Molecular detection device

Abstract

A transverse flow testing apparatus is disclosed, comprising a conjugate pad having a movable bound analyte molecule containing one or more analyte molecules bound to a detectable label. A first test area includes an immobilized analyte-binding molecule that defines a first binding site, or the conjugate pad includes a movable analyte-binding molecule and the first test area includes an immobilized capture molecule that immobilizes the analyte-binding molecule, the immobilized capture molecule defining a first binding site, or the conjugate pad includes a movable analyte-binding molecule and the first test area includes an immobilized analyte-binding molecule that binds a conjugate analyte but not a non-conjugate analyte molecule, defining a second binding site. The number of bound analyte molecules is less than or equal to the number of second binding sites. Also disclosed are a method for detecting the presence of analyte molecules in a test sample, a method for diagnosing a disease or condition, a computer-implemented method for detecting the presence of analyte molecules in a test sample, and a kit.

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for conducting transverse flow tests, and a kit including such apparatus. In particular, the apparatus and method are for conducting quantitative transverse flow tests. [Background technology]

[0002] Transverse flow testing (LFT) can be used to detect a wide range of molecules and is useful for the rapid diagnosis of diseases. LFT typically consists of a nitrocellulose membrane with biomolecules immobilized in different regions (Figure 1a). The sample is applied to a sample pad, dissolving the deposited conjugate and moving along the membrane by capillary action. As the sample moves along the membrane, the conjugate or target analyte is captured by binding molecules (usually proteins) in the test region (e.g., the test line), creating a visible colored line indicating the presence or absence of the target analyte in the sample.

[0003] There are several types of LFTs, the two most common being the sandwich method and the competitive method. In the sandwich method, the conjugate pad consists of a movable labeled conjugate molecule (a target analyte-specific antibody bound to a detectable label), the test line consists of a target analyte-specific immobilized antibody, and the control line consists of an immobilized secondary antibody that binds to the detectable conjugate (Figure 1a). The sample is applied to the sample pad of the testing device, and the target analyte in the sample forms an analyte-conjugate complex with the detectable conjugate, which is movable on the conjugate pad. The analyte-conjugate complex moves from the conjugate pad to the test line, where it is captured by the immobilized anti-analyte antibody. This generates a visible line indicating a positive reaction (Figure 1b). Detectable conjugates that did not bind to the target analyte bind to the control line (Figure 1b). However, the sandwich method is only useful for large analytes with two or more binding sites, and allows both the conjugate and anti-analyte antibody to bind in the first test area. The sandwich method is not suitable for small molecules that do not allow for the simultaneous bonding of two different molecules.

[0004] The competitive method is designed to handle small molecules with only one binding site. In the competitive method, the conjugate pad contains a labeled conjugate that specifically binds to the target analyte in the test sample, and the test line contains immobilized capture molecules specific to the labeled conjugate. When a test sample without the target analyte is applied to the sample pad of the test apparatus, the labeled conjugate ascends the membrane and binds to the test line, generating a detectable signal at the test line. If the target analyte is present in the sample, the labeled conjugate binds to the target analyte, thereby preventing the labeled conjugate from binding to the immobilized capture molecules at the test line. Therefore, the presence of the target analyte in the sample is indicated by the absence of a detectable signal at the test line.

[0005] In sandwich-type and competitive LFTs, the control line is positioned after the test line in the sample flow direction, indicating that the labeled compound has flowed normally. Therefore, in a functioning and valid test, the control line will always produce a detectable signal regardless of the test result. In other words, the signal intensity of the control line is independent of the signal intensity of the test line and serves only to indicate that the LFT is valid.

[0006] Both types of LFTs provide quantitative results and can be used to quantify the concentration of analytes in a test sample. For example, Insudex(R) employs a sandwich method, where quantification is achieved by the intensity of the visible line in the first test area. A higher intensity indicates a higher concentration of the analyte in the sample. Users must analyze the test lines using a dedicated reader after they have been developed.

[0007] Patent Document 1 discloses a competitive LFT instrument for detecting the presence of caffeine. This instrument uses a conjugate consisting of an anti-caffeine antibody and a second antibody bound to a particle, and a test line consisting of molecules that bind to the anti-caffeine antibody. Thus, molecules on the first test line compete for binding to caffeine in the test sample and to the anti-caffeine antibody. The instrument includes a control line consisting of an antibody that binds to the second antibody in the conjugate.

[0008] Patent Document 2 discloses a semi-quantitative barcode-type sandwich LFT instrument having two test lines for detecting β-trace protein (βTP). Each test line includes a binding site that binds to a complex formed between βTP and a detectable conjugate, where the detectable conjugate includes a detectable label and a βTP-binding molecule (e.g., an antibody).

[0009] One drawback of competitive LFTs is that the observed signal intensity is inversely proportional to the concentration of the target analyte present (Non-Patent Literature 1). This means that a detectable signal exists in all samples except the highest concentration. This characteristic leads to confusion in interpreting results, necessitates extensive calibration to obtain accurate quantitative results, and reduces sensitivity. Furthermore, negative results associated with the competitive format mean that the presence of the target analyte is detected in the least sensitive region of the dose-response curve (i.e., near the saturation point (maximum signal intensity) of the test line). This characteristic makes it difficult to improve the sensitivity of competitive LFTs.

[0010] Both types of LFTs (i.e., sandwich and competitive assays) have the drawback that the first test region can become saturated when used as a quantitative assay. In the sandwich assay, this occurs when the concentration of the analyte in the test sample exceeds the amount of immobilized antibody, causing the immobilized antibody in the first test region to become saturated with the labeled analyte. This means that once the amount of analyte in the sample exceeds a certain level, the signal intensity of the first test region indicating its presence cannot increase any further. In a competitive assay, saturation occurs when the amount of the target analyte in the sample equals or exceeds the amount of immobilized analyte on the test line.

[0011] One solution to prevent saturation is to increase the amount of antibody immobilized in the first test area of ​​a sandwich-type LFT, thereby expanding the concentration range measurable by the transverse flow test. However, this negatively impacts the quantitative resolution of the LFT.

[0012] In particular, in both sandwich and competitive LFTs, quantification is achieved by inferring the concentration of the analyte in the test sample from the visual response produced by the conjugate (i.e., secondary inference). However, expanding the detection range of the LFT (i.e., increasing the amount of antibody immobilized in the first test area of ​​the sandwich assay) means that the proportion of the change in the visual response to the overall visual response becomes smaller. For example, in a sandwich assay, when one analyte-conjugate complex binds to the first test area with 100 antibodies, the intensity of the visible line in the first test area changes by 1%. However, when one analyte-conjugate complex binds to the first test area with 1000 antibodies, the intensity of the visible line changes by 0.1%. Therefore, expanding the detection range of quantitative transverse flow assays comes with a decrease in the resolution, and consequently the accuracy, of the transverse flow assay.

[0013] In quantitative LFTs, errors can occur when reading the intensity of the visible line due to the binding (or unbinding) of the conjugate in the first test area. In particular, errors can occur due to the degradation of the antibody used in the assay, causing sandwich LFTs to be biased towards negative results and competitive LFTs towards positive results, as antibodies bound to the detectable label may not bind to the test line. Errors can also occur due to systematic errors in the LFT reader or because ambient light affects the signal intensity measured in the test area. For example, Insudex® detects C-peptide (for diabetes diagnosis) in the range of 0.17 ng / ml to 12 ng / ml, but the hook effect (a phenomenon that causes false negative results when the analyte is at a very high concentration) begins to occur when the C-peptide concentration reaches approximately 80 ng / ml. When measuring a C-peptide concentration of approximately 0.17 ng / ml, the variation of Insudex® is approximately 0.02 ng / ml, or the coefficient of variation is approximately 11%. This means that quantitative measurements can lead to misdiagnosis, and there are limitations to the ability of this product to be used for mass screening.

[0014] A further drawback of competitive LFT is that its quantification ability is significantly limited by the presence of a control line. Due to the presence of the control line, it is necessary to add an excessive amount of labeled compound to the sample so that a sufficient amount of the labeled compound reaches the control line. However, this results in the loss of information regarding the amount of the compound that does not bind to the test line, and inhibits the ability to accurately quantify the outlier concentration of the target analyte.

[0015] The ELISA method can be implemented to quantify the target analyte in a test sample, but ELISA requires a considerable amount of time, is costly, and requires specialized equipment and personnel for implementation.

[0016] Non-Patent Document 2 discloses a lateral flow test for detecting a specific strain of influenza virus composed of an antibody and a nucleic acid aptamer. This lateral flow test device includes a first test line containing streptavidin and a control line containing an anti-mouse antibody. The antiviral antibody is bound to gold nanoparticles, and the aptamer is bound to biotin, respectively. The aptamer can be selected to have high strain specificity. When the target virus is contained in the sample, both the aptamer and the binding antibody bind to the virus, and the complex binds to the streptavidin on the test line by the biotin bound to the aptamer. Thereafter, gold nanoparticles are detected on the test line. Regardless of the presence or absence of the target virus, the gold nanoparticle-binding antibody binds to the anti-mouse antibody on the control line. However, this LFT is not quantitative.

[0017] Therefore, there is a need to provide a quantitative lateral flow test that more accurately quantifies the target analyte in a sample. In particular, there is a need to provide a quantitative LFT that addresses the problems of saturation and resolution and reduces the reading error of signal intensity. Furthermore, such an LFT reduces the need for the expensive and time-consuming ELISA test kits as described above.

Prior Art Documents

Patent Documents

[0018] [Patent Document 1] U.S. Patent No. 8,137,984 [Patent Document 2] U.S. Patent Publication No. 2022 / 0146507 [Patent Document 3] International Publication No. 2023026043 [Patent Document 4] International Publication No. 2001076583 [Patent Document 5] International Publication No. 2009022933 [Non-Patent Document]

[0019] [Non-Patent Document 1] Delmulle et al., J. Agric. Food Chem., 2005, 53, 3364 - 3368 [Non-Patent Document 2] Le et al., Anal. Chem, 2017, 89, 6781 - 6786 [Summary of the Invention]

[0020] This invention is based on the remarkable discovery that more accurate quantitative or semi-quantitative lateral flow tests can be achieved by pre-loading a lateral flow test with a predetermined amount of detectable labeled analyte. This makes it possible to calculate the concentration of the target analyte based on the signal generated by the detectable labeled analyte. Specifically, the first test region is capable of binding to both the labeled analyte and the target analyte molecule, and the detectable bound analyte consists of the analyte molecule bound to the detectable label. The number of binding sites in the second test region is equal to or greater than the number of bound analyte molecules. If the target analyte molecule is not present in the test sample, the bound analyte binds to the first test region and optionally to the second test region, generating a detectable signal in the test region to which the bound analyte is bound. If the target analyte molecule is present in the test sample, the amount of bound analyte that binds to the first test region decreases due to competition between the target analyte molecule and the bound analyte, thus reducing the detectable signal in the first test region. Unbound analytes (i.e., those not bound to the first test region) can bind to the second test region, thereby increasing the intensity of the detectable signal in the second test region (either an increase from the absence state or an increase in intensity exceeding the signal intensity when the target analyte is absent). Therefore, due to competition between the target analyte molecules in the sample and the bound analytes pre-loaded into the instrument, the signal intensity in the first test region is inversely proportional to the signal intensity in the second test region (i.e., the signal intensities of the first and second test regions are interdependent). As a result, the response in the test region changes stepwise depending on the amount of bound analyte. Thus, the signal intensity in the second test region is useful in confirming the signal intensity in the first test region.

[0021] This invention also arises from the unexpected discovery that more accurate quantitative lateral flow testing can be achieved when the signal intensity in the first test area is inversely proportional to the signal intensity in the second test area. In particular, by comparing the signal intensities in the first and second test areas, a ratio of signal intensities can be generated, which can be used to more accurately calculate the concentration of the target analyte in the sample. This ratio also reduces systematic errors caused by the device that reads the signal intensity in the test areas, as well as errors due to ambient light. In contrast, conventional quantitative LFTs consider only the presence or absence of a detectable signal in the first test area when calculating the concentration of the target analyte in the sample, and do not consider the detectable signal in the second test area. In conventional LFTs, the second test area is used only as a control to indicate that its conjugate has functioned and moved above the test strip. However, the lateral flow testing apparatus of this invention measures the change in signal intensity in both the first and second test areas, thus providing two parameters for calculating the concentration of the target analyte in the sample.

[0022] Furthermore, the present invention arose from the unexpected discovery that more accurate quantitative transverse flow testing can be achieved by providing a ratio of the signal intensity of the first test area to the signal intensity of the second test area. In particular, when calculating the ratio of the signal intensity of the first test area to the second test area and using this ratio to calculate the concentration of the target analyte in the sample, considering the sum of the signal intensities of the first and second test areas as 100% can reduce errors due to the background environment. Moreover, errors can be further reduced by calculating the signal intensity ratio multiple times and calculating the average ratio using multiple ratios.

[0023] Quantitative errors due to ambient light can be further reduced by comparing the average signal intensity ratio of the first and second test regions, calculated based on predicted signal intensity, with the actually measured signal intensity ratio of the first and second test regions. Specifically, the signal intensity of the first test region is measured, and the signal intensity of the second test region is predicted based on the assumption that the sum of the signal intensity is 100% (e.g., maximum signal intensity), and the first ratio of the signal intensity of the first and second test regions is determined. Next, the signal intensity of the second test region is measured, and the signal intensity of the first test region is predicted using the same criteria, and the second ratio of the signal intensity of the first and second test regions is determined. Then, the average ratio of the signal intensity is determined from the first and second ratios. Subsequently, the ratio of the measured signal intensity is determined. The ratio of the measured signal intensity provides a ratio measurement calibration to the average ratio of the signal intensity. If the average ratio of the signal intensity does not correspond to the ratio of the measured signal intensity, the average ratio of the signal intensity can be redetermined using new measured values ​​of the signal intensity recalibrated based on an adjusted scaling (or calibration) coefficient.

