted signal strength ratio. The first and second measured signal strength: predicted signal strength ratios can be calculated in any order. A calibration factor (also called a scaling factor) can be applied to the first and second measured signal strengths, respectively. This calibration factor can be used to recalibrate the measurements (see below). The calibration factor can be initialized to an initial value (e.g., 1) and can be adjusted according to calibration checks. When scaling is performed using a calibration factor equal to 1, the scaled measurement can be equal to (e.g., the same as the unscaled measurement) the unscaled measurement.

[0156] Next, the first and second measured signal intensity:predicted signal intensity ratios are averaged (e.g., the average of the first and second measured signal intensity:predicted signal intensity ratios is calculated) to obtain the average first test area signal intensity:second test area signal intensity ratio. Then, the ratio of the measured signal intensities is determined using both the first and second test areas. The measurements used to determine the ratio can be the same as those used in the previous prediction. Alternatively, different measurements can be used (e.g., from different samples of the same image, or from different images).

[0157] The ratio of average first test area signal strength to second test area signal strength is compared with the measured signal strength ratio to provide a ratioometric calibration, which checks whether the signal strength used to calculate the average first test area signal strength to second test area signal strength ratio is accurate.

[0158] If the average first test area signal strength:second test area signal strength ratio does not match the measured signal strength ratio (e.g., within a preset range), the calibration factor for the measured signal strength can be adjusted and the quantification method repeated. That is, the first and second measured signal strengths can be recalculated based on the adjusted calibration factor to obtain a new measured signal strength:predicted signal strength ratio, which in turn yields a new average first test area signal:second test area signal. This new average first test area signal:second test area signal can then be compared with the new measured signal strength ratio (based on the adjusted calibration factor). The recalculated first and second measured signal strengths can be based on the same intensity measurements as before, or new measurements can be obtained.

[0159] If the ratio of the average signal strength in the first test area to the signal strength in the second test area matches the ratio of the measured signal strength, then the ratio of the average signal strength in the first test area to the signal strength in the second test area can be used as the signal strength ratio of the test sample without recalibration.

[0160] This method can be repeated iteratively multiple times until the average signal strength ratio of the first test area to the second test area matches the measured signal strength ratio (or the maximum number of iterations is reached).

[0161] The scaling direction (i.e., the direction in which the calibration factor is adjusted) may vary depending on whether the sum of the measured signal intensities is greater than or less than the maximum signal intensity (e.g., exceeding 100% of the maximum signal intensity). For example, if the sum of the first and second measured signal intensities is greater than the maximum signal intensity, the calibration factor can be decreased (e.g., decreased by a predefined step size or factor). Similarly, if the sum of the first and second measured signal intensities is not greater than or is less than the maximum signal intensity, the calibration factor can be increased (e.g., increased by a predefined step size or factor). This helps to recalibrate signal intensity measurements to accommodate different ambient lighting conditions.

[0162] In some implementations, the average first signal strength:second signal strength ratio matches the measured signal strength when it falls within a predefined range of the measured signal strength ratio. The predefined range can be, for example, within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the measured signal strength ratio, i.e., the allowable error range does not exceed 10%. Preferably, the average first signal strength:second signal strength ratio matches the measured signal strength ratio when it falls within 5% of the measured signal strength ratio.

[0163] A defined sample signal intensity ratio (e.g., whether scaled or not) indicates the concentration of analyte molecules in the test sample. Therefore, the signal intensity ratio can be used to determine the concentration of analyte molecules in the test sample (e.g., based on a calibration curve of the signal intensity ratio versus analyte concentration). This method of quantifying analyte molecules in a sample allows for reduction of systematic errors caused by the LFT device reader and any errors caused by ambient light. Specifically, the first and second measured signal intensities:predicted signal intensities determine the precision of the quantification (i.e., the repeatability of the results), while the ratio of the measured signal intensities and its comparison with the average first test area signal intensity:second test area signal intensity ratio ensures the accuracy of the quantification (i.e., the true quantification of the target analyte in the test sample).

[0164] Similar to existing quantitative LFT, the calibration curve is used to calculate the concentration of analyte molecules based on a defined signal intensity ratio.

[0165] The apparatus and method of the present invention also solve the problem of conventional LFT test line saturation. Specifically, conventional quantitative LFT uses only the test line (not the control line) to quantify the concentration of analyte molecules in a sample. Therefore, when the test line is saturated with analyte molecules from the test sample, any excess analyte molecules from the test sample cannot be quantified. However, in the apparatus and method of the present invention, the use of two test zones (e.g., test lines) and the ratio of the signal intensities of the two test zones means that analyte molecules in the sample exceeding the normal saturation point of the corresponding conventional LFT can be quantified. Figure 21 This demonstrates the saturation mechanism of conventional sandwich LFTs. Specifically, because the number of analyte molecules in the test sample exceeds the number of conjugates or anti-analyte antibodies available on the test line, there are unbound analyte molecules that cannot be quantified. In contrast, the apparatus and method of the present invention rely on the competition between analyte molecules and conjugate analytes in the test sample to generate a ratio of signal intensities in the two test regions, which is then used to quantify the analyte in the test sample. Figure 22 The results show that when the amount of analyte molecules in the test sample is the same as the amount of conjugated analyte and the number of binding sites on the first test line (i.e., similar to the saturation point of a conventional sandwich LFT; in this example, there are 200 units each of analyte, conjugated analyte, and first binding site), the competition between the analyte molecules from the test sample and the conjugated analyte is equal, resulting in equal binding to the first test line. The remaining conjugated analyte (i.e., 50% of the initial conjugated analyte on the conjugation pad) binds to the second test line, resulting in equal signal intensities on the first and second test lines (i.e., a signal intensity ratio of 50%:50% (1:1)). Figure 23 The results show that even when the amount of analyte molecules in the test sample exceeds the amount of conjugated analyte and binding sites on the first test line (i.e., exceeding the saturation point of a conventional sandwich LFT; in this example, the analyte molecules (i.e., 400 units) are twice the amount of both the conjugated analyte and the first binding site (i.e., 200 units each)), quantification of the analyte molecules in the test sample is still possible. This is because this situation causes a change in the signal ratio between the first and second test lines due to increased competition for binding sites on the first test line from the analyte molecules in the test sample. Specifically, in this example LFT, the signal intensity ratio of the first test line to the second test line is 33%:67% (1:2). Therefore, using the apparatus and method of the present invention, quantification of analyte molecules in the test sample at concentrations higher than the normal saturation level of a corresponding conventional LFT is still possible.

[0166] In conventional LFT, one way to overcome the saturation problem is to increase the number of binding sites on the test line. However, this leads to a decrease in the resolution of the analyte in the quantified sample. Specifically, as the number of binding sites on the test line increases, the percentage contribution of each binding site to the maximum signal intensity of the test line decreases. For example, assuming an LFT test line contains 200 binding sites, each binding site accounts for 0.5% of the maximum signal intensity. When the number of binding sites increases to 400, each binding site accounts for only 0.25% of the maximum signal intensity. Figure 24 This illustrates a hypothetical conventional sandwich LFT with 1000 binding sites on the test line. The number of analyte molecules in the test sample ranges from 400 (…). Figure 24 a) Increased to 415 ( Figure 24 b) caused the signal strength on the test line to drop from 40% ( Figure 24 c) changed to 41.5% Figure 24 d). Figure 25 An example of LFT according to the present invention is shown, and the number of analyte molecules in the test sample is shown to be from 400 ( Figure 25 a) Increased to 415 ( Figure 25 b) This results in an overall change of 2% in the signal strength ratio (the first test line changes from 50% to 49%, and the second test line changes from 50% to 51%). Figure 25 (c and d). Specifically, the change in signal intensity of the first test line is mirrored by an inverse relationship with the signal intensity of the second test line. This effectively doubles the change in signal intensity compared to conventional LFT. Larger changes in signal intensity are easier to detect and measure, thus the LFT device of the present invention improves the accuracy of analyte quantification compared to conventional LFT.

[0167] For the same reasons as with signal intensity detection, the quantification of analyte molecular concentration in the test sample is based on the signal present in the first and second test areas after a minimum time has elapsed since the test sample was applied to the sample receiving area. Similar to signal intensity detection, quantification can be performed on the first and second test areas themselves, or on replicas of the first and second test areas (e.g., one or more photographs). Therefore, the minimum possible time for signal intensity detection described above also applies to the quantification of analyte molecular concentration in the test sample.

[0168] Similar to detecting signal intensity (as described above), the quantification of the concentration of analyte molecules in the test sample can be based on the signals present in the first and second test regions before the longest possible time has elapsed since the test sample was applied to the sample receiving region. This longest possible time is the same as the signal intensity detection time discussed above.

[0169] Ratio of each component

[0170] In the LFT device of this invention, the number of molecules of the conjugated analyte is equal to or less than the number of second binding sites in the second test region. This ensures that the signal intensity in the test region always changes when the test sample contains analyte molecules.

[0171] The specific amounts of the conjugated analyte, the first binding site, and the second binding site are not critical to the functionality of the apparatus and method of the present invention, as long as the amounts added to the apparatus during manufacturing are known. These amounts can be used to calibrate the readout device for detecting and measuring the signal intensity of the first and second test regions. In particular, different analyte molecules (i.e., different target analytes) require different amounts of each component because different types of target analytes have different threshold levels and target concentrations.

[0172] In some embodiments, the ratio of the conjugated analyte to the second binding site is 1:1 to 1:100, 1:1 to 1:50, 1:1 to 1:20, 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, or 1:1 to 1:2, or 1:1. In some embodiments, the number of conjugated analyte molecules is the same as the number of second binding sites in the second test region (i.e., the ratio is 1:1).

[0173] The number of first binding sites can be greater than, equal to, or less than the number of second binding sites. Specifically, the number of first binding sites can be selected such that a minimum threshold concentration of the analyte in the test sample required to change the signal intensity of the first test region can be set. In some embodiments, the number of first binding sites is equal to or less than the number of second binding sites. In these embodiments, the ratio of the first binding sites to the second binding sites is 1:100 to 1:1, 1:1 to 1:50, 1:1 to 1:20, 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, or 1:1 to 1:2, or 1:1.

[0174] The ratio of any of the conjugated analytes to the second binding site can be combined with any of the ratios of the first binding site to the second binding site. In some embodiments, the ratio of the conjugated analyte to the second binding site is 1:1, and the ratio of the first binding site to the second binding site is 1:1 (i.e., the ratio of the conjugated analyte to the first binding site to the second binding site is 1:1:1). Specifically, the advantage of a 1:1:1 ratio of conjugated analyte:first binding site:second binding site is that the signal intensity change in the second test region is more pronounced when analyte molecules are present in the test sample (i.e., the signal intensity transition between the first and second test regions is clearer). Specifically, when analyte molecules are absent from the test sample, the signal intensity of the first test region will reach its maximum value (i.e., 100% of the possible signal intensity), while the signal intensity of the second test region will reach its minimum value (i.e., 0% of the possible signal intensity). Therefore, the presence of analyte molecules in the test sample will be indicated by a decrease in the signal intensity of the first test region from its maximum value of 100% and a corresponding increase in the signal intensity of the second test region from its maximum value of 0%.

[0175] In some embodiments, the conjugation pad includes a movable conjugated analyte, a first test region includes immobilized analyte-binding molecules, and a second test region includes immobilized conjugated analyte-binding molecules, wherein the ratio of conjugated analyte to analyte-binding molecules is from 1:1:1 to 1:100:100. Preferably, the ratio of conjugated analyte to analyte-binding molecules is 1:1:1.

[0176] In some embodiments, the conjugation pad comprises a mobile analyte-binding molecule and a mobile conjugated analyte, and the first test region comprises a trapping molecule. The ratio of the conjugated analyte to the second binding site, the ratio of the first binding site to the second binding site, and the ratio of the conjugated analyte to the first binding site to the second binding site can all be independently any of the relevant ratios disclosed above. The ratio of the conjugated analyte to the mobile analyte-binding molecule to the first binding site can be any suitable ratio to adjust for the effect of different flow rates of the target analyte molecule and the conjugated analyte from the conjugation pad to the first and second test regions. In some embodiments, the ratio of the conjugated analyte to the mobile analyte-binding molecule can be 1:1 to 1:100, 1:1 to 1:50, 1:1 to 1:20, 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, or 1:1 to 1:2, or 1:1. In some implementations, the ratio of the conjugated analyte to the mobile analyte-binding molecule to the first binding site can be from 1:1:1 to 1:100:100.

[0177] In some embodiments, the conjugation pad comprises a conjugated analyte and a mobile analyte-binding molecule, and the first test region comprises an immobilized analyte-binding molecule. The ratio of the conjugated analyte to a second binding site, the ratio of the first binding site to the second binding site, and the ratio of the conjugated analyte to the first binding site and the second binding site can all be independently any of the relevant ratios disclosed above. The ratio of the conjugated analyte to the mobile analyte-binding molecule can be any ratio suitable as a concentration buffer to set a minimum concentration threshold before a positive result is displayed in the LFT.

[0178] Test sample

[0179] In the method of this invention, the test sample added to the device can be any target sample. The test sample can be a liquid containing or composed of bodily fluids, a liquid sample derived from a swab or other test (e.g., cells and non-cellular material suspended in a liquid), or a liquid sample derived from a solid sample (e.g., a solution of a solid sample or some of its components). The test sample can be obtained from a living organism, such as a human, animal (i.e., a non-human animal), or plant; or the test sample can be an environmental sample obtained from a natural or artificial environment; or the test sample can be a food or beverage sample (i.e., obtained from food or beverage). In some embodiments, the test sample contains or comprises bodily fluids, such as urine, blood, plasma, serum, saliva, or mucus.

[0180] Specific LFTs—NHPA, caffeine, creatinine

[0181] In some embodiments, the apparatus and method of the present invention are used to detect and / or quantify caffeine (i.e., the analyte molecule is caffeine), for example, in blood. Specifically, testing a patient's blood for caffeine prior to a cardiac MRI scan is particularly useful because caffeine inhibits the vasodilatory effect of adenosine applied during cardiac MRI scans (J. Majd-Ardekani, P. Clowes, V. Menash-Bonsu and TO Nunan, Nucl. Med. Commun., 2000, 21, 361–364), leading to abnormal and / or false-negative results. This can delay the diagnosis of a patient's heart problem (E. Reyes, CY Loong, M. Harbinson, J. Donovan, C. Anagnostopoulos and SRUnderwood, J. Am. Coll. Cardiol., 2008, 52, 2008–2016). In these embodiments, the conjugated analyte comprises one or more caffeine molecules conjugated to a detectable label. This detectable label may be gold nanoparticles. The analyte-binding molecule is capable of binding caffeine; preferably, the molecule is an anti-caffeine antibody. The conjugated analyte-binding molecule can be avidin, streptavidin, or polystreptavidin. The conjugated analyte preferably contains a linker, which can be BSA-biotin. In some embodiments, the conjugated analyte comprises one or more caffeine molecules conjugated to gold nanoparticles via a BSA-biotin linker, the analyte-binding molecule being an anti-caffeine antibody, and the conjugated analyte-binding molecule being avidin, streptavidin, or polystreptavidin. In methods for detecting and / or quantifying caffeine, the sample can be a blood, plasma, or serum sample.

[0182] In some embodiments, the apparatus and method of the present invention are used to detect and / or quantify NHPA (i.e., the analyte molecule is NHPA). In these embodiments, the conjugated analyte comprises one or more NHPA molecules conjugated to a detectable label. The detectable label may be a gold nanoparticle. Preferably, each NHPA molecule is conjugated to the detectable label via a linker, preferably BSA-biotin. The analyte-binding molecule is capable of binding NHPA, preferably an anti-NHPA antibody. In some embodiments, the analyte-binding molecule is an anti-3-nitrotyrosine (3-NTyr) antibody, which has been shown to cross-react with BSA conjugates of NHPA (Wisastra et al., “Antibody-free detection of protein tyrosine nitration in tissue sections”, ChemBioChem, 2011, 12, 2016-2020). The conjugated analyte-binding molecule can be avidin, streptavidin, or polystreptavidin. In some embodiments, the conjugated analyte comprises one or more NHPA molecules conjugated to gold nanoparticles via a BSA-biotin linker, the analyte-binding molecule being an anti-3NTyr antibody, and the conjugated analyte-binding molecule being avidin, streptavidin, or polystreptavidin. In methods for detecting and / or quantifying NHPA, the sample can be a urine sample.

[0183] In some embodiments, the apparatus and method of the present invention are used to detect and / or quantify creatinine (i.e., the analyte molecule is creatinine). In these embodiments, the conjugated analyte comprises one or more creatinine molecules conjugated to a detectable label. The detectable label may be gold nanoparticles. Preferably, each creatinine molecule is conjugated to the detectable label via a linker, which is preferably BSA-biotin. The analyte-binding molecule is capable of binding creatinine, preferably an anti-creatinine antibody. The conjugated analyte-binding molecule may be avidin, streptavidin, or polystreptavidin. In some embodiments, the conjugated analyte comprises one or more creatinine molecules conjugated to gold nanoparticles via a BSA-biotin linker, the analyte-binding molecule is an anti-creatinine antibody, and the conjugated analyte-binding molecule is avidin, streptavidin, or polystreptavidin. In methods for detecting and / or quantifying creatinine, the sample may be a blood, plasma, or serum sample.

[0184] Use of apparatus and method

[0185] The apparatus of the present invention is used to detect the presence of analyte molecules in a test sample and optionally to detect their quantity. The present invention also includes a method for detecting the presence of analyte molecules in a test sample and optionally to detect their quantity. The method and apparatus of the present invention can be used to detect the presence of analyte molecules in test samples obtained from various sources and optionally to detect their quantity, thus having broad application prospects.

[0186] For example, the apparatus and method of the present invention can be used to diagnose diseases or conditions using test samples obtained from humans, animals, or plants. Specifically, the method according to the present invention for detecting the presence of analyte molecules in a test sample and optionally detecting their quantity can be used to inform (i.e., aid) a diagnosis. The diagnosis may rely solely on the results of the method of the present invention, or the results of the method of the present invention may be one of several factors leading to the diagnosis. Therefore, the present invention includes a method for diagnosing a disease or condition comprising performing a method for detecting the presence of analyte molecules in a test sample obtained from humans, animals, or plants and optionally detecting their quantity.

[0187] In another instance, the apparatus and method of the present invention can be used to detect the presence of analyte molecules in environmental samples or food or beverage samples, and optionally to detect their quantity.

[0188] The apparatus and method of the present invention can also be used to detect the presence of health markers and unhealthy markers (i.e., analyte molecules are markers indicating health or unhealth), and optionally to detect their quantity, without necessarily leading to a specific diagnosis of a disease or condition.

[0189] Furthermore, methods for detecting the presence of analyte molecules and optionally their quantity can also be used to monitor the presence or quantity of analyte molecules in an individual (e.g., a person, animal, or plant) or in the environment. This can be used, for example, to monitor an individual's health status or disease or disease predisposition, to monitor the progression of an individual's disease, to monitor the effects of treatment on an individual, or to monitor the rate of change in the quantity of analyte molecules in an individual or in the environment. When the purpose is to monitor the rate of change in the quantity of analyte molecules, this can be used, for example, to monitor and / or calculate drug clearance rates. Therefore, the method of the present invention for detecting the presence of analyte molecules and optionally their quantity can be a method for monitoring the presence and / or quantity of analyte molecules in an individual or in the environment. In some embodiments, the presence of analyte molecules is monitored by acquiring test samples at two or more different time points and detecting the presence of analyte molecules in each test sample. In some embodiments, the step of detecting the presence of analyte molecules in each test sample includes quantifying the concentration of analyte molecules in each test sample. The amount of analyte molecules in each test sample can be compared with a baseline amount and / or the amount in a prior test sample. The baseline quantity can be the number of analyte molecules measured in a baseline test sample obtained at or before the start of the monitoring method, or it can be a reference quantity of analyte molecules calculated as an average quantity measured in one or more control samples. Alternatively, the baseline quantity can be a threshold quantity or range of analyte molecules known in the art. In some embodiments, the presence and / or quantity of analyte molecules in each test sample can be used to monitor an individual's health or condition. For example, the presence or absence of analyte molecules can indicate an individual's quality of health (e.g., good or poor health, or a predisposition to disease / condition), or it can indicate disease progression or the effect of treatment on an individual. Changes in the presence and / or quantity of analyte molecules between two or more test samples can indicate changes in an individual's quality of health, or it can indicate disease progression, the effect of treatment on an individual, or the individual's clearance of a substance (e.g., analyte molecules).

[0190] Measurement System

[0191] Figure 26 A schematic diagram of a computing system 100 according to one embodiment is shown, which is used to quantitatively test the concentration of analyte molecules in a sample based on the signal intensity of a first test region and a second test region of an LFT.

[0192] The computing system 100 includes a processor 110, a memory 120, a non-volatile memory 130, and an input / output (I / O) interface 140. Optionally, the computing system 100 may include an optical sensor 150 (e.g., a digital camera). The computing system 100 can be any form of computing system, such as a personal computer, server, smartphone, tablet computer, etc.

[0193] The computing system 100 is controlled by a processor 110. The processor 110 is configured to quantify the concentration of analyte molecules in a sample based on executable code stored in non-volatile memory 130 and loaded into memory 120 (e.g., random access memory, RAM) for execution.

[0194] The quantitative concentration is determined based on one or more signal strength measurements in the first test area and one or more signal strength measurements in the second test area. These measurements can be acquired from one or more LFT images (e.g., one or more photographs) displaying the first and second test areas. The image can be obtained using an optical sensor (e.g., a digital camera). The optical sensor can be integrated within the computing system (e.g., optical sensor 150) or in an external system. That is, the computing system 100 can be configured to acquire one or more LFT images via the integrated optical sensor or to acquire one or more LFT images from an external system (e.g., via a network, such as the Internet).

[0195] For example, a second system (e.g., a second computing system) may be configured to acquire one or more images displaying a first test area and a second test area, or one or more light intensity measurements of the first test area and the second test area. The computing system 100 may be configured to analyze signal intensity measurements acquired from said one or more images or said one or more light intensity measurements to quantitatively analyze the concentration of the analyte molecules.

[0196] The second system can determine one or more signal strength measurements from one or more images or one or more light intensity measurements, and send the one or more signal strength measurements to the computing system 100. Alternatively, the second system can send one or more images or one or more light intensity measurements to the computing system 100, and then the computing system 100 can determine the one or more signal strength measurements.

[0197] Alternatively, the computing system 100 can acquire one or more images or one or more light intensity measurements, and can also perform subsequent analyses to quantify the concentration of the molecule.

[0198] Therefore, when a user acquires LFT images using a mobile device (e.g., a smartphone or tablet), the mobile device can be configured via software to perform the steps described herein, thereby quantifying the concentration of analyte molecules in the sample. Alternatively, the images can be transmitted to another computing system (computing system 100), such as the cloud, for quantification.

[0199] The quantitative concentration determined by processor 110 can be output via I / O interface 140 (e.g., screen or via network (e.g. Internet) to an external system), and can be used by processor 110 for further calculations, or can be stored in non-volatile memory 130 for later use.

[0200] Other components

[0201] The LFT device of the present invention may further include components for LFT optimization, consistent with conventional practice in the art. These additional components may be any suitable components known in the art for LFT or ELISA assays. For example, the LFT device may include a buffer for regulating the flow rate of the sample along the solid support structure, or a blocking agent for reducing nonspecific binding of conjugated analytes to the solid support structure and lowering background signal. The buffer for regulating the flow rate may be any suitable buffer, such as sucrose or glycerol. In some embodiments, the LFT device includes a buffer for regulating the flow rate, which is 5% glycerol. The blocking agent may be any suitable blocking agent known in the art, such as a blocking agent comprising BSA, PBS, and Tween-20.

[0202] Reagent test kit

[0203] The present invention also provides a kit comprising one or more of the above-described LFT devices. The kit may also include instructions for use of the LFT devices, and / or other reagents and / or components for carrying out the methods of the present invention. These other reagents and / or components may be, for example, swabs for obtaining samples (e.g., mucus or saliva samples), and mixing tubes or liquids for preparing liquid samples (e.g., mixing the mucus or saliva sample with a solution in a mixing tube before application to the LFT device). These other reagents and / or components may be packaged separately from the LFT devices within the kit, for example, in individual vials, test tubes, or pouches. Furthermore, the kit may also include an LFT reader for measuring the signal intensity of each test zone as described above. Alternatively, or additionally, the kit may include instructions for downloading and / or using software on a user device (e.g., a smartphone) to measure the signal intensity of each test zone.

[0204] Example

[0205] Example 1 – Detection of Caffeine

[0206] This embodiment demonstrates the use of an improved competitive LFT method ( Figure 3A method for colorimetric detection of caffeine is described. Unlike methods with only one test line, the LFT employs two test lines, both with signal intensities correlated with the concentration of caffeine present in the sample. The assay is based on an AuNP-BSA-caffeine-biotin conjugate as the detection complex, with immobilized anti-caffeine antibody and avidin serving as two separate test lines. Free caffeine in the sample competes with the gold-labeled conjugate for binding to the anti-caffeine antibody on the first test line, resulting in a signal intensity on the first test line that is inversely proportional to the concentration of caffeine present. The displaced conjugate is then captured by avidin on the second test line, thus the signal intensity on the second test line is positively correlated with the caffeine concentration. This improves the maximum sensitivity for detecting low concentrations of caffeine.

[0207] method

[0208] General Materials and Methods

[0209] NHS-LC-Biotin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), avidin, HRP-labeled anti-mouse secondary antibody, and HRP-labeled avidin were all purchased from Thermo Fisher Scientific. Monoclonal anti-caffeine antibody produced in mice was purchased from Stratech Scientific. Gold(III) chloride hydrate, bovine serum albumin (BSA), theophylline-7-acetic acid, caffeine, 2-(4-hydroxyphenylazo)benzoic acid (HABA), 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution, and phosphate-buffered saline (PBS) tablets were all purchased from Sigma Aldrich. All buffers were prepared with Milli-Q water. PD-10 columns pre-loaded with Sephadex G-25 media were purchased from GE Healthcare Life Sciences. Costa 3370 high-binding 96-well plates for enzyme-linked immunosorbent assay (ELISA) were purchased from Corning. The Hi-Flow Plus membrane HFB07502 used for lateral flow measurements was purchased from Merck Millipore. Test lines were fabricated on the nitrocellulose membrane by applying the appropriate antibody or avidin solution using a SciFlexArrayer instrument and a delivery piezoelectric dispensing capillary (PDC).

[0210] General technology

[0211] i) Ultraviolet-Vis Spectroscopy

[0212] UV-Vis spectroscopy analysis was performed using a Spectramax M3 microplate reader, with a wavelength range of 350 to 750 nm and a resolution of 1 nm. Blank values ​​were subtracted from the UV-Vis spectra as background correction, depending on the buffer solution used for each sample.

[0213] ii) Nanoparticle tracking analysis (NTA)

[0214] Nanoparticle tracking analysis (NTA) was performed using a Nanosight Halo LM10 detector equipped with a 635 nm laser. Samples were filtered through a 0.2 μm PTFE membrane and then injected into the sample chamber using a 1 mL syringe. The captured video (60 s) was processed using Halo 2.3 analysis software.

[0215] iii) Dynamic Light Scattering (DLS)

[0216] Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer Nano S instrument equipped with a 633 nm laser and employing DTS1061 disposable folded capillary cells. Each sample underwent two measurements, with each measurement run 20 times for 10 seconds. The z-mean and zeta potential of the samples were analyzed using Malvern Instruments dispersion technology 7.11 software.