[0024] Accordingly, in a first aspect of the present invention, a transverse flow testing apparatus is provided comprising a solid support structure including a sample receiving region, a conjugate pad, a first test region, and a second test region. This solid support structure is configured to allow liquid to flow sequentially from the sample receiving region through the conjugate pad to the first test region, and then to the second test region. i) The binding pad contains a mobile binding analyte containing one or more analytical molecules bound to a detectable label; ii) a) The first test area contains an immobilized analyte-binding molecule that defines the first binding site, b) The composite pad comprises a movable analyte-binding molecule, the first test area comprises an immobilizing capture molecule that immobilizes the analyte-binding molecule, the immobilizing capture molecule defines the first binding site, or c) The composite pad contains a movable analyte-binding molecule, and the first test area contains an immobilized analyte-binding molecule defining the first binding site, iii) The second test area contains an immobilized bound analyte molecule that binds the bound analyte but does not bind the unbound analyte molecule, and defines the second binding site. Here, the number of molecules of the target molecule that are bound is less than or equal to the number of secondary binding sites.

[0025] Preferably, the number of molecules in the binding analyte is less than the number of second binding sites on the second test region.

[0026] Advantageously, the number of primary bonding sites is equal to the number of molecules in the analyte.

[0027] Preferably, the binding analyte of i), the immobilized analyte binding molecule of ii)a), and the binding analyte binding molecule of iii) are present in a ratio of 1:1:1. Alternatively, the binding analyte of i), the mobile analyte binding molecule of ii)b), the capture molecule of ii)b), and the binding analyte binding molecule of iii) are present in a ratio of 1:1:1:1.

[0028] Preferably, the detectable label is a nanoparticle, particularly a gold nanoparticle.

[0029] Preferably, each analyte molecule of the conjugated analyte is bound to a detectable label via a linking molecule, preferably the linking molecule being biotin-BSA.

[0030] Advantageously, the bound analyte-binding molecule is specific to the binding molecule and binds to it. Preferably, the bound analyte-binding molecule is avidin, streptavidin, or polystreptavidin.

[0031] Conveniently, either the analyte or the analyte-binding molecule is an antibody specific to the other analyte or analyte-binding molecule.

[0032] Preferably, the analyte-binding molecule is an anti-analyte antibody.

[0033] In a second embodiment of the present invention, a method for detecting the presence of an analyte molecule in a test sample is provided, the method comprising: i) Prepare a transverse flow inspection device according to the first side, ii) Apply the test sample to the sample receiving area of ​​the transverse flow inspection device, allowing the test sample to move to the conjugate pad and mix with the conjugate-labeled analyte and, optionally, the mobile analyte-binding molecule. iii) The analyte molecule bound to the test sample, and optionally the mobile analyte molecule bound to it, move sequentially to the first and second test areas, and come into contact with the immobilized molecules in each of the first and second test areas. iv) Detect a signal in both the first and second test areas. Here, a change in signal intensity in both the first and second test areas indicates the presence of the analyte molecule in the test sample.

[0034] Preferably, the signal is an optical signal.

[0035] Advantageously, this method further includes: v) quantifying the concentration of the analyte in the test sample based on the ratio of the signal intensity in the first test area to the signal intensity in the second test area.

[0036] Preferably, in step iv), a decrease in signal intensity in the first test area and an increase in signal intensity in the second test area indicate the presence of the analyte molecule in the test sample.

[0037] Conveniently, signal strength is measured as a percentage of the maximum signal strength, and step iv) includes measuring the signal strength in either the first or second test area and predicting the signal strength in the other test area based on the fact that the sum of the signal strengths in the first and second test areas equals 100% of the maximum signal strength.

[0038] Preferably, step iv) includes: a) Measure the signal intensity in the first test area, predict the signal intensity in the second test area based on the fact that the sum of the signal intensities of the first and second test areas equals 100% of the maximum signal intensity, and calculate the ratio of the first measured value to the predicted value of signal intensity. b) Measure the signal intensity in the second test area, predict the signal intensity in the first test area based on the fact that the sum of the signal intensities of the first and second test areas equals 100% of the maximum signal intensity, and calculate the ratio of the second measured value to the predicted signal intensity. c) The average signal intensity in the first and second test areas is calculated based on the measured value:predicted value signal intensity ratio of the first and second test areas, and the average signal intensity ratio of the first:second test area is determined. d) Calculate the ratio of the measured signal intensity of the first test area to the second test area. e) Compare the average signal intensity ratio of the first and second test regions with the measured signal intensity ratio, and f) If the average signal intensity ratio of the first to second test areas matches the measured signal intensity ratio, the concentration of the analyte molecule in the test sample is quantified based on the average signal intensity ratio of the first to second test areas. Here, steps a) and b) can be performed in any order.

[0039] Preferably, the signal intensity in the first test area and the signal intensity in the second test area are scaled using a calibration coefficient during measurement. Step IV) further includes: g) If the average first:second test domain signal intensity ratio does not match the measured signal intensity ratio calculated in step d), adjust the calibration coefficient and repeat steps a) through f) using the adjusted calibration coefficient.

[0040] When scaling using a calibration coefficient, the default or initial calibration coefficient may be used for one or more of the initial intensity measurements before the calibration coefficient is adjusted. This default or initial calibration coefficient may be equal to 1 (although other values ​​are also possible). If the calibration coefficient is equal to 1, the scaled intensity may be equal to the pre-scaling intensity.

[0041] For convenience, the adjustment of the calibration coefficients includes the following: Determine whether the measured signal intensity in the first and second test areas exceeds the maximum signal intensity; If the measured signal intensity in the first and second test areas exceeds the maximum signal intensity, reduce the calibration coefficient; and If the measured signal intensity in the first and second test areas does not exceed the maximum signal intensity, increase the calibration coefficient.

[0042] Preferably, at least step a) of step iv) is carried out using an optical signal reader, preferably a smartphone.

[0043] Test samples may be obtained from human, animal, or plant subjects, environmental samples, or food or beverage samples.

[0044] A third aspect of the present invention provides a method for diagnosing a disease or condition, comprising carrying out the method according to the second aspect, wherein the test sample is obtained from a human, animal, or plant subject.

[0045] A fourth embodiment of the present invention presents a computer-implemented method for detecting the presence of an analyte molecule in a test sample. The method includes the following: The process involves applying a test sample to a transverse flow inspection device and then acquiring signal intensity measurements in the first and second test areas of the transverse flow inspection device from a first side view; Determine whether there was a change in signal intensity in both the first and second test areas; If a change in signal intensity is detected in both the first and second test areas, an indication will be output to show the presence of the analyte molecule in the test sample.

[0046] The acquisition of signal intensity measurements may include receiving signal intensity measurements from an external system. For example, this method may be implemented by a first computing system. Signal intensity measurements may be measured by a second computing system (e.g., a mobile device, optical sensor, or camera). Signal intensity measurements may be acquired by the second computing system and transmitted to the first computing system for further analysis to determine whether there is a change in signal intensity in both the first and second test areas. Alternatively, signal intensity measurements may be determined by the first computing system from one or more optical measurements for each of the first and second test areas, and / or one or more images showing the first and second test areas of the lateral flow test apparatus. One or more optical measurements and / or one or more images may be acquired by the first computing system (e.g., through the use of an optical sensor and / or camera). Alternatively, one or more optical measurements and / or one or more images may be acquired by a second computing system (e.g., a mobile device, optical sensor, or camera) and transmitted to the first computing system to determine whether or not there is a change in signal intensity in both the first and second test areas.

[0047] Preferably, determining whether there has been a change in signal intensity in both the first and second test areas includes the following: Determining whether a change has occurred in either the first or second test area, depending on whether the signal intensity in either test area is less than the maximum signal intensity; and A change in the other test region is determined when the signal intensity of the other test region exceeds the minimum signal intensity of the first and second test regions.

[0048] Preferably, the signal intensity measurements are obtained from one or more optical measurements for each of the first and second test areas, and more preferably, the signal intensity measurements are obtained from one or more images showing the first and second test areas of the transverse flow test apparatus.

[0049] In a fifth aspect of the present invention, a kit is provided that includes a transverse flow testing apparatus according to the first aspect.

[0050] In some embodiments, the bound analyte includes two analyte molecules bound to a detectable label, or one analyte molecule.

[0051] As used herein, the term “immobilization” means that molecules are held or fixed to the relevant parts of the apparatus by any suitable means well known to those skilled in the art. Immobilized molecules are not released or moved by contact with the test sample or by the flow of the test sample. In particular, “immobilized” analyte-binding molecules or capture molecules mean molecules that are typically held in the test area during use and therefore bind to relevant complementary molecules contained in the liquid that comes into contact with the test area.

[0052] As used herein, the term “mobile” means that the molecule is reversibly held or fixed to a relevant part of the apparatus before use and released from that part of the apparatus upon contact with the test sample. Movable molecules can be carried away from their original holding / fixing point by the lateral flow of the test sample.

[0053] In this specification, "binding site" refers to a region or spot in which a molecule can be immobilized, particularly by complementary bonding with a binding molecule or a capturing molecule.

[0054] In this specification, the term "unbound" as used with respect to analytes means that the analyte is not bound to a bound molecule. For example, an unbound analyte may be an analyte derived from a test sample.

[0055] In this specification, the term "polypeptide" refers to a polymer of amino acid residues. This term also applies to amino acid polymers in which one or more amino acid residues are modified residues or unnaturally occurring residues such as artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers.

[0056] In this specification, the term "polynucleotide" refers to a polymer of nucleic acid residues, including DNA and RNA.

[0057] In this specification, the term "antigen" refers to a molecule that has the ability to induce an immune response in an individual.

[0058] As used herein, the term “environmental sample” refers to a sample taken from a natural or artificial environment. For example, an environmental sample may be a water sample taken from a natural or artificial water source, a soil sample, or a swab sample taken from a natural or artificial surface. [Brief explanation of the drawing]

[0059] [Figure 1] This is a schematic diagram of results obtained using a typical transverse flow assay test strip (a) and a standard sandwich (b) test format. [Figure 2] This is a schematic diagram showing the format of a capture ELISA used to test whether the BSA-biotin-caffeine complex binds to both anti-caffeine antibodies and avidin. [Figure 3] This is a schematic diagram of an improved LFT design featuring two test lines: an anti-caffeine antibody test line and an avidin test line. [Figure 4]This is a schematic diagram of the synthesis reaction of the BSA-biotin-caffeine complex used in Example 1. [Figure 5] This graph shows the inhibition curve of a competitive ELISA using a BSA-biotin-caffeine complex. The data points were obtained from three repeated measurements, and the error bars indicate the standard deviation relative to the mean. [Figure 6] No explanation provided. [Figure 7] This is a schematic diagram of the initial design of the LFA, which has two test lines: an anti-mouse antibody test line and an avidin test line. [Figure 8] This is a schematic diagram illustrating the operating principle of the improved LFT design when scanning a caffeine-free sample (a) and a caffeine-containing sample (b). The expected test results corresponding to the presence or absence of caffeine in the samples are shown in Figure 8(c). [Figure 9] These are photographs of the improved LFT strip design, tested with varying glycerol concentrations in the buffer solution, both in the presence of caffeine (100 μg / ml) and in the absence of caffeine (0 μg / ml). [Figure 10] This is a photograph of an LFT strip with an improved design that varied the caffeine concentration. The scanning buffer consisted of 5% (v / v) glycerol and 2% (w / v) BSa in PBS buffer containing 0.05% (w / v) Tween-20. [Figure 11] Figure 10 shows a semi-logarithmic graph of the signal intensity of test lines 1 and 2 of the LFT strip, analyzed using ImageJ software. The results were obtained by repeating the measurements three times. [Figure 12] These are images of MFT strips scanned at different caffeine concentrations after one week of storage. The scanning buffer was 5% glycerol and 2% BSA in PBS buffer containing 0.05% Tween-20. [Figure 13] This is a schematic diagram of the LFT design used for performance testing of a lateral flow strip in a buffer solution. [Figure 14] These are images of LFT strips scanned using synthetic urine containing different concentrations of NHPA. The solution volume was 200 μl. [Figure 15] Figure 14(a) shows a graph of the pixel gray level of each test line in the strip analyzed using ImageJ, and a graph showing the line intensity (calculated by subtracting the minimum peak value from the background) of each test line (upper panel: streptavidin line, lower panel: antibody line) at each NHPA concentration. [Figure 16] These are images of LFT strips scanned with synthetic urine containing different concentrations of NHPA. For each concentration, three test strips prepared from the same batch of conjugates were assembled and tested. [Figure 17] Figure 16 shows graphs of the signal intensity of antibody lines against different concentrations of NHPA used in the previous image, analyzed using ImageJ. Each data point represents the average value of five LFT strips. [Figure 18] The images show photographs of LFT test strips performed with and without NHPA-containing synthetic urine (a) and with NHPA-containing synthetic urine (b), as well as schematic diagrams showing the positions of the analyte binding line (A) and the streptavidin line (S). [Figure 19] These are photographs of LFT test strips performed using different concentrations of creatinine. [Figure 20] Figure 19 shows a semi-logarithmic graph of the signal intensity of test lines 1 and 2 of the LFT strip, analyzed using ImageJ software. The results were obtained from three measurements. [Figure 21] A schematic diagram of the saturation mechanism in sandwich LFT is shown. Since twice the amount of analyte for each conjugate antibody and anti-analyte antibody is present on the test line, some analyte cannot bind to the test line, resulting in unquantified analyte. [Figure 22] This is a schematic diagram showing an example of the LFT of the present invention, in which the molecular weight of the analyte in the test sample is equal to the molecular weight and amount of each bound analyte and binding site on the first test line. [Figure 23] This is a schematic diagram illustrating an example of the simplified testing method of the present invention, in which the amount of analyte molecules in the test sample is greater than the amount of bound analyte and binding sites on the first test line. [Figure 24]A schematic diagram of a conventional sandwich-type LFT in which the number of analyte molecules in the test sample increases from 400 (A) to 415 (B) is shown, along with the results obtained therefrom (C and D). [Figure 25] This is a schematic diagram of an exemplary LFT of the present invention, in which the number of analyte molecules in the test sample increases from 400 (A) to 415 (B), and the results obtained therefrom (C and D) are shown. [Figure 26] A schematic diagram of a calculation system that quantifies the concentration of analyte molecules in a test sample based on the signal intensity of the first and second test regions of the LFT according to the embodiment is shown. [Figure 27] This graph shows absorbance against BSA-biotin-creatinine concentrations. Each data point represents a three-measurement, and the error bars indicate the standard deviation. [Figure 28] This graph shows absorbance as a function of free creatinine concentration. Data points were obtained from three measurements, and the error bars indicate the standard deviation. [Modes for carrying out the invention]