[0217] iv) Transmission electron microscopy (TEM)

[0218] Nanoparticles were characterized using TEM on a JEOL JEM-2100F instrument operating at 200 kV. Samples (10 μL) were dropped onto a porous carbon membrane on a 300-mesh copper grid purchased from Agar Scientific. Excess sample was removed by bringing the edges of the copper grid close to filter paper. For samples requiring negative staining, 10 μL of 5% (w / v) ammonium molybdate solution was added after the sample, and excess solution was removed with filter paper. TEM experiments on the prepared samples were performed by the Department of Materials Science and Engineering, Imperial College London. Dimensions were measured using ImageJ software, with at least 50 particles measured for each sample and the average value calculated.

[0219] Synthesis of gold nanoparticles

[0220] A modified Frens method was employed. Aqua regia was prepared by mixing concentrated nitric acid and concentrated hydrochloric acid in a 1:3 volume ratio. All glassware used in the synthesis was thoroughly cleaned with aqua regia, rinsed with Milli-Q water, and dried overnight in an oven. Gold(III) chloride hydrate (30 mg, 0.0762 mmol) was dissolved in Milli-Q water (250 mL) to obtain a pale yellow solution. The solution was refluxed for 30 min. Then, sodium citrate (500 mg, 1.94 mmol) from Milli-Q water (5 mL) was rapidly added to the boiling solution. The solution exhibited a red wine-like color and was heated for another 10 min. It was then cooled to room temperature and filtered through a 0.2 μm PTFE membrane. The solution was then characterized by UV-Vis spectroscopy, NTA, DLS, and TEM.

[0221] Preparation and testing of BSA-Biotin-Caffeine conjugates

[0222] i) Biotinylation

[0223] The standard biotinylation protocol was followed. BSA (12 mg, 0.180 μmol) was dissolved in 1.2 mL of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 solution, and added to 25 μL of NHS-LC-biotin (1 mg, 2.20 μmol) in N,N-dimethylformamide (DMF). The reaction was allowed to proceed at room temperature for 3 hours, followed by overnight at 4°C. The sample was purified by gel filtration using 3 mL of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 solution as the elution buffer. Fractions containing biotinylated BSA were identified by recording absorbance at 286 nm. Each fraction was combined, and the concentration of BSA was determined using a standard curve.

[0224] ii) HABA determination

[0225] 4'-Hydroxyazobenzene-2-carboxylic acid (HABA) (24.2 mg, 0.100 mmol) was dissolved in Milli-Q water (9.9 mL). 1 M NaOH solution (100 μL) was added, and the solution was filtered through a 0.22 μm Millipore membrane. Avidin (10 mg) and HABA solution (600 μL) were added to phosphate-buffered saline (PBS) (19.4 mL) to form an orange solution. Each biotinylated sample (20 μL) was added to the HABA / avidin solution (180 μL). The absorbance at 500 nm was recorded and used to determine the biotinylation level according to the following formula:

[0226] Where ΔA 500 The absorbance change at 500 nm, ε HABA-亲和素 is the extinction coefficient of the HABA-avidin complex, l is the pool path length, and [BSA] is the concentration of BSA.

[0227] iii) Hapten conjugation

[0228] A theophylline-7-acetic acid solution (1.49 μmol, 89 μL, 4 mg / mL dissolved in DMSO) was added to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (20 mg). Biotinylated BSA solution (0.090 μmol, 600 μL, 10 mg / mL dissolved in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) was added. The reaction mixture was reacted at room temperature for 2 hours, then stored at 4 °C and subjected to gel filtration. The sample was purified by gel filtration using a solution (3 mL) of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 as the elution buffer. Fractions containing the highest protein concentrations were identified and combined by measuring absorbance at 286 nm.

[0229] iv) TNBS determination

[0230] The assay method reported by Sashidhar et al. (RB Sashidhar, AK Capoor and D. Ramana, J. Immunol. Methods, (1994, 167, 121-127). The BSA conjugate was diluted with 0.1 M NaHCO3 (pH 8.0) to obtain a final concentration of 200 μg / mL (1 mL). 0.01% (w / v) 2,4,6-trinitrobenzenesulfonic acid (TNBS) (500 μL, dissolved in 0.1 M NaHCO3) was added to each sample. The samples were incubated at 37.0 °C for 2 hours. 10% (w / v) SDS (500 μL) and 1 M HCl (250 μL) were added to each sample. The absorbance at 335 nm and 420 nm was recorded. Standard curves were obtained for comparison using the same method with L-lysine and L-glutamic acid. The concentration of amino groups in the samples was calculated using the absorbance at 335 nm. The degree of conjugation was determined by the difference in the number of amino groups per BSA before and after conjugation.

[0231] v) Capture enzyme-linked immunosorbent assay (ELISA)

[0232] A 96-well ELISA plate was coated with anti-caffeine antibody (50 μL, 6 μg / mL, dissolved in PBS buffer, pH 7.4) after incubation at room temperature for 1 hour. The plate was then blocked with 2% (w / v) BSA (in PBS, pH 7.4) containing 0.05% (v / v) Tween-20 and incubated at room temperature for 2 hours. Different concentrations of BSA-biotin-caffeine conjugates (50 μL) were added to the plate, and the plate was incubated at room temperature for 1 hour. HRP-labeled avidin (50 μL, diluted 1:5000 with blocking buffer) was added, and the plate was incubated at room temperature in the dark for 1 hour. After thorough washing, the plate was developed using a 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate system (50 μL). The reaction was terminated by adding 1 M H₂SO₄ (50 μL), and the absorbance at 450 nm was recorded.

[0233] vi) Competitive ELISA

[0234] The general operating procedure is the same as that for direct ELISA. The plate was coated with anti-caffeine antibody (50 μL, 6 μg / mL, in PBS, pH 7.4) and blocked with 2% (w / v) BSA (in PBS, pH 7.4) containing 0.05% (v / v) Tween-20. A mixture of BSA-biotin-caffeine conjugate (25 μL, 40 mg / mL) and different concentrations of caffeine (25 μL) was added to the plate and incubated at room temperature for 1 hour. HRP-labeled avidin (50 μL, 1:5000 dilution) was added, followed by TMB liquid substrate system (50 μL) for color development. The reaction was terminated by adding 1 M H2SO4 (50 μL), and the absorbance at 450 nm was recorded.

[0235] Preparation of AuNP-BSA conjugates

[0236] i) Salt-induced aggregation method

[0237] The minimum amount of BSA required to stabilize colloidal gold was determined using a salt-induced aggregation method. BSA solution (15 mg / mL in 0.05 M sodium phosphate buffer, pH 7.0) was serially diluted to obtain BSA solutions (25 μL) with concentrations ranging from 0.15 mg / mL to 5 mg / mL. Colloidal gold (250 μL) was added to each sample, and the solution was allowed to react for 5 min. Subsequently, 1.7 M NaCl solution (250 μL) was added, and the UV-Vis spectrum of each sample was recorded after 5 min.

[0238] ii) Adsorption method

[0239] Colloidal gold (5 mL) was added to BSA (3 mg) in 0.05 M sodium phosphate solution (200 μL). 1% (w / v) polyethylene glycol (PEG, MW 20000) (125 μL) was added to stabilize the reaction mixture, and the reaction mixture was reacted at room temperature for 2 h. The mixture was centrifuged at 12000 rpm for 40 min. The supernatant was removed, and the precipitate was reconstituted with 0.05 M sodium phosphate solution (5 mL) and 1% PEG (125 μL). The AuNP-BSA conjugate was characterized by UV-Vis spectroscopy.

[0240] iii) Reduction method

[0241] 2-Mercaptoethanol (0.50 μL, 25 mM) was added to the BSA-biotin-caffeine conjugate (300 μL, 0.697 mg, 2.32 mg / mL). The reaction mixture was thoroughly mixed and reacted overnight at 4°C. The sample was purified by gel filtration using a PD10 column with 3 mL of a solution of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 as the elution buffer. Fractions containing the protein conjugate were detected by UV-Vis spectroscopy at 286 nm and combined (600 μL) for the next step. The reduced conjugate was slowly added to colloidal gold (500 μL). The reaction mixture was reacted at 4°C for 2 days. The mixture was then centrifuged at 13400 rcf for 30 min, repeated three times, and washed each time with 10 mM Tris containing 3% (w / v) BSA. The precipitate was reconstituted in 10 mM Tris containing 3% (w / v) BSA and filtered through a 0.2 μm PTFE syringe filter. The conjugate was characterized by TEM using 5% (w / v) ammonium molybdate and 0.1% (w / v) trehalose as negative staining agents. Furthermore, the conjugate was characterized using UV-Vis spectroscopy, NTA, DLS, and TEM.

[0242] iv) Ellman assay

[0243] A assay method reported by Aitken and Learmonth was used (A. Aitken and M. Learmonth, The Protein Protocols Handbook,Humana Press, Totowa, NJ, 3rd edition, 2009. The Ellman reagent solution was prepared as follows: 5',5'-dithiobis(2-nitrobenzoic acid) (DTNB) (0.40 mg, 1.08 μmol) was dissolved in 5 mL of 0.1 M sodium phosphate and 1 mM EDTA solution, pH 8.0. Serial dilutions (0.25 mM to 1.5 mM) of cysteine ​​hydrochloride solutions were prepared using these solutions as standard solutions. 200 μL of the Ellman reagent solution was added to each sample (20 μL), and the mixture was allowed to react for 15 min. The absorbance at 412 nm was recorded, and a standard curve was generated using the cysteine ​​standard solution to determine the thiol content of each sample.

[0244] v) Assay using HABA (protease digestion method)

[0245] The AuNP-BSA-biotin-caffeine conjugate (25 μL) was heated at 56 °C for 10 min. 1% (w / v) proteinase K solution (2.5 μL) was added to the sample, and the mixture was left to stand overnight at room temperature. The sample was centrifuged at 13400 rcf for 5 min. 20 μL of the supernatant was added to 180 μL of HABA-avidin solution, and the absorbance at 500 nm was recorded.

[0246] Preparation for Lateral Flow Measurement (LFA)

[0247] i) Assembly of LFA

[0248] LFAs were assembled using a nitrocellulose membrane attached to a plastic backing card. Anti-caffeine antibody and avidin were immobilized on test lines 1 and 2 of the detection band, respectively. The membrane was dried at room temperature for 1 hour, then immersed in PBS solution of 2% (w / v) BSA (containing 0.05% (v / v) Tween-20) at pH 7.4 for 5 minutes to block excess binding sites. The membrane was dried at room temperature for 2 hours. An absorbent core was attached to the membrane (overlapping by 2 mm), and the plastic card was cut into strips (5 mm × 60 mm). A glass fiber conjugate pad was immersed in a 30% (w / v) sucrose Milli-Q aqueous solution and dried in an oven at 37°C. It was then immersed in an AuNP-BSA-biotin-caffeine conjugate and dried at room temperature for 2 hours. The conjugate pad was attached to the membrane, and the prepared strips were stored in a desiccator until use.

[0249] ii) LFA test strip test

[0250] Different concentrations of caffeine (dissolved in 2% (w / v) BSA) were prepared in each well of a low-binding 96-well microplate. Test strips were placed vertically into each well, removed after 3 minutes, and allowed to dry. Images of the test strips were photographed and analyzed using ImageJ software. The grayscale value of each test line pixel was measured and subtracted from the background.

[0251] Results and Discussion

[0252] Synthesis and characterization of BSA-biotin-caffeine conjugates

[0253] i) Synthesis Strategy

[0254] The conjugation of biotin to caffeine derivatives requires a carrier protein as a scaffold. This carrier protein must be relatively large, with a molecular weight exceeding 20,000 Da, and possess small-molecule conjugation sites. Furthermore, it is crucial that the chosen carrier protein differs from that used to generate the anti-caffeine antibody. Since the anti-caffeine antibody used in the lateral flow assay is generated in mice using keyhole hemocyanin (KLH)-3-caffeine, BSA was chosen as the carrier protein for the biotin-caffeine derivative conjugation. BSA (MW 67,000 Da) is highly water-soluble and stable, and has approximately 30 to 35 accessible lysine residues, making it suitable for the conjugation reaction. It is sold in pure form, is relatively inexpensive, and its use as a carrier protein is well-documented (B. Law and WN Jenner, in...). Immunoassay: A Practical Guide, (UK Taylor & Francis, London, 2005, pp. 16–21).

[0255] Figure 4 A synthetic strategy for preparing BSA-biotin-caffeine conjugates is shown. Biotin and caffeine are attached to BSA via primary amino groups on lysine residues.

[0256] ii) Biotinylation

[0257] BSA was biotinylated using NHS-LC-Biotin and purified by gel filtration. NHS-LC-Biotin contains a biotin moiety with an attached active ester group, which reacts with the ε-amino group of a lysine residue in BSA to form an amide bond.

[0258] The presence of the 6-aminocaproic acid spacer arm reduces steric hindrance and improves the accessibility of the biotin moiety to avidin binding (D. Kim and AE Herr, Biomicrofluidics 2013, 7, 41501).

[0259] Excess NHS-LC-biotin and N-hydroxysuccinimide (NHS) byproducts were removed by filtration of the reaction mixture through a desalting resin. Fractions containing the BSA-biotin conjugate were identified by absorbance at 286 nm in the gel elution profile.

[0260] Biotinylation levels were determined by measuring 4'-hydroxyazobenzene-2-carboxylic acid (HABA). HABA interacts with avidin at its biotin-binding site to form an orange complex with an absorption peak at 500 nm. This is due to the high binding affinity of biotin to avidin (1.3 × 10⁻⁶). 15 M -1 It is much higher than the binding affinity of HABA to avidin (6.0 × 10⁻⁶). 6 M -1 Therefore, adding biotin to the HABA-avidin complex can easily displace HABA from the biotin binding site (GTHermanson, Bioconjugate Techniques (Elsevier, 3rd edition, 2013). Upon addition of the complete BSA-biotin conjugate to the HABA-avidin complex, a decrease in absorbance observed at 500 nm compared to the HABA-avidin complex indicated successful biotinylation. The change in absorbance at 500 nm was used to calculate the concentration of biotin in the sample and the degree of biotinylation.

[0261] Different concentrations of biotinylation reagent were used, but when the molar excess of NHS-LC-biotin was large, BSA aggregation was observed. The optimal molar excess ratio of biotinylation reagent to BSA was 12, at which point each BSA molecule bound 1.8 biotin molecules, and no obvious aggregation was observed.

[0262] iii) Hapten conjugation

[0263] Since caffeine lacks suitable functional groups for covalent attachment to BSA, a caffeine derivative must be used for conjugation. Theophylline-7-acetic acid was chosen as the caffeine derivative and conjugated to BSA-biotin via its free carboxylic acid group. Water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was used as the coupling agent. EDC reacts with the carboxylic acid group on theophylline-7-acetic acid to form a highly reactive O-acylisourea intermediate. The ε-amino group of the lysine residue in BSA-biotin then reacts with the activated group to form an amide bond. The ideal pH for EDC-activated carboxylate is reported to be 3.5 to 4.5 (GT Hermanson, ...). Bioconjugate TechniquesElsevier, 3rd edition, 2013), but BSA-biotin precipitation was observed in MES buffer at pH 4.0. Li et al. (R. Li, Z. Wu, Y. Wangb, L. Ding and Y. Wang, Biotechnol. Reports, A study (2016, 9, 46–52) reported that the hydrophobicity of BSA increases at low pH values ​​because protein unfolding leads to increased accessibility of hydrophobic groups. Furthermore, biotinylation of BSA results in hydrophobic aliphatic biotin modification, thereby increasing the tendency of proteins to aggregate in solution. No aggregation was observed when BSA was used for conjugation in a MES buffer at pH 4.0, but aggregates appeared when the same conjugation was performed using BSA-biotin, indicating a need to increase the pH of the reaction buffer. Using sodium phosphate buffer at pH 7.2 as the reaction buffer resulted in successful amide formation without any protein aggregation.

[0264] The conjugate was purified by gel filtration to remove excess hapten, EDC, and isourea byproducts. Fractions containing the BSA-biotin-caffeine conjugate were identified by absorbance at 286 nm in the gel elution profile.

[0265] Hapten density is affected by the stoichiometry of reagents, coupling conditions, and the carrier protein used (GTHermanson, Bioconjugate Techniques Elsevier, 3rd Edition, 2013. Determining hapten density is crucial for optimizing the performance of BSA conjugates in immunoassays. Direct methods, such as measuring changes in protein absorbance, fluorescence, or mass, are commonly used to assess hapten density (R. Lemus and MH Karol, in...). Allergy Methods and Protocols, (MG Jones and P. Lympany, eds., Humana Press, Totowa, NJ, 2008, pp. 167–182). Although theophylline-7-acetic acid is a strong chromophore, its maximum absorption wavelength is 275 nm (JAOwen and K. Nakatsu, ). Clin. Chem., The absorbance wavelengths of the hapten (1978, 24, 367–368) significantly overlap with those of BSA. Therefore, absorbance in the ultraviolet region cannot be used to quantify the degree of conjugation. The amount of conjugated hapten was determined using an indirect spectroscopic method employing 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS reacts with primary amines on the protein surface to generate an orange derivative.

[0266] Successful conjugation of a hapten via lysine residues on BSA reduces the number of accessible primary amines in the BSA. The concentration of primary amines in the sample can be determined using absorbance at 335 nm by comparing it with a standard curve generated using L-lysine and L-glutamate. The difference in the number of free amines in the BSA before and after conjugation is used to determine the degree of conjugation.

[0267] According to the TNBS assay, the number of accessible primary amine groups in each BSA molecule before conjugation was 40.8. BSA contains 59 lysine residues, of which 30 to 35 are considered available for conjugation due to their proximity to the surface. Since the TNBS assay requires heating to 37°C, the unfolding of the BSA protein structure may result in more accessible lysine residues, leading to a higher-than-expected number of surface lysine residues. All samples underwent the same heat treatment; therefore, the degree of unfolding should be fairly consistent across samples, provided that conjugation does not affect protein stability. The degree of conjugation was determined by comparing the calculated values ​​of the three samples. The difference in the number of accessible primary amine groups between BSA and BSA-biotin was 2.4, consistent with the value of 1.8 obtained using the HABA assay. By comparing the absorbance of the BSA-biotin and BSA-biotin-caffeine conjugates, it was found that this conjugation reaction bound an average of 3.1 caffeine molecules per BSA molecule.

[0268] Synthesis and Characterization of AuNP-BSA Conjugates

[0269] i) Synthesis of AuNP

[0270] AuNP (G. Frens, G. Frens) was synthesized using the citrate reduction method reported by Frens. Nat. Phys. Sci., (1973, 241, 20–22). Citrate ions act as reducing agents, reducing Au(III) to Au(0), thereby promoting the growth of gold colloids. The negatively charged citrate ions also stabilize the surface of AuNPs during their formation (J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, and A. Plech, ). J. Phys. Chem. B, (2006, 110, 15700–15707). The size of the formed gold colloid depends on the concentration of the stabilizing ligand in the solution. The higher the concentration of the stabilizing ligand, the smaller the formed AuNP, because the ligand can stabilize the surface of the gold colloid and prevent its size from increasing further.

[0271] AuNPs were observed to be red using UV-Vis spectroscopy, with an absorption peak at 519 nm. Assuming the citrate coating was not visible, the average nuclear diameter of the AuNPs was determined to be 16 (±2) nm using transmission electron microscopy (TEM). This result is consistent with previous reports (N. Sosibo, F. Keter, A. Skepu, R. Tshikhudo, and N. Revaprasadu, Nanomaterials 2015, 5, 1211–1222).

[0272] ii) Synthesis of AuNP-BSA conjugates

[0273] The binding of proteins to gold nanoparticles mainly occurs through three types of interactions: hydrophobic interactions via tryptophan residues, electrostatic interactions via lysine residues, and gold-thiol bonds via cysteine ​​residues (M. Valcárcel and Á. López-Lorente, Gold Nanoparticles in Analytical Chemistry, Elsevier, 1st edition, 2014. Brewer et al. (SH Brewer, WR Glomm, MC Johnson, MK Knag, and S. Franzen, Langmuir, 2005, 21, 9303–9307) reported that passive adsorption of BSA onto AuNPs occurs primarily through electrostatic interactions between positively charged lysine residues on BSA and the negatively charged citrate coating. Ensuring sufficient protein conjugation to the colloidal gold surface is crucial. The stability of AuNPs depends on the balance between electrostatic repulsion and van der Waals attraction between AuNPs. Insufficient surface protein coating leads to instability of AuNPs in high-salt solutions.

[0274] Salt-induced aggregation method (C. Fang, Z. Chen, L. Li and J. Xia, J. Pharm. Biomed. Anal., (2011, 56, 1035–1040) was used to determine the minimum amount of BSA required to stabilize colloidal gold. Colloidal gold was added to solutions of different concentrations of BSA, followed by the addition of 1.7 M NaCl solution. Since the adsorption kinetics of BSA and the maximum amount of BSA bound to the AuNP surface depend on the BSA concentration, AuNPs with low BSA concentrations are unstable after the addition of NaCl. The salt solution causes flocculation of unstable AuNPs by shielding their negative charge and inducing aggregation. Flocculation can be manifested by a change in solution color from red to purple. With decreasing BSA concentration, a red shift of the absorption peak from 523 nm to 620 nm was observed after the addition of 1.7 M NaCl.

[0275] The absorbance of each sample at 523 nm and 620 nm was recorded, and its relationship with BSA concentration was plotted. The results showed that the minimum BSA concentration required to stabilize AuNP was 2 mg / mL, at which point no significant shift of the absorption peak from 523 nm to 20 nm was observed.

[0276] A BSA-biotin-caffeine conjugate was added to AuNP and incubated overnight for passive adsorption. However, the formed complex was observed to be unstable in solution. Even with a significant excess of the conjugate, a black, insoluble solid was observed at the bottom of the sample tube after the BSA-biotin-caffeine conjugate complexed with AuNP. The presence of the black solid indicates aggregation due to the poor stability of AuNP. Since biotin and caffeine derivatives are conjugated to BSA via lysine residues, the positive charge on the BSA surface is reduced. Therefore, the electrostatic interaction is weak, and the stability of AuNP is also poor. This adsorption method is considered unsuitable for conjugating the BSA-biotin-caffeine conjugate onto the AuNP surface.

[0277] Kaur et al. (J. Kaur, KV Singh, R. Boro, KR Thampi, M. Raje, GCVarshney, and CR Suri, Environ. Sci. Technol., (2007, 41, 5028–5036) reported that AuNP-protein conjugates formed via gold-thiol bonds are more stable than conjugates formed via electrostatic interactions between the protein and the AuNP surface. BSA has one free cysteine ​​residue and 17 disulfide bonds, which can be cleaved to provide free thiol groups for conjugation with AuNP (I. Rombouts, B. Lagrain, KA Scherf, MALambrecht, P. Koehler, and JA Delcour). Sci. Rep., (May 12, 2015, 2210). 2-Mercaptoethanol was used to reduce the intrachain disulfide bonds of BSA to generate free thiol groups that can bind to AuNP. The reduced BSA-biotin-caffeine conjugate was purified by gel filtration, and the concentration of thiol groups was determined spectroscopically using Ellman's reagent 5',5'-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB reacted with the free thiol groups in the sample to produce mixed disulfide bonds and the yellow product 2-nitro-5-thiobenzoic acid (TNB). The thiol concentration was calculated by comparing the absorbance of the solution at 412 nm with a standard curve generated using L-cysteine.

[0278] The difference in the number of thiol groups after BSA reduction was determined. After treatment with 2-mercaptoethanol, the number of free thiol groups on each BSA molecule increased from 0.9 to 2.7.

[0279] The free thiol group on BSA replaces the citrate ion stabilizing AuNP and interacts directly with the AuNP surface. No black solid was observed after the reduced BSA conjugated with AuNP, indicating that the AuNP is sufficiently stable. The resulting AuNP-BSA-biotin-caffeine conjugate remained stable after two months of storage at 4°C.

[0280] iii) Characterization of AuNP and AuNP-BSA conjugates

[0281] a) Ultraviolet-Visible Spectrum

[0282] Due to the oscillation of surface conductive electrons after light absorption, AuNP exhibits localized surface plasmon resonance (LSPR) (C. Larosa, E. Stura, R. Eggenhöffner and C. Nicolini, Materials (Basel), 2009, 2, 1193-1204). LSPR signals can be detected as strong absorption peaks in the visible region of the UV-Vis spectrum. The intensity and wavelength of the observed LSPR peak depend on a variety of factors, including the size and shape of the AuNP and the dielectric constant of the solution (X. Huang and MA El-Sayed, J. Adv. Res., 2010, 1, 13–28).

[0283] The maximum absorbance of citrate-stabilized AuNP was observed at 519 nm. A redshift from 519 nm to 523 nm after reaction with BSA indicates successful protein adsorption onto AuNP. The redshift of the LSPR peak is consistent with the results reported by Balog et al. (S. Balog, L. Rodriguez-Lorenzo, CA. Monnier, M. Obiols-Rabasa, B. Rothen-Rutishauser, P. Schurtenberger, and A. Petri-Fink). Nanoscale, 2015, 7, 5991–5997). Mie theory predicts that an increase in refractive index will cause a redshift of the maximum absorption peak (S. Dominguez-Medina, S. McDonough, P. Swanglap, CF. Landes and S. Link, Langmuir, 2012, 28, 9131–9139). The redshift of the peak wavelength may be due to the increase in the refractive index of AuNP after the protein is adsorbed onto the surface.

[0284] b) Dimensional characterization

[0285] Due to their high electron density, AuNPs can be visualized using TEM to characterize their size. AuNPs are spherical, monodisperse, and have a size of 16 (±2) nm. The size and quality of AuNPs are crucial for determining the sensitivity of lateral flow measurements (LFA) (C. Fang, Z. Chen, L. Li, and J. Xia, J. Pharm. Biomed. Anal., 2011, 56, 1035–1040). Small AuNPs with a diameter less than 10 nm migrate rapidly, but produce weak visible signals on the test line, limiting the sensitivity of the assay. AuNPs with a diameter greater than 40 nm produce strong red signals on the test line, but their slow migration through the membrane pores can cause steric hindrance to ligand-acceptor binding and result in poor flocculation stability (S. Lou, J. Ye, K. Li, and A. Wu, 2011, 56, 1035–1040). Analyst, (2012, 137, 1174). The obtained 16nm AuNPs were stable and provided a clearly visible signal in LFA. Due to their narrow size distribution and uniform shape, the synthesized AuNPs were considered to be of good quality.

[0286] Due to the poor electron scattering ability of proteins, heavy metals are needed as negative staining agents to visualize the BSA-biotin-caffeine conjugate bound to AuNPs. 5% (w / v) ammonium molybdate was used as the negative staining agent. A white halo was observed around each AuNP, indicating the presence of the BSA conjugate. The conjugation of BSA to the AuNP surface results in the formation of a protein shell around each AuNP, resulting in a spherical AuNP-BSA-biotin-caffeine conjugate. The size of the AuNP-BSA-biotin-caffeine conjugate is 19 (±2) nm. The shell thickness is approximately 3 nm, which is consistent with the reported diameter of BSA (M. Su, C. Wang, and C. Bai, ...). Chinese Sci. Bull., (1998, 43, 1882–1886). TEM measurements showed that a BSA monolayer formed around each AuNP. Since no particle aggregation was observed by TEM, the AuNP-BSA-biotin-caffeine conjugate exhibited good stability.