[0060] The present invention generally relates to a transverse flow testing apparatus comprising a detectable bound analyte containing an analyte molecule bound to a detectable label, and two test regions. The first test region has one or more primary binding sites capable of binding both the bound analyte and the analyte molecule from the test sample, and the second test region has one or more secondary binding sites capable of binding the bound analyte but not the target analyte. The number of molecules of the bound analyte in the apparatus is less than or equal to the number of secondary binding sites in the second test region. When the analyte is not present in the test sample, the signal intensity of the second test region is never 100%, and when the analyte is present in the test sample, a change in the signal intensity of the second test region is reliably produced.

[0061] solid support structure A transverse flow testing apparatus includes a solid support structure on which other components and reagents for transverse flow testing are arranged. For example, the solid support structure may be a support card on which other components and reagents for transverse flow testing are arranged. This support card may be housed within a housing. In some cases, the solid support structure itself may constitute the housing, with other components and reagents for transverse flow testing arranged inside. The solid support structure may be formed from any suitable material well known to those skilled in the art, such as plastic.

[0062] The solid support structure is configured so that the liquid (e.g., test sample and other liquid reagents or components added to the apparatus) flows sequentially from the sample receiving area to the conjugate pad, then to the first test area, and then to the second test area. The apparatus is configured to allow the liquid to come into contact with and mix with the conjugated analyte on the conjugate pad, and with the analyte-binding molecules, if present, and further come into contact with various molecules in the first and second test areas.

[0063] Sample receiving region The sample receiving area is configured to receive test samples, preferably in liquid form. The sample receiving area is also configured to receive other liquids, such as liquid reagents, that may be added to the apparatus of the present invention.

[0064] The sample receiving region may include other components necessary or beneficial for the LFT. These components may be movable or fixed on the region. In some embodiments, the sample receiving region contains buffering components for transverse flow testing before the device is put into use. These buffering components are movable upon addition of the test sample and may be movable and fixed on the sample receiving region by any suitable means known to those skilled in the art. For example, the buffering components may be present in a dry state on the sample receiving region, and upon addition of a liquid (such as a liquid test sample), the buffering components may be released from the sample receiving region and mixed with the test sample. Suitable buffering components known to those skilled in the art (e.g., sucrose or glycerol) may be used.

[0065] The sample receiving area can be formed from any suitable material known to those skilled in the art. For example, the sample receiving area can be formed from a paper base, a woven mesh, or cellulose, nitrocellulose, or glass fiber. Preferably, the sample receiving area is formed from nitrocellulose or glass fiber. The sample receiving area can be part of a single structure including a conjugate pad and, optionally, first and second test areas. Alternatively, the sample receiving area can be a separate structure from the conjugate pad and the first and second test areas. In some embodiments, the sample receiving area is part of the conjugate pad, and there is no distinction between the sample receiving area and the conjugate pad (i.e., the test sample is added to the conjugate pad).

[0066] Conjugate Pad The conjugate pad contains a movable conjugate analyte, which is positioned on or inside the conjugate pad, and is configured such that the conjugate analyte is released and mixed by a test sample coming into contact with the conjugate pad. For example, the conjugate analyte can be dried on the conjugate pad so that it is released or movable and mixed upon contact with the test sample.

[0067] In some embodiments, the conjugate pad further includes a movable analyte-binding molecule, and the first test area includes immobilization-capturing molecules for immobilizing the analyte-binding molecule, with each immobilization-capturing molecule defining a first binding site. These embodiments offer the advantage of reducing the effect of differences in flow rates between the bound analyte and the analyte molecules in the test sample, thereby improving the accuracy of the LFT. In particular, the flow rate of a molecule is determined, at least in part, by its size. If there is a large size difference between the bound analyte and the unbound analyte (i.e., the analyte molecules in the test sample), the bound and unbound analytes will flow at different velocities, resulting in one of them reaching the first test area before the other (usually the unbound analyte flows faster). This means that the bound analyte may not optimally compete with the analyte molecules in the test sample (i.e., the unbound analyte) for binding to the analyte-binding molecule, which can result in a decrease in the quantitative accuracy of the analyte molecules in the test sample. By mixing the detection molecule-binding molecule with the test sample and the bound detection substance in the binding pad, the detection molecule-binding molecule is simultaneously exposed to both the bound detection substance and the detection substance in the test sample. This reduces the influence of the difference in flow rate between the bound detection substance and the unbound detection substance (derived from the test sample).

[0068] In some embodiments, the conjugate pad comprises a mobile analyte binding molecule and a conjugate analyte, and the first test area comprises an immobilized analyte binding molecule. These embodiments offer the advantage that the mobile analyte binding molecule acts as a concentration buffer, introducing a minimum threshold concentration before a positive result is displayed on the LFT.

[0069] In some embodiments, the conjugate pad may be an area of ​​the same structure including the first and second test areas, or it may be a structure separate from the first and second test areas. If the conjugate pad is a structure separate from the test areas, it can be attached to the structure including the first and second test areas by any suitable means known to those skilled in the art.

[0070] Conjugate pads can be manufactured from any suitable material known to those skilled in the art. For example, conjugate pads can be manufactured from a paper base, a woven mesh, or from cellulose, nitrocellulose, or glass fiber. In some embodiments, conjugate pads are manufactured from nitrocellulose or glass fiber.

[0071] Examination areas 1 and 2 The first and second test areas may be different areas of a single structure, or they may be separate structures or areas of separate structures. If the first and second test areas are separate structures or areas of separate structures, the structures may be fixed to each other by any suitable means known to those skilled in the art in order to allow the flow of liquid 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 positioned such that, during the use of the LFT, the liquid flowing through / along the transverse flow test apparatus reaches the first test area before reaching the second test area.

[0072] Preferably, the first and second test regions are separate regions of a single structure, which is a permeable or semi-permeable membrane that allows liquid to pass through or penetrate. In some embodiments, the first and second test regions are separate regions of a single nitrocellulose membrane.

[0073] In some embodiments, the first test area includes immobilized analyte-binding molecules. Each immobilized analyte-binding molecule defines a first binding site on the first test area.

[0074] In some embodiments, the conjugate pad includes a movable analyte binding molecule and a movable conjugate analyte, and the first test area includes an immobilized capture molecule that binds to and immobilizes the analyte binding molecule. Each immobilized capture molecule defines a first binding site on the first test area. These embodiments offer the advantage of reducing the effect of the difference in flow rate between the bound analyte and the analyte molecules in the test sample, as described above, thereby improving the accuracy of the LFT.

[0075] In some embodiments, the conjugate pad includes a movable conjugate analyte and a movable analyte binding molecule, and the first test area includes an immobilized analyte binding molecule. Each immobilized analyte binding molecule defines a first binding site on the first test area. These embodiments offer the advantage that the movable binding analyte acts as a concentration buffer, thereby introducing a minimum threshold concentration before a positive result is displayed on the LFT. In particular, analyte molecules in the test sample compete with the bound analyte for the movable analyte binding molecule on the binding pad. Once an analyte molecule binds to an analyte binding molecule, that analyte molecule can no longer bind to the immobilized analyte binding molecule on the first test area. In the first test area, competition then occurs between analyte molecules that are not bound to the movable analyte binding molecule and the bound conjugate analyte.

[0076] In each configuration of the binding pad and the first test area, the second test site contains an immobilized binding analyte molecule that binds to the binding analyte but not to the unbinding analyte (i.e., analyte molecules derived from the test sample). Each immobilized binding analyte molecule defines a second binding site. Therefore, binding analytes that do not bind to the first test area bind to the second test area. As the concentration of analyte molecules in the test sample increases, the number of binding analyte molecules that can bind to the first test area decreases, reducing the signal intensity generated in the first test area, while more binding analyte molecules bind to the second test area, increasing the signal intensity generated in the second test area. Therefore, the signal intensity in the second test area depends on and is inversely proportional to the signal intensity in the first test area. For the signal intensity to increase when one or more analyte molecules are present in the test sample, the number of second binding sites in the second test area must be equal to or greater than the number of binding analyte molecules in the transverse flow analyzer. In particular, when a transverse flow test is performed in the absence of analyte molecules in the test sample, the first test region always contains at least one first binding site, and the second test region always contains at least one second binding site that is not bound to the first binding site. Therefore, the presence of analyte molecules in the sample can increase the amount of labeled analyte bound to the second test region by at least one molecule, thereby increasing the signal intensity in the second test region.

[0077] The first and second test areas can each be any suitable shape (e.g., square, rectangle, circle, triangle, ellipse, linear). Preferably, the first and second test areas are linear, formed by linearly fixing the relevant analyte-binding or capture molecule and the bound analyte-binding molecule. For example, if the first and second test areas are areas on a membrane, the first and second test areas can each be linear areas on the membrane. Preferably, the first and second test areas each extend along / through the membrane across the entire width of the liquid channel (i.e., perpendicular to the direction of liquid flow). This provides the advantage that the analyte molecule and bound analyte molecule must come into contact with the first test area before reaching the second test area. This prevents false positive reactions in the second test area due to binding of the bound analyte molecule. Furthermore, this configuration also prevents tampering with test results, as the test sample and bound analyte molecule cannot bypass the test areas.

[0078] binding analyte Each molecule of the bound analyte used in the apparatus and method of the present invention comprises an analyte molecule bound to a detectable label. One or more analyte molecules are bound to each detectable label. In some embodiments, only one analyte molecule is bound to each detectable label.

[0079] The analyte molecule bound to the detectable label must compete with the analyte molecule in the test sample for the analyte-bound molecule in the conjugate pad and / or first test area. Preferably, the analyte molecule bound to the detectable label is of the same type as the analyte molecule in the test sample. For example, if the analyte molecule detected in the test sample is caffeine, the analyte molecule bound to the detectable label may also be caffeine.

[0080] The minimum concentration threshold of the analyte molecule in the test sample required to obtain a positive result can be adjusted by changing the number of analyte molecules bound to each detectable label. For example, increasing the number of analyte molecules bound to each detectable label causes the bound analyte molecules to compete more strongly with the bound analyte molecules. This is because the ratio of molecules that the bound analyte molecules can bind to, i.e., the ratio of analyte molecules to bound analyte molecules in the test sample, becomes biased in favor of the bound analyte molecules. Therefore, in some embodiments, each bound analyte includes two or more analyte molecules bound to each detectable label, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 analyte molecules bound to each detectable label.

[0081] In some embodiments, two analyte molecules are bound to each detectable label, the conjugate pad includes a mobile analyte-binding molecule, and the first test area includes an immobilized analyte-binding molecule. Preferably, the mobile analyte-binding molecule is the same as the immobilized analyte-binding molecule. Embodiments having a mobile analyte-binding molecule and an immobilized analyte-binding molecule offer the advantage of reducing the effect of different flow rates between the analyte molecules and the bound analyte in the test sample, as well as for the reasons stated above. Furthermore, the mobile analyte-binding molecule functions as a concentration buffer, as well as for the reasons stated above. Moreover, the two analyte molecules bound to the detectable label allow the bound analyte molecules to bind to both the mobile and immobilized analyte molecule conjugates. On the other hand, analyte molecules from the test sample can only bind to either the mobile or immobilized analyte molecule conjugate. This further enhances the concentration buffering effect of this embodiment.

[0082] In some embodiments, each detectable label becomes saturated with analyte molecules (i.e., the number of analyte molecules bound to each detectable label is the maximum number that can be bound to that label). The specific number of analyte molecules bound to a detectable label varies for each target molecule, as it depends on the size of the target analyte molecule.

[0083] The analyte molecule is bound to a detectable label using any suitable means well known to those skilled in the art. In particular, the detectable label remains detectable while the analyte molecule is bound to it. In some embodiments, the analyte molecule is bound to the detectable label via a linker. The linker may be any suitable linker well known to those skilled in the art. For example, the linker may include biotin and / or BSA. In some embodiments, the linker is biotin-BSA.