[0287] Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) have also been used as alternative techniques for characterizing the size of synthesized AuNP and AuNP-BSA-biotin-caffeine conjugates. NTA relies on tracking the Brownian motion of individual nanoparticles to calculate the nanoparticle size based on the Stokes-Einstein equation, while DLS relies on the intensity fluctuations of scattered light caused by the Brownian motion of the nanoparticles to determine the nanoparticle size (BJ Frisken, Appl. Opt., (2001, 40, 4087). The average diameter of AuNPs was measured to be 17 nm and 24 nm using NTA and DLS, respectively. The particle size reported using NTA is more consistent with the particle size determined by transmission electron microscopy (TEM). Since light scattering intensity is proportional to the sixth power of the nanoparticle diameter, DLS is more sensitive to larger particles, thus leading to an overestimation of the average nanoparticle size.

[0288] An increase in hydrodynamic diameter was observed when the BSA-biotin-caffeine conjugate bound to AuNP. The change in hydrodynamic diameter was measured to be 5 nm using NTA, and 7 nm using DLS. (Y. Li, G. Yang, and Z. Mei, ...) Acta Pharm. Sin. B, DLS measurements (2012, 2, 53–59) revealed a hydrodynamic diameter of 7.2 nm for BSA, while NTA and DLS measurements of the AuNP-BSA-biotin-caffeine conjugate supported the formation of a BSA monolayer around each AuNP core.

[0289] The average surface area of ​​16 nm AuNP is 804.25 nm. 2 Based on the diameter of the BSA molecule (3 nm) and the overall diameter of the AuNP, the theoretical maximum number of BSA molecules bound per AuNP with monolayer coverage was calculated to be 119. The number of BSA-biotin-caffeine molecules conjugated around each AuNP was experimentally determined using a proteinase K-digested HABA assay. Because the absorption region of AuNP overlaps with that of the HABA avidin complex, the HABA assay cannot be directly applied to AuNP-BSA-biotin-caffeine conjugates. The concentration of AuNP was determined by UV-Vis spectroscopy, a method based on the AuNP size and its extinction coefficient (ε) at 450 nm. 450 The correlation between (W. Haiss, NTK Thanh, J. Aveyard and DG Fernig, Anal. Chem., 2007, 79, 4215-4221). Using A ε 450 Value 2.67 × 10 8The concentration of AuNP was calculated from its absorbance at 450 nm. The AuNP-BSA-biotin-caffeine conjugate was digested with proteinase K to cleave the biotin moiety from the AuNP conjugate. AuNP was removed by centrifugation, and the supernatant was analyzed using HABA assay to determine the biotin concentration. The ratio of biotin moiety to AuNP was used to calculate the ratio of BSA molecules to AuNP. On average, each AuNP bound to 56 BSA molecules, which is lower than the theoretical maximum calculated based on the AuNP size. This corresponds to a surface coverage of 6.96 × 10⁻⁶. 12 BSA molecules / cm 2 , higher than Shi et al. (X. Shi, D. Li, J. Xie, S. Wang, Z. Wu and H. Chen, Chinese Sci. Bull., The minimum surface cover required to avoid AuNP aggregation, as reported in 2012, 57, 1109-1115, is 3.8 × 10⁻⁶. 12 BSA molecules / cm 2 This indicates that the BSA-biotin-caffeine conjugate on the AuNP surface plays a good stabilizing role.

[0290] c) Zeta potential characterization

[0291] The zeta (ζ) potentials of citrate-stabilized AuNP and AuNP-BSA-biotin-caffeine conjugates were characterized using DLS. Zeta potential measurements can indicate the stability of charge-stabilized nanoparticles. Particles with zeta potentials above 25 mV or below -25 mV exhibit sufficient electronic repulsion to prevent aggregation (SH Brewer, WR Glomm, MC Johnson, MK Knag, and S. Franzen, Langmuir, 2005, 21, 9303–9307). The synthesized AuNP had a zeta potential of -37.2 mV. The highly negative surface charge indicates that the citrate coating layer effectively stabilizes the AuNP. Upon conjugation of AuNP with a BSA-biotin-caffeine conjugate, the zeta potential increased from -37.2 mV to -31.5 mV. This suggests that when BSA binds to AuNP via a thiol-gold bond, the negatively charged citrate molecule undergoes a degree of displacement, supporting successful conjugation. Since BSA carries a negative charge at pH values ​​above its isoelectric point of 4.7, the overall surface charge remains negative.

[0292] d) Studies using avidin aggregation

[0293] The binding affinity of the AuNP-BSA-biotin-caffeine conjugate to avidin was examined by incubating the conjugate overnight. Avidin is a tetrameric protein with four biotin-binding sites. Kim et al. (WJKim, S.-H. Choi, Y.-S. Rho and D.-J. Yoo, Bull. Korean Chem. Soc., A report (2011, 32, 4171-4175) states that the specific interaction between biotin and avidin leads to the aggregation of biotinylated AuNPs. Upon addition of avidin, a slow color change from red to purple was observed. The degree of aggregation depends on the concentration of avidin present. Aggregation is observed when avidin bound to a biotin moiety on one AuNP can crosslink with adjacent AuNPs having unsaturated biotin sites. At low concentrations of avidin (0.1 μg / mL), no aggregation was observed because avidin was insufficient to bind to the biotin moiety on AuNPs. At high concentrations of avidin (1 μg / mL), AuNP aggregation was observed because each avidin bound to at least two biotin moieties on two different AuNPs. After overnight incubation, a significant broadening of the absorption peak and a red shift from 523 nm to 548 nm were observed. The redshift of the absorption peak is attributed to the LSPR coupling interaction caused by the conduction electron oscillation polarization between adjacent AuNPs linked by avidin molecules (Y. Yang, S. Matsubara, M. Nogami, and J. Shi, Mater. Sci. Eng. B, 2007, 140, 172–176). Peak broadening is due to variations in coupling interactions, as they are highly dependent on aggregate size and the orientation of AuNPs within the aggregate (K. Aslan, CC Luhrs, and VH Pérez-Luna, 2007, 140, 172–176). J. Phys. Chem. B, 2004, 108, 15631-15639).

[0294] TEM was used to observe the aggregation induced by the binding of avidin molecules to biotin on AuNP. The results indicate that the binding of the BSA-biotin-caffeine conjugate to AuNP did not hinder the binding of biotin to avidin. This also confirms that treatment of BSA-biotin-caffeine with 2-mercaptoethanol does not affect the binding of biotin to proteins.

[0295] Enzyme-linked immunosorbent assay (ELISA)

[0296] i) Capture ELISA

[0297] A capture enzyme-linked immunosorbent assay (ELISA) was performed to determine the binding affinity of the BSA-biotin-caffeine conjugate to anti-caffeine antibodies and avidin. A schematic diagram of the capture ELISA method used is shown below. Figure 2 As shown.

[0298] The wells were coated with anti-caffeine antibody, and excess binding sites were blocked with BSA. Other blocking agents, such as Tween-20 and the commercially available synthetic blocking agent (ELISA SynBlock), were tested, but the signal-to-noise ratios were low due to excessive non-specific binding. Casein-based blocking agents were avoided because trace amounts of biotin in them could interfere with the specific biotin-avidin interaction in the assay. Using 2% (w / v) BSA as the blocking agent ensured that the obtained signal was due to the specific binding of the BSA-biotin-caffeine conjugate to the anti-caffeine antibody, rather than to hydrophobic or electrostatic interactions between the conjugate and the wells.

[0299] Different concentrations of BSA-biotin-caffeine conjugates were added to the corresponding wells, along with HRP-labeled avidin. In the presence of horseradish peroxidase (HRP), the 3,3',5,5'-tetramethylbenzidine (TMB) substrate reacted to generate a chromogenic derivative. The degree of color development was proportional to the amount of BSA-biotin-caffeine conjugate bound. The absorbance of the solution at 450 nm was recorded, and a curve was plotted as a function of the BSA-biotin-caffeine conjugate concentration. The presence of the signal confirmed that the synthesized BSA-biotin-caffeine conjugate could successfully bind both anti-caffeine antibody and avidin simultaneously. With increasing BSA-biotin-caffeine conjugate concentration, more HRP-labeled avidin was captured by the biotin-avidin interaction, leading to an increase in absorbance observed at 450 nm. Because the saturation of the anti-caffeine antibody binding site increased at higher BSA-biotin-caffeine conjugate concentrations, the absorbance versus concentration curve was non-linear.

[0300] ii) Competitive ELISA

[0301] The same ELISA method was used to test the competitive binding of BSA-biotin-caffeine conjugate to free caffeine on anti-caffeine antibodies coated on wells of a plate. A fixed amount of BSA-biotin-caffeine conjugate (20 μg / mL) was used, while the concentration of free caffeine varied. Figure 5 As shown, a semi-logarithmic plot was plotted showing the relationship between absorbance at 450 nm and caffeine concentration. A dose-response model was used to fit the data, and the IC50 was found to be... 50 The value was 1 ng / mL. The limit of detection was determined to be 0.03 ng / mL by measuring the caffeine concentration at which the inhibition rate reached 10%.

[0302] Repeated competitive ELISA assays were performed using the AuNP-BSA-biotin-caffeine conjugate to determine whether the AuNP conjugate affects the binding affinity of the BSA-biotin-caffeine conjugate to anti-caffeine antibodies and avidin. Plots were drawn showing the binding affinity between the AuNP and BSA-biotin-caffeine conjugate and anti-caffeine antibodies and avidin. Figure 6 A similar semi-logarithmic plot. Fitting the data using a dose-response model revealed IC... 50 The value was 7 ng / mL. The detection limit was 0.4 ng / mL. The assay sensitivity decreased slightly when using the AuNP-BSA-biotin-caffeine conjugate, likely due to steric hindrance of the AuNP conjugate. Because the AuNP-BSA-biotin-caffeine conjugate is relatively large, binding of the AuNP conjugate to the antibody may hinder the binding of other AuNP conjugates to adjacent antibodies. However, the assay is sensitive enough for the intended application, as the range of caffeine detectable in blood is 0.1 μg / mL to 10 μg / mL.

[0303] ELISA results showed that the AuNP-BSA-biotin-caffeine conjugate retained its binding ability with both the anti-caffeine antibody and avidin. The competition established between the AuNP-BSA-biotin-caffeine conjugate and free caffeine can be used to accurately determine the caffeine concentration in the sample with a low detection limit.

[0304] Lateral flow measurement (LFA)

[0305] i) Optimization of measurement design

[0306] An LFA was developed by using AuNP-BSA-biotin-caffeine conjugate to competitively bind to anti-caffeine antibodies against free caffeine. Figure 7 The initial design of an LFA for caffeine detection is demonstrated. An anti-caffeine antibody was incubated with an excess of the AuNP-BSA-biotin-caffeine conjugate. After centrifugation to remove excess antibody, the resulting antibody-AuNP-BSA-biotin-caffeine complex was deposited on the conjugate pad. An anti-mouse antibody was coated onto the first test line to capture all anti-caffeine antibodies. Free caffeine present in the sample displaces the AuNP-BSA-biotin-caffeine conjugate from the anti-caffeine antibody, and the displaced AuNP-BSA-biotin-caffeine conjugate is captured by the second test line via a specific biotin-avidin interaction.

[0307] Preliminary tests using this method yielded an extremely weak signal at test line 1. This indicates poor binding of the anti-caffeine antibody-AuNP-BSA-biotin-caffeine complex to the secondary antibody. Partial dissociation of the anti-caffeine antibody from the complex may compete with the antibody-AuNP-BSA-biotin-caffeine complex, leading to saturation of binding sites on the anti-mouse antibody. The binding of the AuNP-BSA-biotin-caffeine conjugate to the anti-caffeine antibody may also cause steric hindrance to the binding of the secondary antibody. Epitopes on the caffeine antibody recognized by the anti-mouse antibody may be blocked by the AuNP label, resulting in a significant decrease in sensitivity. Therefore, the binding kinetics of this method are considered too slow and unsuitable for caffeine detection.

[0308] like Figure 3 As shown, an improved competitive LFA method without the need for a secondary antibody is proposed. Unlike previous methods that involved drying anti-caffeine antibodies and AuNP-BSA-biotin-caffeine conjugates on the conjugate pad, this method deposits the anti-caffeine antibody as the first test line. Therefore, the process of the AuNP-BSA-biotin-caffeine conjugate competing with free caffeine in the sample for caffeine antibodies occurs on the test line, rather than on the membrane.

[0309] ii) Working principle of the measurement

[0310] Figure 7 The working principle and expected test results of the LFA design are illustrated in the diagram. Figure 8 In the middle, the AuNP-BSA-biotin-caffeine conjugate is deposited on the conjugate pad. After the sample is applied, the liquid flows along the membrane and causes the gold conjugate to move. When caffeine is not present in the sample ( Figure 8 a) The AuNP-BSA-biotin-caffeine conjugate was captured by the anti-caffeine antibody on the first test line. When caffeine is present in the sample ( Figure 8 (b) Free caffeine competes with the AuNP-BSA-biotin-caffeine conjugate for binding to anti-caffeine antibodies. The AuNP-BSA-biotin-caffeine conjugate displaced from the first test line binds to avidin on the second test line. The signal intensity of the second test line is proportional to the concentration of caffeine in the sample.

[0311] iii) Optimization of LFA conditions

[0312] To determine performance, various conditions were optimized, such as the amount of conjugate, the concentrations of antibody and avidin on the test line, and the blocking of nonspecific binding.

[0313] a) Membrane sealing

[0314] A high background signal was observed when no blocking agent was used in the sample buffer. The AuNP-BSA-biotin-caffeine conjugate nonspecifically bound to the negatively charged nitrocellulose membrane via hydrophobic and electrostatic interactions, resulting in a pale pink background on the remainder of the nitrocellulose membrane after each run. Washing the membrane with PBS buffer after the run did not remove the background signal, thus requiring the use of a blocking agent to reduce nonspecific binding. The same blocking buffer used for ELISA (2% (w / v) BSA in PBS containing 0.05% (v / v) Tween-20) was tested in LFA and found to significantly reduce the background signal.

[0315] b) Lateral flow velocity

[0316] The capillary flow rate decreases exponentially through the membrane (GA Posthuma-Trumpie, J. Korf and A. vanAmerongen, Anal. Bioanal. Chem., 2008, 393, 569–582), therefore, test lines farther from the sample pad allow more time for interaction between the ligand and receptor. Because the anti-caffeine antibody test line is positioned close to the sample application site, excessively high sample flow rates may result in insufficient time for the AuNP-BSA-biotin-caffeine conjugate to react with the anti-caffeine antibody. The migration rate of the AuNP-BSA-biotin-caffeine conjugate can be slowed by introducing an active agent that reduces the rate of lateral water absorption from the membrane. For example, sucrose is commonly used in paper-based assays to reduce flow rates (B. Lutz, T. Liang, E. Fu, S. Ramachandran, P. Kauffman and P. Yager, in 16 th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Okinawa, Japan, 2012, Vol. 20, pp. 788-790), because of its excellent water solubility and flow resistance characteristics (WO 2005069007 A1, 2005, 1-25). A 30% (w / v) sucrose was dried on the conjugated pad to form a sucrose glaze. When the sample was applied to the membrane, the sucrose glaze dissolved, thus slowing the flow rate along the membrane. A decrease in the intensity of the second test line was observed relative to the first test line; however, the reduced flow rate was still insufficient to capture all AuNP-BSA-biotin-caffeine on the first test line. Higher concentrations of sucrose were not tested because concentrations above 50% (w / v) were found to be too viscous and difficult to handle. After drying on the conjugated pad, the sucrose glaze significantly hardened the conjugated pad and may interfere with the capture and release of the AuNP-BSA-biotin-caffeine conjugate on the conjugated pad.

[0317] Hua et al. (F. Hua, P. Zhang, F. Zhang, Y. Zhao, C. Li, C. Sun, X. Wang, R. Yang, C. Wang, A. Yu and L. Zhou, Sci. Rep., A report dated May 2015, 17(178) stated that the use of PEG and glycerol could affect the signal intensity of the test and control lines in LFA due to the high viscosity of the solution. Therefore, the possibility of using glycerol in LFA optimization was explored. Figure 9 As shown, different concentrations of glycerol were tested. Two anti-caffeine antibody lines were added to the first test zone, and one avidin line was added to the second test zone. The second anti-caffeine antibody line was intended to capture any excess AuNP-BSA-biotin-caffeine conjugate that migrated past the first anti-caffeine antibody line. However, this still failed to capture all of the AuNP-BSA-biotin-caffeine conjugate before it reached the avidin test line. When no glycerol was used, three lines were observed on the test strips run with only the buffer solution. Although the addition of 2.5% (v / v) glycerol reduced the signal intensity of the second test zone, it was still insufficient to capture all the conjugates in the first test zone. Figure 9 ).

[0318] It was observed that the addition of 10% (v / v) glycerol was sufficient to capture all AuNP-BSA-biotin-caffeine conjugates on the first test line. However, tests using a sample containing 100 μg / mL caffeine failed to produce a positive signal on the second test line. Figure 9 The presence of glycerol excessively slows the migration of the AuNP-BSA-biotin-caffeine conjugate, preventing free caffeine from competing with the conjugate for anti-caffeine antibodies in the first assay line. The molecular weight of free caffeine molecules in the sample is much lower than that of the AuNP-BSA-biotin-caffeine conjugate, therefore, even in the presence of glycerol, they migrate across the membrane much faster than the conjugate. Kim et al. (YA Kim, EH Lee, KO Kim, YTLee, BD Hammock, and HS Lee, Anal. Chim. Acta, (2011, 693, 106-113) It was previously reported that the relative migration rate between the two competing molecules is crucial for the sensitivity of competitive LFAs. Although the analyte in the sample should migrate before the conjugate, the migration rate of the analyte only needs to be slightly higher than that of the conjugate in order to achieve sufficient overlap between the two competing molecules on the membrane.

[0319] The study found that 5% (v / v) glycerol was the optimal concentration for slowing the migration of the AuNP-BSA-biotin-caffeine conjugate without adversely affecting the competition between the conjugate and free caffeine. Figure 9 Most of the AuNP-BSA-biotin-caffeine conjugates were successfully captured by the anti-caffeine antibody in the first test band. When running samples containing 100 μg / mL caffeine, the migrating AuNP-BSA-biotin-caffeine conjugates competed with free caffeine, resulting in a strong signal in the second test band.

[0320] iv) Sensitivity test

[0321] To determine the working range and sensitivity of LFA in detecting caffeine, the effect of applying different concentrations of caffeine on LFA was studied. LFA test strips were prepared according to... Figure 3 The assay design shown was prepared, and caffeine solutions with concentrations ranging from 1 ng / mL to 100 μg / mL were used for detection. The optimized run buffer contained glycerol as an active agent to regulate the water absorption rate; and BSA and Tween-20 as blocking agents to reduce nonspecific binding. The test strip results are shown below. Figure 10 As shown.

[0322] As caffeine concentration increases, the intensity of test line 1 decreases accordingly, consistent with expectations for competitive assays. No color development occurs at test line 1 when caffeine concentration exceeds 10 μg / mL. This is the visual detection limit for caffeine in a sample without calibration. This is an inherent limitation of traditional competitive assays, as the test line always has a signal unless the analyte concentration is sufficiently high to fully saturate it. Without a standard curve to calibrate the relationship between signal intensity and target analyte concentration, this method can only provide qualitative results for the presence of caffeine at concentrations above 10 μg / mL. This is below the effective working range required for detecting caffeine in blood. By using silver (R.-H. Shyu, H.-F. Shyu, H.-W. Liu and S.-S. Tang, ...), ... Toxicon, 2002, 40, 255–258) or gold enhancement methods (J. Kaur, KV Singh, R. Boro, KR Thampi, M. Raje, GC Varshney and CRSuri, Environ. Sci. Technol., (2007, 41, 5028–5036) Amplifying the signal can improve the sensitivity of the measurement. However, this method is not ideal because it loses the convenience of one-step LFA.

[0323] This limitation was overcome by setting up a second test line to capture the AuNP-BSA-biotin-caffeine conjugate displaced from its binding to the anti-caffeine antibody by free caffeine in the sample. The signal intensity of test line 2 increased accordingly with increasing caffeine concentration in the sample. Figure 10 Using the second test line increases the visual detection limit to 10 ng / mL. This improves the sensitivity of caffeine detection by 1000 times. Compared to negative results produced by traditional competitive assays, the positive signal of test line 2 makes qualitative analysis of caffeine content in samples more direct when caffeine concentration is above 10 ng / mL. The increased sensitivity makes this assay suitable for testing blood samples with caffeine concentrations between 0.1 μg / mL and 10 μg / mL.

[0324] In addition to visual inspection, ImageJ software was used to analyze the signal strength of the test leads. Figure 11 The signal intensities of test lines 1 and 2 at different caffeine concentrations are shown, and semi-logarithmic plots are plotted (three copies of the measurement results). IC50 values ​​for test lines 1 and 2 are also shown. 50 The values ​​were 5 ng / mL and 8 ng / mL, respectively. These IC50 values... 50 The values ​​are comparable to those obtained by competitive ELISA. As shown in Table 1, the caffeine content in the sample can be semi-quantitatively analyzed by comparing the ratio of the signal intensities of the two test lines.

[0325] Table 1 – Semi-quantitative analysis of caffeine concentration based on the signal intensity ratio of test lines 1 and 2.

[0326]

[0327] v) Stability of the test strip

[0328] The shelf-life stability of LFA test strips is crucial because the strips are typically mass-produced and stored for a period of time before use. This study investigated the stability of the reagents printed on a nitrocellulose membrane and the AuNP-BSA-biotin-caffeine conjugate deposited on the conjugated pad. After preparation, the test strips were placed in a desiccator and stored at room temperature for 7 days. Tests were performed using different concentrations of caffeine, and the results are as follows: Figure 12 As shown. For test line 2, the visual detection limit for caffeine concentration remained at 10 ng / mL.

[0329] The signal strengths of test lines 1 and 2 at different caffeine concentrations are plotted as semi-logarithmic graphs, and the IC values ​​of test lines 1 and 2 are... 50The values ​​were 3 ng / ml and 11 ng / ml, respectively. After one week of storage, the assay sensitivity did not change significantly, indicating that the reagents maintained good stability and reactivity after drying on the membrane. Traditional competitive and sandwich LFAs both include a positive control line to verify the validity of the test results by ensuring the reactivity of the reagents present in the assay. Although this method does not include a control line, the two test lines can serve as cross-validation; the presence of at least one test line indicates that the assay is valid. If the reagents on the test strip degrade, the reactivity of both the anti-caffeine antibody and avidin will decrease, and neither test line will develop color.

[0330] in conclusion

[0331] A competitive light-emitting fiber (LFA) for the chromogenic detection of caffeine was successfully developed, with signal intensity proportional to the caffeine concentration in the sample. The binding affinity of the AuNP-BSA-biotin-caffeine conjugate to avidin and anti-caffeine antibodies was verified using ELISA. By optimizing the design, blocking conditions, and flow rate of the improved competitive LFA method, an LFA suitable for caffeine concentration detection was developed.

[0332] The main advantage of this LFA method lies in its high sensitivity and positive reading. Introducing a second test line increases sensitivity by 1000 times, bringing the visual detection limit for caffeine down to 10 ng / mL. Compared to commercially available LFAs like caffeine detection 74, which lack the required sensitivity, this method's measurement range is more suitable for detecting caffeine concentrations in blood samples. Furthermore, this test method is rapid and convenient, providing qualitative results by visually observing the test strip just 5 minutes after sample application. No special equipment or additional cleaning steps are required, and only a small amount of sample is needed for testing. Therefore, this test method can easily be implemented using finger-prick blood sampling.

[0333] This embodiment also serves as a proof-of-concept for a competitive assay capable of detecting small molecule target analytes with a positive reading. This overcomes the limitations of traditional competitive assays, which typically suffer from low sensitivity and narrow analyte detection ranges. By modifying a BSA-conjugated hapten and immobilizing an antibody on the first test line, this competitive assay can be applied to the detection of other small molecules. Using two test lines allows for cross-referencing of results, and the signal intensity ratio of the two test lines can provide semi-quantitative data. This one-step assay requires no washing, enhancement, or calibration steps, is relatively low-cost, and is convenient for use as a point-of-care test (POCT).

[0334] Example 2 – Detection of NHPA

[0335] The aim of this study was to develop an in vitro diagnostic assay to measure NHPA in urine in the form of an improved competitive lateral flow immunoassay, and to demonstrate its performance (sensitivity, limit of detection, linearity) using laboratory (i.e., non-clinical) samples.

[0336] The focus of this work is to provide an instant (POC) test for detecting NHPA in solution using a 3-nitrotyrosine (3-NTyr) antibody and lateral flow assay (LFA).

[0337] Currently, no specific antibodies against NHPA have been reported; however, BSA conjugates of NHPA have been reported to bind to monoclonal antibodies against 3-nitrotyrosine (3-NTyr), therefore such antibodies are expected to bind to both NHPA and 3-NTyr. Although 3-NTyr is also present in urine, its concentration is hundreds of times lower than that of NHPA, and therefore is not expected to interfere with the assay. The following work aims to evaluate the feasibility of detecting NHPA in urine using a 3-NTyr antibody in an improved competitive assay.

[0338] A-Conjugate Synthesis

[0339] i) Coupling of biotin and BSA

[0340] 10% BSA (20 mg, 0.3 μmol) was dissolved in a 2 mL solution of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, and added to NHS-LC-biotin (1.12 mg, 2.46 μmol) in 40 μL of dimethyl sulfoxide (DMSO). The reaction was carried out on ice for 2 hours. The sample was then purified by gel filtration using 20 mM borax buffer (pH 8.0) as the elution buffer. Fractions containing biotinylated BSA were identified and combined by recording absorbance at 280 nm.

[0341] To quantify the biotinylation level, a solution containing the biotinylated protein was added to a mixture of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) and avidin. Due to its higher affinity for avidin, avidin displaces HABA, resulting in a proportional decrease in absorbance at 500 nm.

[0342] The amount of biotin present in the solution was quantified by measuring the absorbance of the HABA-avidin solution before and after the addition of the biotin-containing sample, using the same equation as in Example 1 above.

[0343] The biotinylation reaction was successful, and calculations showed that the synthesized product contained an average of 3.3 biotin molecules per BSA molecule.

[0344] ii) Coupling of NHPA with biotinylated BSA

[0345] A solution of 3-nitro-4-hydroxyphenylacetic acid (NHPA) (1.43 μmol, 5.64 μL, 50 mg / mL, dissolved in ethanol) was added to a deionized aqueous solution (3000 μL, pH 4.5) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (6.85 mg). Then, the biotinylated BSA solution prepared in step 1 (0.24 μmol, 3000 μL, 5.28 mg / mL, dissolved in 20 mM borax buffer, pH 8.0) was added. The reaction mixture was placed on a vortex mixer and reacted at room temperature for 2 hours, followed by overnight reaction at 4°C. The sample was purified by gel filtration using 2 mM borax buffer (pH 8.0) as the elution buffer. Fractions containing the highest protein concentrations were identified and combined by measuring absorbance at 280 nm.

[0346] Spectroscopic analysis using calibration curves was performed to quantitatively assess the success of the reaction. Calculations showed that the synthesized product contained an average of 3.6 NHPA molecules per BSA molecule.

[0347] iii) Conjugation of NHPA-Biotin-BSA with gold nanoparticles

[0348] Spectroscopic analysis of the gold nanoparticles (PBS-stabilized) showed a distinct peak at 523 nm, consistent with expectations for 20 nm nanoparticles.

[0349] First, the gold nanoparticles (GNP) (1 mL, 1 OD) were centrifuged at 14000 rpm for 5 min. The solution became clear, and a nanoparticle precipitate formed at the bottom of the Eppendorf tube. The supernatant was carefully removed, avoiding disturbing the precipitate. BSA-biotin-NHPA conjugate (400 μg / mL, 100 μL) in 2 mM borax buffer (pH 8.0) was added to the GNP precipitate. The mixture was incubated by rotation at room temperature for 4 h.