[0084] A detectable label can be any suitable molecule known to those skilled in the art that generates a detectable signal having a detectable or measurable intensity. For example, a detectable label can be a dye particle, a carbon particle, a fluorescent label, a latex particle, a gold particle, a magnetic particle, or similar. In some embodiments, the detectable label generates an optical signal, such as a change in color or the appearance and / or disappearance of a visible marker. Thus, the signal intensity can be a change (either positive or negative) in the light intensity detected in a given test area. For example, a detectable label emits red light when bound to a test site. An increase in measurable intensity may not actually be related to an increase in the intensity of red light, but rather to a relative increase in the intensity of red light with respect to other light frequencies (e.g., an increase in absorption of other light frequencies). Alternatively, an increase in signal intensity may be related to a decrease in the intensity of reflected light. For example, in the case of a red dye bound to a white test area, an increase in signal intensity may be related to a decrease in the overall intensity of reflected light across all visible frequencies (due to absorption of non-red light by the dye), or to a decrease in the intensity of one or more non-red frequencies.

[0085] In a preferred embodiment, the detectable label is a detectable nanoparticle, preferably a gold nanoparticle.

[0086] The minimum concentration of the analyte molecule in the test sample required to generate a detectable signal in the second test area can be preset using a predetermined amount of bound analyte and a predetermined number of binding sites in the first test area. For example, as the amount of bound analyte in the transverse flow inspection device increases, the competition between the bound analyte and the analyte molecules in the test sample becomes more favorable to a larger number of bound analytes, thus increasing the minimum concentration of the target analyte required to cause a change in the signal intensity in the first and second test areas.

[0087] analyte molecule The analytes include any analytes of interest, particularly those related to diseases or pathological conditions. Other analytes of interest include hormones, antibiotics, contaminants (e.g., food and beverage contaminants), and other pollutants. The analytes may be polypeptides, polynucleotides, or organic or inorganic compounds.

[0088] In some embodiments, the molecule to be analyzed is an antigen, such as a protein, polypeptide, or a fragment thereof. Examples of antigens include bacterial or viral proteins or fragments thereof, such as coronavirus proteins or fragments thereof. Proteins of interest include, for example, enzymes or protein hormones.

[0089] The analyte is a biomarker of a disease or condition, or a biomarker of immunity to a disease, and the detection and quantification of this biomarker is useful for the diagnosis and / or monitoring of the associated disease or condition, or immunity to it. The analyte may be, for example, a marker of pregnancy, renal function, bacterial infection, or viral infection. The biomarker may be a metabolite, lipid, steroid, protein, polypeptide, polynucleotide, or a fragment thereof. In some embodiments, the analyte is 3-nitro-hydroxyphenylacetic acid (NHPA). In some embodiments, the analyte is an antibody. In some embodiments, the analyte is creatinine.

[0090] The analyte may be a pharmaceutical, dietary supplement, or other organic or inorganic compound for which detection and quantification are useful. In some embodiments, the analyte is caffeine.

[0091] analyte binding molecule The analyte molecule in the analyte sample and the molecule that binds to the analyte molecule in the bound analyte can be used as the analyte binding molecule. Therefore, the analyte binding molecule is suitable for binding to the analyte molecule of the test sample and, separately, to the analyte molecule of the bound analyte (i.e., the analyte binding molecule is suitable for binding to the analyte molecule of the test sample and the analyte molecule of the bound analyte, but not to binding them simultaneously). The analyte binding molecule is preferably specific to the analyte molecule (of the test sample and the bound 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 is an antibody specific to 3-nitrotyrosine (3-NTyr), which is known to cross-react with NHPA (Wisastra et al., "Antibody-free detection method for protein tyrosine nitration in tissue sections", ChemBioChem, 2011, Vol. 12, pp. 2016-2020). As another example, if the analyte is caffeine, the analyte-binding molecule may be an anti-caffeine antibody, as is well known to those skilled in the art. As yet another example, if the analyte is creatinine, the analyte-binding molecule may be an anti-creatinine antibody.

[0092] In some embodiments, the analyte is an antibody, as described above, and the analyte-binding molecule is a molecule to which that antibody specifically binds. For example, the analyte-binding molecule may be an antigen, and the analyte may be an antibody specific to that antigen. In other examples, the analyte is an antibody, and the analyte-binding molecule may be an antibody specific to the analyte (i.e., an anti-antibody).

[0093] capture molecule In embodiments where the first test area includes a capture molecule, the capture molecule can be any suitable molecule capable of binding to and immobilizing the analyte-binding molecule, thereby retaining the analyte-binding molecule in the first test area. In some embodiments, the capture molecule has the ability to bind to a complex formed by the analyte-binding molecule and the labeled analyte molecule or the analyte molecule from the test sample (i.e., the capture molecule is specific to the complex formed by the analyte-binding molecule and the labeled analyte, or the complex formed by the analyte-binding molecule and the analyte molecule from the test sample). Preferably, in these embodiments, the capture molecule does not bind to an analyte-binding molecule that is not bound to the labeled analyte or the analyte molecule from the test sample. In some embodiments, the immobilized capture molecule is an antibody specific to the analyte-binding molecule. In embodiments where the analyte-binding molecule is an antibody, the capture molecule may be an anti-antibody complex antibody specific to the analyte-binding molecule bound to the labeled analyte or the analyte molecule from the test sample.

[0094] Conjugated analyte binding molecules The conjugated analyte-binding molecule in the second test area binds the conjugated analyte, rather than the unconjugated analyte molecule (i.e., the analyte molecule derived from the test sample). Any suitable conjugated analyte-binding molecule can be used, such as a detectable label or a molecule that specifically binds to the linker of the conjugated analyte, if present.

[0095] In some embodiments, the linking linker contains biotin, preferably biotin-BSA, and the bound analyte binding molecule is avidin, streptavidin, or polystreptavidin.

[0096] Signal detection The signal generated by the detectable label of the combined analyte can be detected by any means suitable for the type of signal to be detected. In some embodiments, the detectable label generates an optical signal, which is detected and its intensity measured using an optical signal sensor and / or reader. For example, the optical signal can be detected and its intensity measured using a smartphone (e.g., using the smartphone's camera). Alternatively, the optical signal can be detected and its intensity measured using a reader that includes a transverse flow test holder configured to accept a transverse flow test for measurement. The holder may include one or more walls configured to surround the transverse flow test (e.g., to block external light). The reading device may include a light source for illuminating the lateral flow test during measurement. This allows control of the amount of light incident on the lateral flow test during measurement. Details of the signal detection means will be described later with reference to Figure 26.

[0097] Detection of analyte molecules in test samples 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. In particular, the presence of analyte molecules in the test sample means that there is competition for binding to the first test region between the analyte molecule and the bound analyte molecule. When the competition reaches a point where the first binding site in the first test region is saturated with analyte molecules and / or bound analyte molecules, the bound analyte molecules that could not bind to the first test region bind to the second binding site in the second test region. The more analyte molecules that bind to the first binding site, the more bound analyte molecules that cannot bind to the first binding site, and therefore the signal intensity in the first test region decreases. The fewer bound analyte molecules that cannot bind to the first binding site, the more bound analyte molecules that bind to the second binding site, and therefore the signal intensity in the second test region increases.

[0098] In some embodiments, the presence of one or more analyte molecules in the test sample is indicated by a decrease in signal intensity in the first test region to less than 100%. Therefore, in these embodiments, if no analyte molecules are present in the test sample, the device either binds all first binding sites in the first test region to the bound analyte molecules (either an excess of labeled analyte or the same number of first binding sites as the number of labeled analyte molecules), or the entire labeled analyte binds to the first binding sites (either an excess of first binding sites or the same number of labeled analyte molecules as the number of first binding sites).

[0099] In some embodiments, the presence of one or more analytes in the test sample is indicated by an increase in signal intensity in the second test region from less than 100%. In these embodiments, if no analytes are present in the test sample, some or all of the second binding sites do not bind to the bound analytes. Therefore, if one or more analytes are present in the test sample, bound analytes that do not bind to the first test region due to competition with other analytes in the test sample bind to the second test region instead, thereby increasing the signal intensity in the second test region. Consequently, the signal intensity in the second test region is inversely correlated with the signal intensity in the first test region.

[0100] In some embodiments, the presence of one or more analyte molecules in the test sample is indicated by an increase in signal intensity from 0% in the second test region. Therefore, in these embodiments, if no analyte molecules are present in the test sample, all bound analyte molecules bind to the first test region and not to the second test region (or substantially not bind, resulting in no detectable signal).

[0101] The detection of signal intensity in the first and second test areas is based on the signals present in the first and second test areas at the minimum time after the test sample has been applied to the sample-receiving area. It will be understood that signal intensity detection can be performed using the first and second test areas themselves (i.e., direct detection from the physical first and second test areas) or using reproductions such as photographs of the first and second test areas. In particular, sufficient time must be allowed before signal intensity detection to allow time for the test sample to move through the conjugate pad, the first test area, and then the second test area. The detection of signal intensity in the first and second test areas is based on any suitable minimum time (e.g., 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 detection of signal intensity in the first and second test areas is 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.

[0102] The detection of signal intensity in the first and second test areas can be based on signals present in the first and second test areas before the maximum time has elapsed since the test sample was applied to the sample-receiving area. For example, the detection of signal intensity can be based on signals present in the first and second test areas within 1 hour, 50 minutes, 45 minutes, 40 minutes, 35 minutes, or 30 minutes after the test sample was applied to the sample-receiving area. In some embodiments, the detection of signal intensity in the first and second test areas is based on signals present in the first and second test areas within 1 hour after the test sample was applied to the sample-receiving area.

[0103] Quantification The apparatus and method of the present invention provide semi-quantitative or quantitative results. In particular, the lateral flow testing apparatus of the present invention is manufactured to have known amounts of each component, i.e., the bound analyte and first and second binding sites, and optionally, a movable analyte-binding molecule, in order to produce an apparatus having an appropriate detection threshold for the target analyte of interest. In particular, each target analyte has a different threshold concentration that indicates a relevant condition (e.g., the presence of a disease or pathological condition, a threshold level of an acceptable contaminant, etc.). Those skilled in the art know how to adjust the amounts of the bound analyte and the first and second binding sites, and optionally the movable analyte-binding molecule, to provide a specific threshold for a positive reaction (i.e., detection of the target analyte in the test sample).

[0104] Therefore, changes in signal intensity in both the first and second test regions indicate the presence of the analyte molecule in the test sample above the threshold concentration. In particular, the changes in signal intensity are due to a decrease in signal intensity in the first test region, resulting from increased competition between the bound analyte and analyte molecules in the test sample, and a decrease in the number of bound analyte molecules binding to the first test region, while the increase in signal intensity in the second test region (and vice versa) is due to an increase in bound analyte molecules that do not bind to the first test region but bind to the second test region.

[0105] Therefore, the method and apparatus of the present invention provide quantitative determination of the concentration of the analyte molecule in a test sample by measuring the change in signal intensity in the first and second test areas. As mentioned above, since the target molecule threshold concentration in the test sample required for a change in signal intensity is known, a further decrease in signal intensity in the first test area and a further increase in signal intensity in the second test area correlate with an increase in the target molecule concentration in the test sample.

[0106] Furthermore, the apparatus and method of the present invention enable more accurate quantification of analyte molecules in the test sample because there is no loss of information regarding the amount of conjugate that does not bind to the first test region and / or binds to the second test region. As mentioned above, conventional LFTs require an excess of labeled conjugate to ensure that the control line is an effective control, which means that information regarding the amount of labeled conjugate that binds to the control line is lost. In contrast, with the apparatus and method of the present invention, the amount of detectable labeled analyte is equal to or less than the number of binding sites in the second test region, so there is no corresponding loss of information.

[0107] It should be noted that the apparatus and method of the present invention do not require a separate control line, as the presence of a detectable signal in either the first or second test area indicates a valid test. The absence of both lines indicates an invalid test.

[0108] In some embodiments, the quantification of analyte molecules in a test sample is performed by measuring the signal intensity in a first test area and a second test area, respectively, and calculating the ratio of the two signal intensities. This ratio of the two signal intensities indicates the number of analyte molecules in the test sample. In particular, as mentioned above, since the apparatus of the present invention is manufactured with known and predetermined amounts of each component, the amount of labeled analyte molecules bound to the first and second test areas, respectively, is known when no analyte molecules are present.

[0109] The signal intensity in each of the first and second test regions can be measured. This can be achieved by acquiring one or more photographs of the first and second test regions. The locations of the first and second test regions in one or more photographs can be obtained using user input or image recognition methods such as edge detection and / or machine learning. The signal intensity in each test region is determined based on the intensity of one or more pixels representing that test region. This may be based on a single pixel or on the accumulation of intensity values ​​of multiple pixels (e.g., average intensity).

[0110] Subsequently, the pixel intensity value is converted to a signal intensity representing the intensity change (e.g., relative to the calibration region). The signal intensity may be expressed as a relative value (e.g., a normalized value) to either the maximum signal intensity or the minimum signal intensity, or both. The maximum signal intensity may be a predefined value (e.g., based on a preceding calibration measurement) or determined from a measurement of the signal intensity in the calibration region of the transverse flow test (e.g., a calibration line with a predefined color (or discoloration) corresponding to a fully saturated test region (e.g., 100% signal intensity)). The signal intensity may be determined as a percentage of the maximum signal intensity. This percentage may be based on the minimum signal intensity. This minimum signal intensity is either predefined or measured (e.g., a calibration region with a predefined color corresponding to a fully unsaturated test region (e.g., 0% signal intensity)). In this embodiment, 100% represents a fully saturated line and 0% represents a fully unsaturated line, but it will be understood that the opposite values ​​may apply depending on the responsiveness of the line to the analyte.

[0111] In some embodiments, the quantification of the analyte molecule in a test sample is performed by measuring the signal intensity in either the first or second test area, and then predicting the signal intensity in the other test area based on the fact that the sum of the signal intensities of the first and second test areas equals 100%. This measurement and subsequent prediction yield the ratio of measured to predicted signal intensities in the first and second test areas.

[0112] In some embodiments, the signal intensity of the first test region is measured, and then the signal intensity of the second test region is predicted based on the fact that the sum of the signal intensities of the first and second test regions equals 100%. This yields the first measured value:predicted signal intensity ratio. Alternatively, the signal intensity of the second test region is measured, and then the signal intensity of the first test region is predicted based on the fact that the sum of the signal intensities of the first and second test regions equals 100%. This yields the second measured value:predicted signal intensity ratio. The first and second measured value:predicted signal intensity ratios can be calculated in either order. A calibration coefficient (also known as a scaling coefficient) can be applied to both the first and second measured signal intensities. This calibration coefficient is used to recalibrate the measured values ​​(described later). The calibration coefficient starts at an initial value (e.g., 1) and may be adjusted based on calibration checks. When scaling with a calibration coefficient of 1, the scaled measured value may be equal to the unscaled measured value (e.g., no change).

[0113] Next, the first and second measured:predicted signal intensity ratios are averaged (for example, the average of the first and second measured:predicted signal intensity ratios is calculated). This generates the average first:second test region signal intensity ratio. Then, the ratio of the measured signal intensity is determined using the measured signal intensity from both the first and second test regions. The measured values ​​used to calculate the measured signal intensity ratio may be the same as those used in the preceding prediction. Alternatively, different measured values ​​may be obtained (for example, from different samples of the same image, or from different images).

[0114] The averaged signal intensity ratio of the first and second test areas is compared to the measured signal intensity ratio. This provides a ratio measurement calibration that verifies the accuracy of the signal intensities used to calculate the averaged signal intensity ratio of the first and second test areas.

[0115] If the average first:second test region signal intensity ratio does not match the ratio of measured signal intensities (e.g., within a predefined range), the calibration coefficient of the measured signal intensities can be adjusted, and the quantification method can be repeated. That is, based on the adjusted calibration coefficient, the initially measured and second measured signal intensities can be recalculated to obtain the new measured-to-predicted signal intensity ratio, the new average signals for the first and second test regions can be obtained, and these new average signals for the first and second test regions can be compared to the new ratio of measured signal intensities (based on the adjusted calibration coefficient). The recalculated first and second measured signal intensities can be based on the same intensity measurements as before, or on new measurements.

[0116] If the mean first:second test area signal intensity ratio matches the ratio of the measured signal intensities, the mean first:second test area signal intensity ratio is used as the signal intensity ratio of the test sample without recalibration.

[0117] This method can be repeated multiple times until the average signal intensity of the first to second test domains matches the measured signal intensity ratio (or until the maximum number of repetitions is reached).

[0118] The scaling direction (the direction of adjustment of the calibration coefficient) may differ depending on whether the measured signal intensity is greater than or less than the maximum signal intensity (e.g., 100% or more of the maximum signal intensity). For example, if the sum of the first and second measured signal intensities exceeds the maximum signal intensity, the calibration coefficient may decrease (e.g., by a predetermined step size or by a predetermined coefficient). Similarly, if the first and second measured signal intensities do not exceed the maximum signal intensity, or are less than the maximum signal intensity, the calibration coefficient can be increased (e.g., by a predetermined step size or by a predetermined coefficient). This allows for recalibration of signal intensity measurements to account for fluctuating ambient lighting conditions.

[0119] In some embodiments, if the average first:second signal intensity ratio falls within a predetermined range of the measured signal intensity ratio, the average first:second signal intensity ratio coincides with the measured signal intensity. 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 intensity ratio. That is, an error tolerance of 10% or less is permitted. Preferably, if the average first:second signal intensity ratio falls within 5% of the measured signal intensity ratio, the average first:second signal intensity ratio coincides with the measured signal intensity ratio.

[0120] The determined signal intensity ratio of a sample (e.g., scaled or unscaled) indicates the concentration of the analyte in the test sample. Therefore, the concentration of the analyte in the test sample can be determined using the signal intensity ratio (e.g., based on a calibration curve of signal intensity ratio and analyte concentration). This method of quantifying the analyte in a test sample allows for the reduction of systematic errors caused by the LFT instrument's reader and errors caused by ambient lighting. In particular, the first and second measured:predicted signal intensity ratios determine the quantitative accuracy (reproducibility of results), and the comparison of the measured signal intensity ratio with the average first:second test area signal intensity ratio ensures quantitative accuracy (true quantification of the target analyte in the test sample).

[0121] Similar to existing quantitative LFTs, a calibration curve is used to calculate the concentration of the analyzed molecule based on the ratio of the determined signal intensities.

[0122] The apparatus and method of the present invention also address the problem of test line saturation in conventional LFTs. In particular, conventional quantitative LFTs use only a test line and not a control line to quantify the concentration of analyte molecules in a test sample. Therefore, when the test line becomes saturated with analyte molecules from the test sample, excess analyte molecules from the test sample cannot be quantified. However, the apparatus and method of the present invention described in [reference] uses two test regions (e.g., lines) and uses the ratio of the signal intensities of these two test regions to quantify analyte molecules in a test sample beyond the normal saturation point of the corresponding conventional LFT. Figure 21 shows the saturation mechanism of a conventional sandwich LFT. In particular, there are unbound, and therefore unquantified, analyte molecules because there are more analyte molecules in the test sample than available conjugates or anti-analyte antibodies on the test line. In contrast, the apparatus and method of the present invention generates a signal intensity ratio of two test regions, depending on the competition between analyte molecules and bound analyte molecules in the test sample, and uses this to quantify the analyte in the test sample. Figure 22 shows that when the molecular weight of the analyte in the test sample is equal to the amount of bound analyte and the number of binding sites on the first test line (i.e., similar to the saturation point in a conventional sandwich LFT; in this example, there are 200 units each of analyte, bound analyte, and first binding site), the competition between the analyte molecules from the test sample and the bound analyte becomes equal, and binding to the first test line is also equal. The remaining bound analyte (i.e., 50% of the bound analyte initially present on the binding pad) binds to the second test line, resulting in equal signal intensity in both the first and second test lines (i.e., a signal intensity ratio of 50%:50% (1:1)). Figure 23 shows the case where the molecular weight of the analyte in the test sample exceeds the amount of bound analyte and binding sites on the first test line (i.e., the saturation point in a conventional sandwich LFT). In this example, even if there are twice as many analyte molecules (400 units) as there are bound analyte molecules and the first binding site (200 units each), quantification of the analyte molecules in the test sample is still possible.This is because increased competition for binding sites on the first test line by analyte molecules from the test sample results in a change in the signal ratio between the first and second test lines. In particular, in this example of LFT, the signal intensity ratio between the first and second test lines is 33%:67% (1:2). Therefore, it is still possible to quantify the concentration of analyte molecules in a test sample that exceeds the normal saturation level of the corresponding conventional LFT using the apparatus and method of the present invention.

[0123] In conventional LFTs, one solution to overcome the saturation problem is to increase the number of binding sites on the test line. However, this leads to a decrease in resolution when quantifying analytes in the test sample. In particular, as the number of binding sites on the test line increases, the proportion that each binding site contributes to the maximum signal intensity of the test line decreases. For example, in a hypothetical LFT with 200 binding sites on the test line, each binding site accounts for 0.5% of the maximum signal intensity. If the number of binding sites is increased to 400, the proportion that each binding site accounts for decreases to 0.25% of the maximum signal intensity. As an example, Figure 24 shows a hypothetical conventional sandwich LFT with 1000 binding sites on the test line. When the number of analytes in the test sample increases from 400 molecules (Figure 24a) to 415 molecules (Figure 24b), the signal intensity on the test line changes from 40% (Figure 24c) to 41.5% (Figure 24d). Figure 25 shows an example of the LFT according to the present invention, demonstrating that an increase in the number of analyte molecules in the test sample from 400 (Figure 25a) to 415 (Figure 25b) results in an overall change of 2% in the signal intensity ratio (from 50% to 49% in the first test line and from 50% to 51% in the second test line; Figures 25c and d). In particular, the change in signal intensity in the first test line is reflected by an inverse correlation with the signal intensity in the second test line. This substantially doubles the change in signal intensity compared to conventional LFTs. Since a larger change in signal intensity makes detection and measurement easier, the LFT apparatus of the present invention improves the quantitative accuracy of analyte molecules compared to conventional LFTs.

[0124] For similar reasons as in the measurement of detection signal intensity, the quantification of the concentration of the analyte molecule in the test sample is performed based on the signals present in the first and second test areas after a predetermined time has elapsed since the test sample was applied to the sample receiving area. Similar to the detection of signal intensity, quantification can be performed on the first and second test areas themselves, or on copies of these areas (such as one or more photographs). Therefore, the aforementioned minimum time requirement for signal intensity detection also applies to the quantification of the concentration of the analyte molecule in the test sample.

[0125] Similar to the detection of signal intensity (described above), the quantification of the concentration of the analyte molecule in the test sample can be performed based on the signals present in the first and second test areas before the maximum time has elapsed since the test sample was applied to the sample-receiving area. The maximum time may be the same as that discussed in relation to the detection of signal intensity described above.

[0126] component ratio In the LFT apparatus of the present invention, the number of molecules of the bound analyte is equal to or less than the number of second binding sites in the second test region. This ensures that when the test sample contains analyte molecules, a change in signal intensity always occurs in the test region.

[0127] The specific amounts of each binding analyte, first binding site, and second binding site are not essential to the function of the device and method of the present invention, as long as the amounts added to the device during manufacturing are known. These amounts can be used to calibrate the reader used to detect signals and measure their intensity in the first and second test areas. In particular, different analytical molecules (i.e., different target analytes) require different amounts of each component. This is because different types of target analytes have different threshold levels and concentrations of interest.

[0128] In some embodiments, the ratio of the bound analyte molecule 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 bound analyte molecules is the same as the number of second binding sites in the second test region (i.e., a 1:1 ratio).

[0129] The number of first binding sites may be greater than, equal to, or less than the number of second binding sites. In particular, the number of first binding sites can be selected to set the minimum threshold concentration of the analyte in the test sample required for the change in signal intensity in the first test region. 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 first to second binding sites is one of the following: 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.

[0130] The ratio of any of the above target molecules to the second binding site can be combined with the ratio of any of the above first binding sites to the second binding site. In some embodiments, the ratio of the target molecule 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 target molecule:first binding site:second binding site is 1:1:1). In particular, when the ratio of bound analyte:first binding site:second binding site is 1:1:1, there is an advantage that the signal intensity of the second test region changes more clearly when the analyte molecule is present in the test sample (i.e., a clear transition in the signal intensity between the first and second test regions). In particular, when the analyte molecule is not present in the test sample, the signal intensity of the first test region is at its maximum value (i.e., 100% of the possible signal intensity), and the signal intensity of the second test region is at its minimum value (i.e., 0% of the possible signal intensity). Thus, the presence of the analyte molecule in the test sample is indicated by a decrease in the signal intensity of the first test region from 100% of its maximum value, and an increase in the signal intensity of the second test region from 0% of its maximum value.

[0131] In some embodiments, the conjugate pad includes a movable conjugate analyte, the first test area includes an immobilized analyte binding molecule, and the second test area includes an immobilized conjugate analyte binding molecule, with the ratio of conjugate analyte to analyte binding molecule to conjugate analyte being 1:1:1 to 1:100:100. Preferably, the ratio of binding analyte to analyte binding molecule to binding analyte binding molecule is 1:1:1.

[0132] In embodiments in which the conjugate pad includes a movable analyte conjugate molecule and a movable conjugate analyte, and the first test area includes a capture molecule, the ratio of the conjugate analyte to the second binding site, the ratio of the first binding site to the second binding site, and the ratio of the conjugate analyte to the first binding site to the second binding site may each be any of the relevant ratios disclosed above independently. The ratio of the conjugate analyte to the movable analyte conjugate molecule to the first binding site can be any ratio suitable for adjusting the effect of different flow rates of the target analyte molecule and conjugate analyte on the first and second test areas from the conjugate pad. In some embodiments, the ratio of the conjugate analyte to the movable analyte conjugate molecule is in the range of 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 ratio of the bound analyte to the freeable analyte-binding molecule to the first binding site is in the range of 1:1:1 to 1:100:100.

[0133] In embodiments in which the conjugated pad comprises a conjugated analyte and a movable analyte-binding molecule, and the first test area comprises an immobilized analyte-binding 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 and second binding sites may each be independently any of the relevant ratios disclosed above. The ratio of the bound analyte to the movable analyte-binding molecule can be any ratio suitable for functioning as a concentration buffer to set a minimum concentration threshold before a positive result is displayed on the LFT.

[0134] Test sample The test sample added to the apparatus in the method of the present invention can be any sample of interest. The test sample may be a liquid containing or consisting of body fluids, a liquid sample obtained from a swab or other test (e.g., cellular and non-cellular substances suspended in liquid), or a liquid sample obtained from a solid sample (e.g., a solution of a solid sample or a part of its components). The test sample may be obtained from living organisms such as humans, animals (i.e., non-human animals), or plants, an environmental sample obtained from a natural or artificial environment, or a food or beverage sample (i.e., obtained from food or beverages). In some embodiments, the test sample may contain or consist of body fluids, such as urine, blood, plasma, serum, saliva, or mucus.