[0350] The conjugate was then centrifuged at 8500 rpm for 15 min, the supernatant was removed, and the GNP was resuspended in 1 mL of buffer containing 20 mM tris, 2% BSA, 4% sucrose, 5% glycerol, 0.05% Tween 20, and pH 7.2.

[0351] B - Urine test strip test

[0352] This section evaluates the performance of conventional LFA for NHPA detection under real-world background conditions.

[0353] i) Lateral flow measurement assembly

[0354] LFA is assembled using nitrocellulose membrane attached to a plastic backing card. Figure 13 Anti-3-nitrotyrosine antibody lines were printed at a concentration of 1 mg / mL, consisting of 20 lines, each 25 μm wide, using 0.1 μL antibody solution / cm / line. Streptavidin lines were printed at a concentration of 1 mg / mL, consisting of 10 lines, each 50 μm wide, using 0.1 μL streptavidin solution / cm / line. The membranes were sealed in aluminum foil bags containing a desiccant silicone bag and refrigerated until use.

[0355] On the day of testing, absorbent pads (5 × 17 mm) were attached to the membrane (overlapping 2 mm), and plastic cards were cut into strips (5 mm wide). Glass fiber conjugate pads (5 × 7 mm) were immersed in AuNP-BSA-Biotin-NHPA conjugate (0.5 OD) and dried in a desiccator for 2 hours. The conjugate pads were then attached to the membrane (overlapping 2 mm). Sample pads (5 x 17 mm) were immersed in running buffer (20 mM Tris, 2% (w / v) BSA, 4% (w / v) sucrose, 5% (v / v) glycerol, 0.05% (v / v) Tween 20, pH 7.2), dried overnight at room temperature, and then attached to the membrane, overlapping the conjugate pads by 2 mm.

[0356] ii) LFA test strip test

[0357] Different concentrations of NHPA in urine (200 μL) were prepared in each well of a low-binding 96-well microplate. Test strips were placed vertically into each well until the liquid flow front reached the end of the absorbent pad, then removed and imaged. The images of the test strips were analyzed using ImageJ software. Figure 14 ImageJ analysis showed two peaks in the streptavidin line and the antibody line. Figure 15 a). Measure the grayscale value of each pixel in the test line and subtract that grayscale value from the background to calculate the intensity of each line and its relationship with the NHPA concentration. Figure 15 b).

[0358] As the concentration of NHPA increases, the amount of AuNP-BSA-biotin-NHPA conjugate captured by the anti-3-nitrotyrosine antibody decreases, resulting in a linear decrease in the signal intensity of the antibody test line. Figure 15 (b) When the NHPA concentration in the urine sample was 250 ng / ml, the intensity of the antibody line decreased by more than 90%.

[0359] The repeatability of the assay was assessed using three test strips for each concentration of NHPA. Figure 16 Then it was compared with two other batches of test strips prepared by AuNP. The results are summarized in Figure 17 The figure shows the results of antibody line analysis using ImageJ software, where each data point is the average of five test strips. Analysis of the test lines on all test strips showed good repeatability. Figure 17 ).

[0360] Figure 17 The reaction showed a linear response with NHPA concentrations ranging from 0 to 150 ng / ml, after which the response plateaued, indicating that all antibody sites were occupied by NHPA in solution.

[0361] Conclusion: These results clearly demonstrate an effective assay for detecting NHPA in urine, with a threshold range of 170–330 ng / ml (250 ± 80 ng / ml). The proposed method is suitable for distinguishing between samples above and below the threshold, classifying them as positive and negative samples, respectively. Figure 18 The results showed that the positive sample only showed a visible band at the streptavidin test line, which was consistent with expectations and indicated that the assay had good repeatability.

[0362] Example 3 – Detection of Creatinine

[0363] Using a method similar to that described in Example 1, Figure 3 The LFT design shown is used to detect creatinine.

[0364] method

[0365] Synthesis of gold nanoparticles

[0366] Gold(III) chloride hydrate (30 mg, 0.0762 mmol) was added to Milli-Q water (250 mL), and the mixture was refluxed for 30 minutes. Then, sodium citrate solution (500 mg, 1.94 mmol) was added. Heating was continued for another 30 minutes, followed by cooling and filtration through a 0.2 mM PTFE membrane to obtain a red solution. The solution was characterized using UV-Vis spectroscopy and NTA. This is an improvement on the Frens method.

[0367] Synthesis of creatinine derivatives

[0368] Ethyl 4-bromobutyrate (26.6 mL, 100 mmol) was added to creatinine (9 g, 80 mmol) in DMF solution (50 mL), and heated at 85 °C for 4 hours. After cooling, ethyl acetate (500 mL) was added, forming a precipitate (12.71 g, 52% yield), which was then separated by filtration. The precipitate (3.1 g, 10 mmol) was added to KOH (10 mmol) in methanol solution (25 mL). Methanol was evaporated to remove the precipitate, and ethanol was added. The salts of the precipitate were removed by vacuum filtration, and the filtrate was heated to evaporate the ethanol. The solution was then added to NaOH (0.5 M, 10 mL), and heated under reflux for 6 hours. Ethanol was removed by heating, and concentrated hydrochloric acid was added to adjust the pH to 3. Water was evaporated to remove the precipitate, giving a yellow oil and NaCl, which was removed by centrifugation. The product was characterized by NMR, mass spectrometry, and IR.

[0369] Biotin conjugation with BSA

[0370] NHS-LC-Biotin (2 mg, 4.40 μmol) was dissolved in DMF (50 ml) and added to BSA (24 mg, 0.360 μmol) dissolved in 1.2 ml buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2). The mixture was incubated at room temperature for 5 hours and then gel filtered using the same buffer. The different fractions were analyzed by 286 nm UV-Vis spectroscopy, and the fractions with the highest absorbance were combined.

[0371] HABA assay

[0372] 4'-Hydroxyazobenzene-2-carboxylic acid (HABA) (25 mg, 0.103 mmol) was added to Milli-Q water containing 1 M NaOH (100 mM). Avidin (12 mg) was dissolved in PBS (19.4 ml, 0.02 mM, pH 7.4), and the filtered HABA solution (600 ml) was added. The mixture was added to the biotinylated sample, and the absorbance at 500 nm was recorded.

[0373] The conjugation of creatinine and BSA

[0374] The creatinine derivative (20 μL) was added to phosphate buffer (80 μmol, pH 7.4) containing 20 mg of EDC. The mixture was incubated at room temperature for 4 hours, followed by gel filtration purification using the same phosphate buffer. Maximum protein concentration was determined using UV-Vis spectroscopy.

[0375] TNBS Measurement

[0376] The BSA conjugate (200 μg / ml) was added to 2,4,6-trinitrobenzenesulfonic acid (TNBS) (500 μL, dissolved in 0.12 M NaHCO3) and incubated at 37 °C for 3 hours. The absorbance at 335 nm was recorded to calculate the concentration of amino groups.

[0377] Capture ELISA

[0378] Anti-creatinine antibody (50 μL, 8 μg / ml) was added to a 96-well ELISA plate. After 1 hour, 2% BSA in PBS solution (10 μL) was added. Different concentrations of BSA-biotin-creatinine (50 μL) were then added. HRP-labeled avidin (50 μL) was added, and the plate was incubated in the dark for 1 hour. The plate was then washed four times with PBS buffer, and TMB (50 μL) was added to develop a blue color. The solution turned yellow after adding H2SO4 (50 μL, 1M). Finally, the absorbance was measured at 450 nm.

[0379] Competitive ELISA

[0380] The same method as described above was used. However, in this case, the volume of the BSA conjugate was maintained at 25 μL, and different concentrations of creatinine were added, with a total volume of 50 μL.

[0381] Conjugation of gold with BSA

[0382] The reduction method described in Example 1 was used. The BSA conjugate was added to 0.50 μL of 2-mercaptoethanol (25 mM) and incubated at room temperature for 6 hours. Gel filtration was then performed using phosphate-buffered saline (PBFS). 1000 μL of the sample was added to the filtered gold particle solution (1 ml) and incubated at 4°C for 3 days. The mixture was centrifuged at 13,400 rcf for 1 hour, and the supernatant was removed. The precipitate was dissolved in 3% BSA solution (1 ml, 10 mM Tris buffer). Characterization was performed using NTA and UV-Vis spectroscopy.

[0383] Ellman assay

[0384] Ellman's reagent was prepared by adding 0.42 mg (1.22 μmol) of 5',5'-dithiobis(2-nitrobenzoic acid) (DTNB) to sodium phosphate buffer (0.1 M, pH 8, 1 mM EDTA, 5 mL). The reduced BSA sample (20 μL) was added to Ellman's reagent (180 μL), and the absorbance at 412 nm was recorded using UV-Vis spectroscopy.

[0385] LFA test strip manufacturing

[0386] Attach the sample pad to the backing card. Attach a nitrocellulose membrane and fix the anti-creatinine antibody and avidin onto two straight lines (the first line is the antibody, the second line is avidin). Add 1% BSA solution to the membrane and dry. Prepare a test strip and attach an absorbent core to the other end. Immerse the conjugated pad in a 30% sucrose solution and add the AuNP-BSA-biotin-creatinine conjugate twice, then dry for 2 hours. Attach the conjugated pad to the test strip.

[0387] LFA test strip test

[0388] Creatinine solutions of different concentrations were prepared and added to the wells of a 96-well plate. The test strips were placed into the wells after the sample pads were immersed in the solutions for 10 minutes. After drying, photographs were taken and analyzed using ImageJ software. The obtained values ​​were plotted as a semi-logarithmic graph. Figure 20 ).

[0389] Results and Discussion

[0390] Biotinylation

[0391] Similar to Example 1, the biotin conjugated with BSA was quantified using the HABA assay. The biotin to BSA ratio was determined to be 2.2 biotin / BSA.

[0392] Synthesis of creatinine derivatives

[0393] Creatinine itself does not contain any carboxylic acid groups to which it is attached; therefore, a derivative containing an acidic group needs to be synthesized, which can then form an amide bond with BSA. The steps for synthesizing the creatinine derivative are as follows, as shown in Scheme 1.

[0394] Step 1: Creatinine reacts with ethyl 4-bromobutyrate to produce a yellow solid intermediate, namely ethyl 4-(2-imino-3-methyl-5-oxoimidazolidine-1-yl)butyrate hydrobromide.

[0395] Step 2: Then neutralize the hydrobromic acid with the base KOH.

[0396] Step 3: Ethyl 4-(2-imino-3-methyl-5-oxoimidazolidine-1-yl)butyrate is heated under reflux with an alkali to remove the ester group.

[0397] Step 4: The sodium carboxylate salt is converted to 4-(2-imino-3-methyl-5-oxoimidazolidine-1-yl)butyric acid using a strong acid. This substance is a creatinine derivative. The generated NaCl is deposited at the bottom.

[0398] Option 1:

[0399] The creatinine derivatives were then characterized by NMR, IR, and mass spectrometry.

[0400] i) Infrared spectroscopy (IR)

[0401] The carboxylic acid group attached to the creatinine molecule was confirmed using IR spectroscopy. (Leftmost point 3375 cm) -1 The broad peak at 1697 cm⁻¹ confirms the stretching vibration of the OH group in the COOH group. -1 The sharp peak at the point confirms the stretching vibration of the C=O group in the COOH group. As shown in Table 2, these values ​​are all within the range reported in the literature.

[0402] Table 2 – IR Values

[0403] ii) NMR

[0404] Performed 1 HNMR and 13 CNMR. The structure of the creatinine derivative was compared with reported values ​​for creatinine and 4-aminobutyric acid (H₂N-CH₂-CH₂-CH₂-COOH). The values ​​of the creatinine moiety in the product were within the reported range. The values ​​of the carboxylic acid moiety in the product were also within the reported range for 4-aminobutyric acid. The value of one CH₂ moiety of the carboxylic acid was outside the reported range—the HNMR range should be 3.25 to 3.75 ppm, but the observed value was 4.36 ppm. This difference is reasonable because the values ​​in the references refer to the CH₂ atom attached to NH₂, while the CH₂ in the creatinine derivative is attached to a nitrogen atom that is part of a five-membered ring. Intra-ring resonances cause electron deshielding of the atoms attached to it. The greater the degree of deshielding, the more leftward the chemical shift. The same phenomenon was observed in the CNMR. All values ​​were within the range except for the CH₂ (70.61 ppm) which was the same as in the discussion related to HNMR. Therefore, this higher chemical shift is due to electron deshielding caused by the resonance of the ring structure.

[0405] No OH groups (COOH) were observed in the 1H NMR spectrum. The 1H NMR uses heavy water (D₂O) as the solvent, and hydrogen (H) and deuterium (D) in the solution can exchange to form COOD and HOD. Deuterium is NMR active, but its signal energy is different, so it cannot be observed in the NMR spectrum. Therefore, no OH signal was observed in the 1H NMR spectrum.

[0406] carbonyl carbon in13 No signal was detected in the CNMR spectrum. However, IR has confirmed the presence of carboxylic acid groups in the creatinine derivative.

[0407] iii) Mass spectrometry analysis

[0408] The creatinine derivative was characterized using electrospray ionization. The molecular weight of the creatinine derivative was 199, and a large peak appeared at 200, confirming the presence of the product.

[0409] Therefore, a creatine derivative containing a carboxylic acid group was successfully synthesized, which can be linked to BSA via an amide bond.

[0410] Creatinine derivative conjugation

[0411] This creatinine derivative containing a carboxylic acid group can bind to BSA. EDC is added to activate the derivative. The carboxylic acid group of the derivative reacts with EDC to generate an intermediate, which can then form an amide bond with a lysine residue in BSA. The mixture is purified by gel filtration to remove excess creatinine derivative, EDC, and generated byproducts.