[0135] Specific LFTs - NHPA, caffeine, creatinine In some embodiments, the apparatus and methods of the present invention are for, for example, the detection and / or quantification of caffeine in the blood (i.e., the analyte molecule is caffeine). In particular, it is useful to test for caffeine in a patient's blood before a cardiac MRI scan because caffeine inhibits the vasodilatory effect of adenosine administered during a cardiac MRI scan (J. Majd-Ardekani, P. Clowes, V. Menash-Bonsu, TO Nunan, Nucl. Med. Commun., 2000, 21, 361-364). This can lead to abnormal results or false negative results, potentially delaying the diagnosis of cardiac disease in the patient (E. Reyes, CY Loong, M. Harbinson, J. Donovan, C. Anagnostopoulos and SR Underwood, 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. The detectable label may be gold nanoparticles. The analyte-binding molecule is capable of binding to caffeine and is preferably an anti-caffeine antibody. The bound analyte-binding molecule may be avidin, streptavidin, or polylistreptoavidin. The bound analyte preferably includes a linker, which may be BSA-biotin. In some embodiments, the bound analyte includes one or more caffeine molecules bound to gold nanoparticles via a BSA-biotin linker, the analyte-binding molecule is an anti-caffeine antibody, and the bound analyte-binding molecule is avidin, streptavidin, or polylistreptoavidin. In a method for detecting and / or quantifying caffeine, the sample may be a blood, plasma, or serum sample.

[0136] In some embodiments, the apparatus and methods of the present invention are for the detection and / or quantification of 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 gold nanoparticles. Preferably, each NHPA molecule is conjugated to the detectable label via a linker, the linker preferably BSA-biotin. The analyte-conjugating molecule is capable of binding to NHPA and is preferably an anti-NHPA antibody. In some embodiments, the analyte-conjugating molecule is an anti-3-nitrotyrosine (3-NTyr) antibody, which has been shown to cross-react with the BSA conjugate of NHPA (Wisastra et al., "Antibody-Free Detection Method for Protein Tyrosine Nitration in Tissue Sections," ChemBioChem, 2011, Vol. 12, pp. 2016-2020). The conjugated analyte-conjugating molecule may 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-conjugated molecule is an anti-3NTyr antibody, and the conjugated analyte-conjugated molecule is avidin, streptavidin, or polystreptavidin. In methods for detecting and / or quantifying NHPA, the sample may be a urine sample.

[0137] In some embodiments, the apparatus and methods of the present invention are for the detection and / or quantification of 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, the linker being preferably BSA-biotin. The analyte-binding molecule is capable of binding to creatinine and is preferably an anti-creatinine antibody. The conjugated analyte-binding molecule may be avidin, streptavidin, or polylistreptoavidin. 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 polylistreptoavidin. In a method for detecting and / or quantifying creatinine, the sample may be a blood, plasma, or serum sample.

[0138] Use of this apparatus and method The apparatus of the present invention is for detecting the presence and optionally the amount of an analyte molecule in a test sample, and the present invention includes a method for detecting the presence and optionally the amount of an analyte molecule in a test sample. The method and apparatus of the present invention are useful for detecting the presence and optionally the amount of an analyte molecule in test samples obtained from various sources, and are therefore useful for a wide range of purposes.

[0139] For example, the apparatus and methods of the present invention are useful for diagnosing diseases or conditions using test samples obtained from humans, animals, or plants. In particular, the methods for detecting the presence and optionally the amount of analyte molecules in a test sample according to the present invention can be used to provide information for diagnosis (i.e., contribute to diagnosis). Diagnosis may depend solely on the results of the methods of the present invention, or the results of the methods 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, which involves performing a method for detecting the presence and optionally the amount of analyte molecules in a test sample obtained from humans, animals, or plants.

[0140] As another example, the apparatus and method of the present invention are useful for detecting the presence and, optionally, the amount of analyte molecules in environmental samples or food and beverage samples.

[0141] Furthermore, the apparatus and method of the present invention are useful for detecting the presence and, optionally, the amount of markers of good health and markers of poor health (i.e., when the analyte molecule is a marker indicating good or poor health), even if they do not necessarily lead to the diagnosis of a specific disease or condition.

[0142] Furthermore, methods for detecting the presence and optionally the amount of analyte molecules are also useful for monitoring the presence or amount of analyte molecules in a subject (e.g., human, animal, plant) or the environment. This can be, for example, for monitoring the health status or disease tendency of a subject, monitoring disease progression in a subject, monitoring the effect of treatment on a subject, or monitoring the rate of change in the amount of analyte molecules in a subject or the environment. When the purpose is to monitor the rate of change in the amount of analyte molecules, it can be used, for example, to monitor and / or calculate the clearance rate of a drug. Thus, methods for detecting the presence (and optionally the amount) of analyte molecules according to the present invention can be methods for monitoring the presence and / or amount of analyte molecules in a subject or the environment. In some embodiments, the presence of analyte molecules is monitored by taking 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 may be compared to a reference amount and / or the amount in a preceding test sample. The baseline amount may be the amount of analyte measured in a reference test sample acquired at or before the start of the monitoring method, or it may be a baseline amount of analyte calculated as the average of amounts measured in one or more control samples. Alternatively, the baseline amount may be a threshold amount or amount range of analyte known in the art. In some embodiments, the presence / absence and / or amount of analyte in each test sample is useful for monitoring the health status or condition of a subject. For example, the presence / absence of analyte may indicate the quality of the subject's health status (e.g., good / poor health status, or tendency to develop a disease / condition), or it may indicate disease progression or the effect of treatment on the subject. Changes in the presence and / or amount of analyte between two or more test samples may indicate changes in the subject's health status, disease progression, the effect of treatment, or clearance of the substance (e.g., analyte) by the subject.

[0143] Measurement system Figure 26 shows a schematic diagram of a calculation system 100 that quantifies the concentration of analyte molecules in a test sample based on the signal intensity of the first and second test regions of the LFT, according to an embodiment.

[0144] The computing system 100 comprises a processor 110, memory 120, non-volatile memory 130, and an input / output (I / O) interface 140. Optionally, the computing system 100 may also 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, or tablet.

[0145] The computing system 100 is controlled by a processor 110. The processor 110 is configured to quantify the concentration of analyte molecules in a test sample based on executable code stored in non-volatile memory 130 and loaded into memory 120 (e.g., random access memory, RAM) for execution.

[0146] The quantified concentration is determined based on one or more signal intensity measurements in the first test area and one or more signal intensity measurements in the second test area. These measurements are obtained from an LFT image (e.g., one or more photographs) showing the first and second test areas. This image is acquired by an optical sensor, such as a digital camera. The optical sensor may be integrated into the computing system (e.g., optical sensor 150) or installed in an external system. That is, the computing system 100 may be configured to acquire one or more LFT images via an integrated optical sensor, or it may be configured to acquire one or more LFT images from an external system (e.g., via a network such as the Internet).

[0147] For example, a second system (e.g., a second computing system) may be configured to acquire one or more images showing the first and second test areas, or one or more light intensity measurements of the first and second test areas. The computing system 100 may be configured to analyze the signal intensity measurements obtained from one or more images or one or more light measurements to quantify the concentration of the analyte.

[0148] The second system can determine one or more signal intensity measurements from one or more images or one or more light intensity measurements and transmit one or more signal intensity measurements to the computing system 100. Alternatively, the second system can transmit one or more images or one or more light intensity measurements to the computing system 100, and the computing system 100 can determine one or more signal intensity measurements.

[0149] Alternatively, the computing system 100 can acquire one or more images or one or more light intensity measurements, and then perform further analysis to quantify the concentration of the analyte molecules.

[0150] Therefore, when a user acquires LFT images using a mobile device (e.g., a smartphone or tablet), the mobile device may be configured via software to perform the steps described herein to quantify the concentration of the analyte molecule in the test sample. Alternatively, the images may be transferred to another computing system (computing system 100) for quantification, for example, via the cloud.

[0151] The quantified concentration determined by the processor 110 is output via the I / O interface 140 (e.g., a screen, or an external system via a network such as the internet) and can be used by the processor 110 for further calculations or stored in the non-volatile memory 130 for later use.

[0152] Other components The LFT device of the present invention may include further components for optimizing the LFT, in accordance with prior art practices. These additional components are any suitable components known in the art for use in LFT or ELISA assays. For example, the LFT device may include a buffer for adjusting the flow rate of the sample along the solid support structure, or a blocking agent for reducing nonspecific binding of the labeled analyte to the solid support structure and decreasing background signal. The buffer for adjusting the flow rate is any suitable buffer, such as sucrose or glycerol. In some embodiments, the LFT device includes 5% glycerol as the buffer for adjusting the flow rate. The blocking agent is any suitable blocking agent known in the art, such as BSA, PBS, or blocking agents containing Tween-20.

[0153] kit The present invention also provides a kit comprising one or more of the above-described LFT devices. The kit may also include instructions for using the LFT devices and / or further reagents and / or components for carrying out the methods of the present invention. Examples of further reagents and / or components include swabs for collecting samples (e.g., mucus or saliva samples), mixing tubes or liquids for preparing liquid samples (e.g., mucus or saliva samples can be mixed with a solution in a mixing tube before being applied to the LFT device). These additional reagents and / or components may be packaged separately from the LFT devices within the kit (e.g., in individual vials, tubes, or packaging materials). Furthermore, the kit may also include the aforementioned LFT readers for measuring signal intensity in each test area. Alternatively, or additionally, the kit may include instructions for downloading and / or using software for measuring signal intensity in each test area on a user device (e.g., a smartphone). [Examples]

[0154] Example 1 - Caffeine detection This example demonstrates the detection of caffeine chromaticity using an improved competitive LFT format (Figure 3). The LFT employs two test lines instead of a single test line, with the signal intensity of both lines corresponding to the caffeine concentration in the sample. This assay uses the AuNP-BSA-caffeine-biotin conjugate as the detection conjugate, employing immobilized anti-caffeine antibody and avidin 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; therefore, the signal intensity of the first test line is inversely proportional to the caffeine concentration. Subsequently, the substituted conjugate is captured by avidin on the second test line, so the signal intensity of the second test line shows a positive correlation with the present caffeine concentration. This improves the maximum sensitivity of the assay in detecting low caffeine concentrations.

[0155] method General materials and methods NHS-LC-biotin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), avidin, HRP-labeled anti-mouse secondary antibody, and HRP-labeled avidin were purchased from Thermo Fisher Scientific. Mouse-derived monoclonal anti-caffeine antibody 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 purchased from Sigma Aldrich. The buffers used were prepared with Milli-Q water. PD-10 columns packed with Sephadex G-25 medium were purchased from GE Healthcare Life Sciences. Costar 3370 high-binding 96-well plates used for enzyme immunosorbent assay (ELISA) were purchased from Corning. For the lateral flow assay, we used the Hi-Flow Plus membrane HFB07502 purchased from Merck Millipore. The test lines on the nitrocellulose membrane were: The samples were prepared using a SciFlexArrayer equipped with a delivery piezodispensing capillary (PDC).

[0156] General technology i) UV-Vis spectrophotometry UV-Vis spectrophotometric measurements were performed using a Spectramax M3 microplate reader in the range of 350–750 nm with a resolution of 1 nm. A blank corresponding to the buffer used for each sample was subtracted from the UV-Vis spectrum as background correction.

[0157] ii) Nanoparticle Tracking Analysis (NTA) 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 injected into the sample chamber using a 1 mL syringe. The acquired video (60 seconds) was processed using Halo 2.3 analysis software.

[0158] iii) Dynamic light scattering (DLS) Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer Nano S instrument equipped with a 633 nm laser, using a DTS1061 disposable foldable capillary cell. Two measurements were performed for each sample, with each measurement consisting of 20 scans of 10 seconds each. The z-mean and zeta potentials of the samples were analyzed using Malvern Instruments Dispersion Technology 7.11 software.

[0159] iv) Transmission electron microscope (TEM) Nanoparticles were characterized by TEM using a JEOL JEM-2100F instrument operating at 200kV. A sample (10 μL) was dropped onto a porous carbon film on a 300-mesh copper grid purchased from Agar Scientific. Excess material was removed by bringing the edge of the copper grid close to the filter paper. For samples requiring negative staining, 10 μL of 5% (w / v) ammonium molybdate solution was added after sample addition, and excess material was removed using filter paper. TEM experiments on the prepared samples were performed at the Department of Materials Science, Imperial College. Size measurements were performed using ImageJ software, and the average size of at least 50 particles was calculated for each sample.

[0160] Synthesis of gold nanoparticles An improved Frens protocol was used. Aqua regia was prepared by mixing concentrated nitric acid and concentrated hydrochloric acid in a volume ratio of 1:3. All glassware used in the synthesis was thoroughly washed with aqua regia, then washed 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. This solution was heated under reflux for 30 minutes. Sodium citrate (500 mg, 1.94 mmol) dissolved in Milli-Q water (5 mL) was rapidly added to the boiling solution. The solution turned red wine color and was heated for a further 10 minutes. After cooling to room temperature, the solution was filtered through a 0.2 μm PTFE membrane. Subsequently, the solution was characterized by UV-Vis spectrophotometry, NTA, DLS, and TEM.

[0161] Preparation and testing of BSA-biotin-caffeine complex i) Biotinylation A standard biotinylation protocol was followed. BSA (12 mg, 0.180 μmol) was dissolved in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 (1.2 mL) and added to NHS-LC-biotin (1 mg, 2.20 μmol) in N,N-dimethylformamide (DMF) (25 μL). The reaction was allowed to stand at room temperature for 3 hours, then kept overnight at 4°C. The sample was purified by gel filtration using 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 (3 mL) as the elution buffer. The fraction containing biotinylated BSA was identified by recording the absorbance at 286 nm. The fractions were combined, and the concentration of present BSA was measured using a standard curve.

[0162] ii) HABA assay 4'-Hydroxyazobenzene-2-carboxylic acid (HABA) (24.2 mg, 0.100 mmol) was dissolved in Milli-Q water (9.9 mL). 1 M NaOH (100 μL) was added, and the solution was filtered through a 0.22 μm Millipore filter. Avidin (10 mg) and HABA solution (600 μL) were added to phosphate-buffered saline (PBS) (19.4 mL) to prepare an orange solution. Each biotinylated sample (20 μL) was added to the HABA / avidin solution (180 μL). The absorbance value at 500 nm was recorded, and the biotinylation level was determined according to the following formula:

number

[0163] Here, ΔA 500 ε is the change in absorbance at 500 nm. HABA-アビジン is the extinction coefficient of the HABA-avidin complex, l is the cell optical path length, and [BSA] is the BSA concentration.

[0164] iii) Hapten bond Theophylline-7-acetic acid solution (1.49 μmol, 89 μL, 4 mg / mL in DMSO) was added to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride (20 mg). Biotinylated BSA solution (0.090 μmol, 600 μL, 10 mg / mL in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2) was added. The reaction mixture was allowed to react at room temperature for 2 hours and then stored at 4°C before gel filtration. The sample was purified by gel filtration using 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 (3 mL) as the elution buffer. The fraction containing the highest protein concentration was identified and bound by absorbance measurement at 286 nm.

[0165] iv) TNBS assay The assay protocol reported by Sashidhar et al. was used (RB Sashidhar, AK Capoor and D. Ramana, J. Immunol. Methods, 1994, 167, 121-127). BSA conjugate was used at 0.1M. NaHCO3 Dilute to (pH 8.0) to a final concentration of 200 μg / mL. Add 0.01% (w / v) 2,4,6-trinitrobenzenesulfonic acid (TNBS) (500 μL, 0.1 M) to each sample. NaHCO3 A medium (BSA) was added. 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. Absorbance at 335 nm and 420 nm was recorded. The same procedure was performed for L-lysine and L-glutamic acid to obtain a standard curve for comparison. The amino group concentration in the samples was calculated using the absorbance at 335 nm. The degree of binding was determined by the difference in the number of amino groups per BSA before and after binding.

[0166] v) Capture enzyme immunosorbent assay (ELISA) A 96-well ELISA plate was coated with anti-caffeine antibody (50 μL, 6 μg / mL, PBS buffer, pH 7.4) and incubated at room temperature for 1 hour. The plate was blocked at room temperature for 2 hours with 2% (w / v) BSA in PBS at 0.05% (v / v) Tween-20, pH 7.4. Different concentrations of BSA-biotin-caffeine conjugates (50 μL) were added to the plate and incubated at room temperature for 1 hour. HRP-labeled avidin (50 μL, 1:5000 dilution with blocking buffer) was added and incubated in the dark at room temperature for 1 hour. After thoroughly washing the plate, a 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate system (50 μL) was added to develop color. The reaction was stopped by adding 1 M H2SO4 (50 μL), and the absorbance at 450 nm was recorded.

[0167] vi) Conflict ELISA The basic protocol was the same as for direct ELISA. Plates were coated with anti-caffeine antibody (50 μL, 6 μg / mL, PBS buffer, pH 7.4) and blocked with 2% (w / v) BSA in PBS containing 0.05% (v / v) Tween-20 (pH 7.4). BSA-biotin-caffeine conjugate (25 μL, 40 mg / mL) was mixed with caffeine (25 μL) at different concentrations and added to plates, which were incubated at room temperature for 1 hour. HRP-labeled avidin (50 μL, 1:5000 dilution) was added, and TMB liquid substrate system (50 μL) was added to develop color. The reaction was stopped by adding 1 M H2SO4 (50 μL), and the absorbance at 450 nm was recorded.

[0168] AuNP-BSA conjugate preparation i) Salt-induced agglutination method To determine the minimum amount of BSA required for colloidal gold stabilization, a salt-induced agglutination method was used. BSA (15 mg / mL in 0.05 M sodium phosphate buffer, pH 7.0) was serially diluted to prepare 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 allowed to react for 5 minutes. 1.7 M NaCl solution (250 μL) was added, and the UV-Vis spectrum of each sample was recorded after 5 minutes.

[0169] ii) Adsorption method Colloidal gold (5 mL) was added to BSA (3 mg) in a 0.05 M sodium phosphate solution (200 μL). 1% (w / v) polyethylene glycol (PEG, molecular weight 20000) (125 μL) was added for stabilization, and the reaction mixture was allowed to react at room temperature for 2 hours. The mixture was centrifuged at 12000 rpm for 40 minutes. The supernatant was removed, and the precipitate was reconstituted with 0.05 M sodium phosphate (5 mL) and 1% PEG (125 μL). The AuNP-BSA complex was characterized by UV-Vis spectroscopy.

[0170] iii) Reduction method 2-mercaptoethanol (0.50 μL, 25 mM) was added to the BSA-biotin-caffeine complex (300 μL, 0.697 mg, 2.32 mg / mL). The reaction mixture was thoroughly mixed and allowed to react overnight at 4°C. The sample was purified by gel filtration using a PD10 column, and eluted with 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 (3 mL) as the elution buffer. The fraction confirmed to contain the protein conjugate at 286 nm by UV-Vis spectrophotometer was pooled (600 μL) and used in the next step. The reduced conjugate was slowly added to colloidal gold (500 μL). The reaction was allowed to stand at 4°C for 2 days. The mixture was then centrifuged three times at 13400 rcf for 30 minutes each time, and washed with 10 mM Tris buffer (containing 3% BSA). The precipitate was reconstituted with 10 mM Tris buffer (containing 3% BSA) and filtered through a 0.2 μm PTFE syringe filter. The conjugates were characterized by TEM using 5% (w / v) ammonium molybdate and 0.1% (w / v) trehalose as negative stains. They were also characterized by UV-Vis spectrophotometry, NTA, DLS, and TEM.

[0171] iv) Elman's method The assay reported by Aitken and Learmonth (A. Aitken and M. Learmonth, The Protein Protocols Handbook, Humana Press, Totowa, NJ, 3rd edn., 2009) was used. Elman's reagent solution was prepared by dissolving 5',5'-dithiobis(2-nitrobenzoic acid) (DTNB) (0.40 mg, 1.08 μmol) in 0.1 M sodium phosphate, 1 mM EDTA, pH 8.0 (5 mL). For use as standard solutions, cysteine ​​hydrochloride (0.25 mM to 1.5 mM) was sequentially diluted in 0.1 M sodium phosphate, 1 mM EDTA, pH 8.0 to prepare different concentrations. Elman's reagent solution (200 μL) was added to each sample (20 μL) and reacted for 15 minutes. Absorbance at 412 nm was recorded, and the hydrogen sulfide content of each sample was measured using a standard curve prepared with a cysteine ​​standard solution.

[0172] v) HABA assay with proteinase digestion AuNP-BSA-biotin-caffeine complex (25 μL) was heated at 56°C for 10 minutes. 1% (w / v) proteinase K solution (2.5 μL) was added to the sample and left overnight at room temperature. The sample was centrifuged at 13400 rcf for 5 minutes. The supernatant (20 μL) was added to HABA-avidin solution (180 μL), and the absorbance at 500 nm was recorded.

[0173] Preparation of Lateral Flow Assay (LFA) i) Assembly of the LFA LFAs were assembled using a nitrocellulose membrane attached to a plastic-backed card. Anti-caffeine antibody and avidin were immobilized on test lines 1 and 2 of the detection zone, respectively. After drying the membrane at room temperature for 1 hour, it was immersed in 2% (w / v) BSA in 0.05% (v / v) Tween-20, pH 7.4 PBS for 5 minutes to block excess binding sites. The membrane was dried at room temperature for 2 hours. A wick was attached to the membrane (with a 2 mm overlap), and the plastic card was cut into strips (5 mm x 60 mm). A glass fiber conjugate pad was immersed in a 30% (w / v) sucrose solution in Milli-Q water and dried in an oven at 37°C. Subsequently, it was 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.

[0174] ii) Testing of LFA strips Caffeine in 2% (w / v) BSA was prepared at different concentrations in each well of a low-adsorption 96-well microplate. Test strips were immersed vertically in each well for 3 minutes, then removed and dried. Images of the test strips were taken and analyzed using ImageJ software. The gray level of the pixels in each test line was measured and subtracted from the background.

[0175] Results and Discussion Synthesis and Characterization of BSA-Biotin-Caffeine Complex i) Synthesis Strategy The binding of biotin to caffeine derivatives requires a carrier protein as a crosslinking protein. The carrier protein must be large, with a molecular weight of 20,000 Da or more, and possess a binding site for small molecules. It is also important that the selected carrier protein is not identical to the one used to produce the anti-caffeine antibody. Since the anti-caffeine antibody used in the transverse flow assay was produced in mice using keyhole limpet hemocyanin (KLH)-3-caffeine, BSA was selected as the carrier protein for binding to biotin and caffeine derivatives. BSA (molecular weight 67,000 Da) is water-soluble, highly stable, and has approximately 30 to 35 accessible lysine residues, making it suitable for binding. It is commercially available in pure form, 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).

[0176] The synthesis strategy for the BSA-biotin-caffeine complex is shown in Figure 4. Biotin and caffeine bind to BSA via the primary amino group of the lysine residue.

[0177] ii) Biotinylation BSA was biotinylated using NHS-LC-biotin and purified by gel filtration. NHS-LC-biotin contains a biotin group bonded to an active ester group, which reacts with the ε-amino group of the lysine residue of BSA to form an amide bond. 6- The presence of an aminocaproic acid spacer arm reduces steric hindrance and improves the binding of the biotin group to avidin (D. Kim and AE Herr, Biomicrofluidics, 2013, 7, 41501).

[0178] Excess NHS-LC-biotin and the byproduct N-hydroxysuccinimide (NHS) were removed by filtering the reaction mixture through desalting resin. Fractions containing the BSA biotin conjugate were identified by absorbance at 286 nm in the gel elution profile.

[0179] The biotinylation level was measured by a 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assay. HABA interacts with the biotin binding site of avidin and forms an orange complex with an absorption peak at 500 nm. Since the binding affinity of biotin for avidin (1.3×10 15 M - 1 ) is much higher than that of HABA for avidin (6. 0×106M - 1 ), adding biotin to the HABA-avidin complex causes HABA to be easily displaced from the biotin binding site (G. T. Hermanson, Bioconjugate Techniques, Elsevier, 3rd edn., 2013). Adding the complete BSA-biotin complex to the HABA-avidin complex resulted in a decrease in absorbance at 500 nm compared to the HABA-avidin complex, indicating successful biotinylation. The change in absorbance at 500 nm was used to calculate the biotin concentration and the degree of biotinylation in the sample.

[0180] Different concentrations of the biotinylation reagent were used, and more aggregation of BSA was observed with a larger molar excess of NHS-LC-biotin. The optimal molar excess of the biotinylation reagent for BSA was determined to be 12, which gives 1.8 biotin molecules per molecule of BSA and was without significant aggregation.

[0181] iii) Hapten binding Since caffeine lacked a functional group suitable for covalent bonding to BSA, a caffeine derivative had to be used for bonding. Theophylline-7-acetic acid was selected as the caffeine derivative to bond to BSA-biotin via a free carboxylate group. Water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was used as the coupling reagent. EDC reacts with the carboxylic acid group of theophylline-7-acetic acid to form a highly reactive O-acyl isourea intermediate. Next, the ε-amino group of the lysine residue of BSA-biotin reacts with the activating group to form an amide bond. The ideal pH for carboxyl group activation by EDC has been reported to be pH 3.5-4.5 (GT Hermanson, Bioconjugate Techniques, Elsevier, 3rd edn., 2013), but precipitation of BSA-biotin 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, 2016, 9, 46-52) reported that at low pH, protein expansion increases accessibility to hydrophobic groups, leading to increased hydrophobicity of BSA. Furthermore, the hydrophobic aliphatic biotin modification resulting from biotinylation of BSA increased the tendency for protein aggregation in solution. While aggregation was not observed when BSA was used for binding in MES buffer at pH 4.0, aggregates were formed when BSA-biotin was used for the same binding, suggesting the need to raise the pH of the reaction buffer. When sodium phosphate buffer at pH 7.2 was used instead as the reaction buffer, amide formation proceeded without protein aggregation.

[0182] The conjugates were purified by gel filtration to remove excess hapten, EDC, and the by-product isourea. The fraction containing the BSA-biotin-caffeine conjugate was identified by absorbance at 286 nm in the gel elution profile.

[0183] Hapten density is affected by the stoichiometric ratio of reagents, coupling conditions, and the carrier protein used (GT Hermanson, Bioconjugate Techniques, Elsevier, 3rd edn., 2013). Determining hapten density is important for optimizing the performance of BSA conjugates for use in immunoassays. Direct methods measuring absorbance, fluorescence, or changes in protein content are commonly used to assess hapten density (R. Lemus and MH Karol, in Allergy Methods and Protocols, eds. MG Jones and P. Lympany, Humana Press, Totowa, NJ, 2008, pp. 167-182). Theophylline-7-acetic acid is a potent chromophore, but its maximum absorbance is 275 nm (JA Owen and K. Nakatsu, Clin. Chem., 1978, 24, 367-368), which significantly overlaps with the absorbance of BSA. Therefore, the degree of binding cannot be quantified using absorbance in the UV region. Instead, the amount of bound hapten was measured by indirect spectroscopy using 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS reacts with primary amines on the protein surface to produce an orange derivative.