[0412] The BSA conjugate content was highest in the first three fractions, so these three fractions were combined for the next step.

[0413] To determine the amount of creatinine conjugation, 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used, and the results are shown in Table 3 below.

[0414] Table 3 – Determination of amino group substitution by TNBS assay

[0415] The absorbance of BSA-Biotin-creatinine is lower than that of BSA-Biotin, which in turn is lower than the absorbance of BSA itself. Because BSA is conjugated to both biotin and creatinine via amide bonds, fewer primary amine groups are available. Consequently, the amount of TNBS bound to BSA is also reduced, leading to a decrease in the formation of the orange product and thus a reduction in color intensity.

[0416] The amine concentration values ​​were obtained from the standard curves of lysine and glutamic acid.

[0417] The BSA molecule has 59 lysine residues on its surface, of which 30-35 are available for conjugation. This is consistent with the determined data that each BSA molecule contains 32.6 primary amines. Biotin conjugation reduces the number of primary amines to 30.35, a net reduction of 2.4. This is consistent with the result determined by HABA that each BSA molecule contains 2.1 biotin molecules. Each BSA molecule binds approximately 2 biotin molecules. Further creatinine conjugation reduces the number of primary amines in each BSA molecule to 26.53, i.e., another net reduction of 3.82. Therefore, on average, approximately 4 creatinine molecules are bound to each BSA molecule.

[0418] Synthesis of gold nanoparticles and their conjugation with BSA

[0419] Example 1 above demonstrates that adsorption is not a suitable method for conjugating BSA onto gold nanoparticles because passive adsorption occurs during incubation, leading to aggregation. Specifically, a black, insoluble solid is deposited. The electrostatic interaction between gold nanoparticles and BSA is insufficient to achieve effective gold-BSA conjugation. Therefore, a reduction method is used to conjugate creatinine-biotin-BSA onto gold nanoparticles.

[0420] The presence of thiol groups in BSA was quantitatively determined using Ellman's reagent containing DTNB, with absorbance at 412 nm. The absorbance of the reduced BSA conjugate was significantly increased compared to the unreduced BSA conjugate. The concentration of thiol groups was calculated based on the L-cysteine ​​standard curve. The number of thiol groups increased significantly from 0.81 to 2.51, a net increase of 1.7, confirming the reduction of the BSA conjugate.

[0421] The gold nanoparticle (AuNP) sample was then added to the reduced BSA conjugate. The thiol groups can form bonds with the gold nanoparticles. The AuNP and AuNP-BSA conjugate were characterized using UV-Vis spectroscopy. The absorption peak of AuNP was located at 520 nm. After binding to the BSA-biotin-creatinine conjugate, a new maximum value was observed at 526 nm.

[0422] Size characterization was performed using NTA (Non-Activated Tolerancing). An aggregation peak was observed near 600 nm when measured using NaCl-Na3PO4 buffer. No aggregation was observed when using MilliQ water for AuNP NTA or when using Tris buffer for the AuNP-BSA-biotin-creatinine conjugate NTA. The aggregation phenomenon is mainly due to the presence of Na+ and Cl- ions in the buffer, which can bind to AuNP to form larger complex molecules. These ions were not present in the other buffers used.

[0423] The NTA of AUNP showed a sharp peak at 80 nm, confirming the size of the gold nanoparticles. The NTA of the AUNP-BSA-creatinine conjugate showed an additional peak at 97 nm, confirming the size of the AUNP-BSA conjugate molecule.

[0424] Capture ELISA

[0425] Perform an ELISA assay to ensure that the binding ratios of creatinine-anticreatinine antibody and biotin-avidin are consistent. Figure 27 As shown, decreasing the BSA concentration resulted in a decrease in absorbance at 450 nm. Significant changes in both color intensity and absorbance were observed when the concentration was reduced from 25 μg / ml to 0.625 μg / ml.

[0426] BSA concentrations above 50 μg / ml were also tested. A concentration of 150 μg / ml was used and diluted to 125, 100, 75, and 50 μg / ml. No significant change in absorbance was observed by decreasing the concentration in this region. This is because all antibodies are saturated with BSA at concentrations above 25 μg / ml. Therefore, the amount of chromogenic TMB product produced is the same across the concentration range of 50 to 150 μg / ml, resulting in no significant change in the absorbance profile.

[0427] Competitive ELISA

[0428] This is a repetition of the previous capture ELISA, but in this experiment, the concentration of the BSA conjugate remained constant, and different concentrations of free creatinine were added. A semi-logarithmic plot was plotted to generate dose-response curves. Figure 28 As the concentration of free creatinine increases, the amount of antibody sites available for binding to BSA-biotin-creatinine conjugates decreases, thus reducing absorbance.

[0429] Lateral flow measurement

[0430] Different concentrations of creatinine were used in each liquid sample to observe its effect on the signal intensity of test lines 1 and 2. The creatinine concentration range tested was 0 ng / ml to 100 μg / ml. The resulting LFT test strips are shown below. Figure 19 As shown. Figure 19 and Figure 20 This indicates that the improved LFT design performs as expected in detecting creatinine. Specifically: At creatinine concentrations of 0 ng / ml and 1 mg / ml, strong signals were generated on the antibody test line (test line 1), while no signal was generated on the avidin test line (test line 2). This indicates that the AuNP-BSA-biotin-creatinine conjugate was not displaced from test line 1, and all conjugates were captured by the antibody.

[0431] -5 ng / ml and 10 ng / ml creatinine slightly decreased the signal of test line 1, while the signal of test line 2 slightly increased, indicating that some AuNP-BSA-biotin-creatinine conjugate was displaced and captured by test line 2.

[0432] - As creatinine concentration increased to 100 ng / ml and 1 μg / ml, the signal intensity of test line 1 decreased significantly, while the signal intensity of test line 2 increased significantly. This indicates that the competition between creatinine and the AuNP-BSA-biotin-creatinine conjugate is intense, resulting in more AuNP-BSA-biotin-creatinine conjugate being displaced from test line 1 and captured by test line 2.

[0433] - At a creatinine concentration of 10 μg / ml, the signal intensity of test line 1 was negligible, while at a creatinine concentration of 100 μg / ml, no signal was observed. At creatinine concentrations of 10 μg / ml and 100 μg / ml, the signal intensity of test line 2 was very high. This indicates that the AuNP-BSA-biotin-creatinine conjugate did not bind to test line 1, but rather to test line 2.

[0434] Figure 20 The results show that as creatinine concentration increases, the signal intensity of test line 2 also increases, exhibiting a direct proportional relationship. Conversely, as creatinine concentration increases, the signal intensity of test line 1 decreases, exhibiting an inverse proportional relationship.

Claims

1. A lateral flow testing device comprising a solid support structure, the solid support structure including a sample receiving area, a conduit pad, a first testing area, and a second testing area, wherein the solid support structure is configured to allow liquid to flow sequentially from the sample receiving area through the conduit pad to the first testing area, and then to the second testing area, wherein... i) The conjugation pad includes a movable conjugation analyte, the conjugation analyte comprising one or more analyte molecules conjugated to a detectable marker; ii)a) The first test region contains an immobilized analyte-binding molecule defining a first binding site. b) The conjugation pad contains a movable analyte-binding molecule, and the first test region contains an immobilized trapping molecule for immobilizing the analyte-binding molecule, wherein the immobilized trapping molecule defines a first binding site, or c) The conjugation pad contains a movable analyte-binding molecule, and the first test region contains an immobilized analyte-binding molecule defining a first binding site. iii) The second test region contains an immobilized conjugated analyte-binding molecule that binds the conjugated analyte but not the unconjugated analyte molecule, and defines a second binding site; The number of molecules of the conjugated analyte is less than or equal to the number of the second binding sites.

2. The lateral flow testing apparatus of claim 1, wherein the number of molecules of the conjugated analyte is less than the number of second binding sites on the second test region.

3. The lateral flow testing apparatus as described in claim 1 or 2, wherein the number of first binding sites is equal to the number of molecules of the conjugated analyte.

4. The lateral flow testing apparatus as described in claim 1 or 4, wherein: i) the conjugated analyte, ii) a) the immobilized analyte-binding molecule and iii) the conjugated analyte-binding molecule exist in a ratio of 1:1:1; or The conjugated analyte (i), the mobile analyte-binding molecule (ii)b), the trapping molecule (ii)b), and the conjugated analyte-binding molecule (iii) are present in a ratio of 1:1:1:

1.

5. The lateral flow testing apparatus according to any one of the preceding claims, wherein the detectable marker is a nanoparticle, preferably a gold nanoparticle.

6. The lateral flow testing apparatus of any of the preceding claims, wherein each analyte molecule of the conjugated analyte is conjugated to a detectable marker via a linker molecule, preferably wherein the linker molecule is biotin-BSA.

7. The lateral flow testing apparatus of claim 6, wherein the conjugated analyte binding molecule is specific to and can bind to the linker molecule, preferably wherein the conjugated analyte binding molecule is avidin, streptavidin, or polystreptavidin.

8. The lateral flow testing apparatus of any of the preceding claims, wherein one of the analyte and the analyte binding molecule is an antibody specific to the other of the analyte and the analyte binding molecule.

9. The lateral flow testing apparatus of any of the preceding claims, wherein the analyte-binding molecule is an anti-analyte antibody.

10. A method for detecting the presence of analyte molecules in a test sample, the method comprising: i) Provide a lateral flow testing apparatus according to any one of claims 1-9, ii) Apply the test sample to the sample receiving area of ​​the lateral flow test device, allowing the test sample to migrate to the conjugation pad and mix with the conjugated analyte, and optionally mix with mobile analyte-binding molecules. iii) Allows the test sample and conjugated analytes, and optionally mobile analyte-bound molecules, to sequentially migrate to the first and second test regions and contact the immobilized molecules in each of the first and second test regions. iv) Detect the signals in the first test area and the second test area respectively, wherein changes in the signal intensity in both the first test area and the second test area indicate the presence of analyte molecules in the test sample.

11. The method of claim 10, wherein the signal is an optical signal.

12. The method of claim 10 or claim 11, further comprising: v) Quantitatively determine the concentration of analyte molecules in the sample based on the ratio of the signal intensity in the first test area to the signal intensity in the second test area.

13. The method of any one of claims 10 to 12, wherein in step iv), a decrease in the signal intensity of the first test region and an increase in the signal intensity of the second test region indicate the presence of analyte molecules in the test sample.

14. The method according to any one of claims 10-13, wherein, Signal strength is measured as a percentage of the maximum signal strength, wherein step iv) includes measuring the signal strength of one of the first test area and the second test area, and predicting the signal strength of the other of the first test area and the second test area based on the sum of the signal strengths of the first test area and the second test area being 100% of the maximum signal strength.

15. The method of claim 14, wherein step iv) comprises: a) Measure the signal strength of the first test area, and predict the signal strength of the second test area based on the fact that the sum of the signal strengths of the first and second test areas is 100% of the maximum signal strength, thereby calculating the ratio of first measured signal strength to predicted signal strength. b) Measure the signal strength of the second test area, and predict the signal strength of the first test area based on the fact that the sum of the signal strengths of the first and second test areas is 100% of the maximum signal strength, thereby calculating the second measured signal strength:predicted signal strength ratio. c) Based on the first and second measured signal strength:predicted signal strength ratio, calculate the average signal strength of each of the first and second test areas to determine the average first test area signal strength:second test area signal strength ratio. d) Calculate the ratio of the measured signal strength in the first test area to that in the second test area. e) Compare the ratio of the average signal strength in the first test area to the signal strength in the second test area, and the ratio of the measured signal strength. f) When the ratio of average signal intensity in the first test region to signal intensity in the second test region matches the ratio of measured signal intensity, the concentration of analyte molecules in the sample is quantitatively determined based on this ratio. Steps a) and b) can be performed in any order.

16. The method of claim 15, wherein: During measurement, the signal strength in the first test area and the signal strength in the second test area are scaled using a calibration factor; and Step iv) also includes: g) When the ratio of average signal strength in the first test area to signal strength in the second test area does not match the ratio of the measured signal strength calculated in step d, adjust the calibration factor and repeat steps a to f using the adjusted calibration factor.

17. The method of claim 16, wherein adjusting the calibration factor comprises: Determine whether the sum of the measured signal strengths in the first test area and the second test area is greater than the maximum signal strength; In response to the sum of the measured signal intensities in the first and second test areas being greater than the maximum signal intensity, the calibration factor is reduced; In response to the fact that the sum of the measured signal strengths in the first and second test areas does not exceed the maximum signal strength, the calibration factor is increased.

18. The method of any one of claims 10-17, wherein at least step a) of step iv) is performed using an optical signal reader, preferably a smartphone.

19. A method for diagnosing a disease or condition, comprising implementing the method as defined in any one of claims 10-18, wherein the test sample is obtained from a human, animal, or plant individual.

20. A computer-implemented method for detecting the presence of analyte molecules in a test sample, the method comprising: After the test sample is applied to the lateral flow testing device according to any one of claims 1-9, the signal intensity measurement values ​​of the first test area and the second test area of ​​the lateral flow testing device are obtained respectively. Determine whether the signal strength in both the first and second test areas changes; In response to the determination that the signal intensity in both the first and second test regions has changed, an indication of the presence of analyte molecules in the test sample is output.

21. The method of claim 20, wherein determining whether the signal strengths of both the first test area and the second test area have changed comprises: In response to a signal strength in one of the first and second test areas being less than the maximum signal strength, it is determined that a change exists in one of the first and second test areas; and In response to the signal strength of the other of the first test area and the second test area being greater than the minimum signal strength, it is determined that there is a change in the other of the first test area and the second test area.

22. The method of claim 20 or claim 21, wherein the signal strength measurement is acquired from one or more light measurement values ​​of each of the first test area and the second test area, and preferably, wherein the signal strength measurement value is acquired from one or more images of the first test area and the second test area of ​​the display lateral flow testing device.

23. A kit comprising a lateral flow testing device as defined in any one of claims 1-9.

Images