[0184] Successful hapten binding via lysine residues in BSA reduces the number of available primary amines within the BSA. Primary amine concentrations in the sample were measured by comparing the absorbance at 335 nm with standard curves created using L-lysine and L-glutamic acid. The degree of binding was determined by the difference in the number of free amines on the BSA before and after binding.

[0185] The number of available primary amines in each BSA molecule before binding was calculated to be 40.8 by the TNBS assay. Although BSA contains a total of 59 lysine residues, only 30-35 are considered available for binding because they are located near the surface. Since the TNBS assay required heating to 37°C, it is possible that the number of available lysine residues increased due to the unfolding of the BSA protein structure, and the obtained value was higher than expected relative to the surface lysine residues. Since all samples were subjected to the same heat treatment, the degree of unfolding among the samples is considered to be nearly uniform, unless binding affected the stability of the protein. The calculated values ​​for the three samples were compared to determine the degree of binding. The difference in the number of available primary amines between BSA and BSA-biotin was 2.4, which was consistent with the value of 1.8 obtained by the HABA assay. By comparing the absorbance of BSA-biotin and the BSA-biotin-caffeine complex, it was found that an average of 3.1 caffeine molecules bound to each BSA molecule through the binding reaction.

[0186] Synthesis and Characterization of AuNP-BSA Conjugates i) Synthesis of AuNP AuNPs were synthesized using the citrate reduction method reported by Frens (G. Frens, Nat. Phys. Sci., 1973, 241, 20-22). Citrate ions act as reducing agents, reducing Au(III) to Au(0). This promotes the growth of the gold colloid. Negatively charged citrate ions 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 particle size of the formed gold colloid is determined by the concentration of stabilizing ligands in the solution. The higher the concentration of stabilizing ligands, the more the ligands stabilize the gold colloid surface and inhibit further growth, resulting in the formation of finer AuNPs.

[0187] AuNPs exhibited a red color, and an absorption peak was observed at 519 nm by UV-Vis spectroscopy. Assuming the citrate coating was not visible, the average core diameter of the AuNPs was measured at 16 (±2) nm by transmission electron microscopy (TEM). This result is consistent with a previous literature report (N. Sosibo, F. Keter, A. Skepu, R. Tshikhudo and N. Revaprasadu, Nanomaterials, 2015, 5, 1211-1222).

[0188] ii) Synthesis of the AuNP-BSA complex Proteins bind to AuNPs primarily through three types of interactions: hydrophobic interactions via tryptophan residues, electrostatic interactions via lysine residues, and gold-thiol bonds via cysteine ​​residues (M. Valcarcel and A. Lopez-Lorente, Gold Nanoparticles in Analytical Chemistry, Elsevier, 1st edn., 2014). Of these, Brewer et al. (SH Brewer, WR Glomm, MC Johnson, MK Knag and S. Franzen, Langmuir, 2005, 21, 9303-9307) reported that the passive adsorption of BSA to AuNPs occurs mainly through electrostatic interactions between positively charged lysine residues on BSA and negatively charged citrate coatings. Ensuring sufficient protein binding to the colloidal gold surface is extremely important. The stability of AuNPs is determined by the balance between electrostatic repulsion and van der Waals attraction between AuNPs. If the protein coating on the surface is insufficient, AuNPs become unstable in high-salt solutions.

[0189] To determine the minimum amount of BSA required to stabilize colloidal gold, the salt-induced agglutination method (C. Fang, Z. Chen, L. Li and J. Xia, J. Pharm. Biomed. Anal., 2011, 56, 1035-1040) was used. Colloidal gold was added to BSA of different concentrations, followed by the addition of a 1.7 M NaCl solution. Since the kinetics of BSA adsorption, and consequently the maximum amount of BSA that binds to the AuNP surface, depend on the BSA concentration, AuNPs with low concentrations of BSA become unstable upon NaCl addition. The salt solution shields the negative charge of the AuNPs, causing agglutination and resulting in aggregation between unstable AuNPs. This aggregation can be visualized by the change in the solution color from red to purple. When the BSA concentration used was reduced, it was observed that the absorbance peak shifted redward from 523 nm to 620 nm upon addition of 1.7 M NaCl.

[0190] The absorbance of each sample at 523 nm and 620 nm was recorded and plotted against BSA concentration. The minimum BSA concentration required to stabilize AuNP was determined to be 2 mg / mL, and at this concentration, no significant shift of the absorption peak from 523 nm to 620 nm was observed.

[0191] The BSA-biotin-caffeine complex was added to AuNPs and incubated overnight to allow passive adsorption to occur. However, the formed complex was observed to be unstable in solution. Even when using an excess of the complex after the BSA-biotin-caffeine complex was attached to AuNPs, a black, insoluble solid was observed at the bottom of the sample tube. The presence of this black solid indicates aggregation due to insufficient stabilization of the AuNPs. Since the binding of biotin and caffeine derivatives to BSA occurs via lysine residues, the positive charge on the BSA surface decreases. Therefore, the electrostatic interaction is weak, and the AuNPs are only slightly stabilized. The adsorption method was deemed unsuitable as a method for attaching the BSA-biotin-caffeine complex to the surface of AuNPs.

[0192] Kaur et al. (J. Kaur, KV Singh, R. Boro, KR Thampi, M. Raje, GC Varshney and CR Suri, Environ. Sci. Technol., 2007, 41, 5028-5036) reported that AuNP-protein complexes formed via gold-thiol bonds are more stable than those 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 that can be used for binding to AuNP (I. Rombouts, B. Lagrain, KA Scherf, MA Lambrecht, P. Koehler and JA Delcour, Sci. Rep., 2015, 5, 12210). The intrachain disulfide bonds of BSA were reduced using 2-mercaptoethanol to generate free sulfhydryl groups that can bind to AuNP. The reduced BSA-biotin-caffeine complex was purified by gel filtration, and the concentration of sulfhydryl groups was measured by spectroscopy using Elman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). DTNB reacted with the free sulfhydryl groups in the sample to produce a mixed disulfide and the yellow product 2-nitro-5-thiobenzoic acid (TNB). The sulfhydryl group concentration was calculated by comparing the absorbance of the solution at 412 nm with a standard curve prepared using L-cysteine.

[0193] The change in the number of sulfhydryl groups after BSA reduction was measured. Treatment with 2-mercaptoethanol increased the number of free sulfhydryl groups per BSA molecule from 0.9 to 2.7.

[0194] The free sulfhydryl groups on BSA substitute for citrate ions that stabilize AuNP and directly interact with the AuNP surface. No black solid was observed during the complexation of the reduced BSA conjugate with AuNP, indicating sufficient stabilization of AuNP. The formed AuNP-BSA-biotin-caffeine conjugate maintained its stability even after 2 months of storage at 4°C.

[0195] iii) Characterization of AuNP and AuNP-BSA complex a) UV-Vis spectroscopy AuNPs exhibit localized surface plasmon resonance (LSPR) due to vibrations of surface conduction electrons during light absorption (C. Larosa, E. Stura, R. Eggenhoffner and C. Nicolini, Materials (Basel), 2009, 2, 1193-1204). This LSPR signal is detected as a strong absorption peak in the visible region of the UV-Vis spectrum. The intensity and wavelength of the observed LSPR peak depend on various factors such as the size and shape of the AuNPs and the dielectric constant of the solution (X. Huang and MA El-Sayed, J. Adv. Res., 2010, 1, 13-28).

[0196] The absorption maximum of citrate-stabilized gold nanoparticles was observed at 519 nm. A redshift from 519 nm to 523 nm was confirmed by the reaction with BSA, indicating that the protein was adsorbed onto the gold nanoparticles. The redshift of the LSPR peak was observed 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). According to Mie theory, an increase in refractive index is predicted to result in a redshift of the absorption maximum peak (S. Dominguez-Medina, S. McDonough, P. Swanglap, CF Landes and S. Link, Langmuir, 2012, 28, 9131-9139). The redshift in the peak wavelength may be due to an increase in the refractive index of gold nanoparticles associated with protein adsorption to the surface.

[0197] b) Size characteristics evaluation Due to the high electron density of AuNPs, they can be visualized using TEM for size characterization. The AuNPs were spherical, monodisperse, and had a size of 16 (±2) nm. The size and quality of AuNPs are important in determining the sensitivity of the transverse flow assay (LFA) (C. Fang, Z. Chen, L. Li and J. Xia, J. Pharm. Biomed. Anal., 2011, 56, 1035-1040). Minute AuNPs less than 10 nm in diameter migrate rapidly but are only weakly visualized in the test line, limiting the sensitivity of the assay. On the other hand, AuNPs larger than 40 nm show a strong red signal in the test line, but they pose problems with slow migration through membrane pores, causing steric hindrance to ligand-receptor binding and low stability against aggregation (S. Lou, J. Ye, K. Li, A. Wu, Analyst, 2012, 137, 1174). The obtained 16nm AuNPs were stable and provided a clear signal in LFA. The synthesized AuNPs were judged to be of high quality due to their narrow size distribution and uniform shape.

[0198] Due to the low electron scattering ability of the protein, negative staining using heavy metals was essential to visualize the BSA-biotin-caffeine complex bound to AuNP. 5% (w / v) ammonium molybdate was used as the negative staining agent. A white halo indicating the BSA complex was observed around each AuNP. BSA binding to the AuNP surface formed a protein shell around each AuNP, resulting in a spherical AuNP-BSA-biotin-caffeine complex. The size of the AuNP-BSA-biotin-caffeine complex was measured at 19 (±2) nm. The shell thickness was approximately 3 nm, which corresponds to the diameter of BSA reported in the literature (M. Su, C. Wang and C. Bai, Chinese Sci. Bull., 1998, 43, 1882-1886). TEM measurements suggest the formation of a monolayer of BSA around each AuNP. The AuNP-BSA-biotin-caffeine complex was sufficiently stabilized, as no particle aggregation was observed during TEM observation.

[0199] Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) were also employed as alternative techniques to evaluate the size of synthesized AuNPs and AuNP-BSA-biotin-caffeine complexes. NTA tracks the Brownian motion of individual nanoparticles and calculates the nanoparticle size based on the Stokes-Einstein equation. DLS, on the other hand, determines nanoparticle size by utilizing the variation in scattered light intensity due to the Brownian motion of nanoparticles (BJ Frisken, Appl. Opt., 2001, 40, 4087). The average diameters of AuNPs measured using NTA and DLS were 17 nm and 24 nm, respectively. The particle sizes reported by NTA were in better agreement with the particle sizes determined by TEM. Since the light scattering intensity is proportional to the sixth power of the nanoparticle diameter, DLS is more sensitive to larger particles, leading to an overestimation of the average nanoparticle size.

[0200] An increase in hydrodynamic diameter was observed when the BSA biotin-caffeine complex bound to AuNPs. The change in hydrodynamic diameter was measured at 5 nm in NTA and 7 nm in DLS. Since the hydrodynamic diameter of BSA was 7.2 nm according to Li et al. (Y. Li, G. Yang and Z. Mei, Acta Pharm. Sin. B, 2012, 2, 53-59), the NTA and DLS measurement results for the AuNP-BSA-biotin-caffeine complex support the formation of a BSA monolayer around each AuNP core.

[0201] Average surface area of ​​16 nm AuNP (804.25 nm 2Using the size of the AuNP and the diameter of the BSA molecule (3 nm), we calculated the theoretical maximum number of BSA molecules that can bind to each AuNP for monolayer coating to be 119 molecules. The number of BSA-biotin-caffeine molecules bound around each AuNP was experimentally determined using a HABA assay with proteinase K digestion. The HABA assay cannot be directly applied to the AuNP-BSA-biotin-caffeine complex because the absorbance region of the AuNP overlaps with the absorbance region of the HABA avidin complex. The concentration of the AuNP was determined by the size of the AuNP and the extinction coefficient (ε) at 450 nm. 450 Determined by UV-Vis spectrophotometric method based on (W. Haiss, NTK Thanh, J. Aveyard and DG Fernig, Anal. Chem., 2007, 79, 4215-4221). ε 450 Value 2. 67×108 Using this method, the AuNP concentration was calculated from the absorbance at 450 nm. The AuNP-BSA-biotin-caffeine complex was subjected to proteinase K digestion, which cleaved the biotin group from the AuNP complex. The AuNP was removed by centrifugation, and the biotin concentration of the supernatant was measured by a HABA assay. The ratio of BSA molecules to AuNP was calculated using the ratio of biotin groups to AuNP. On average, 56 BSA molecules were bound to each AuNP, a value below the theoretical maximum calculated based on the size of the AuNP. This corresponds to a surface coverage of 6.96 × 10⁻⁶. 12 This corresponds to BSA molecules / cm2, as reported by Shi et al. (X. Shi, D. Li, J. Xie, S. Wang, Z. Wu and H. Chen, Chinese Sci. Bull., 2012, 57, 1109-1115). This indicates that AuNPs are sufficiently stabilized by the BSA-biotin-caffeine complex on their surface.

[0202] c) Zeta potential characteristic evaluation The zeta (ζ) potentials of citrate-stabilized AuNPs and AuNP-BSA-biotin-caffeine complexes were evaluated by DLS. Zeta potential measurement is an indicator of the stability of charge-stabilized nanoparticles. When the zeta potential is 25 mV or higher or less than -25 mV, sufficient electronic repulsion exists between particles, preventing aggregation (SH Brewer, WR Glomm, MC Johnson, MK Knag and S. Franzen, Langmuir, 2005, 21, 9303-9307). The zeta potential of the synthesized gold nanoparticles was -37.2 mV. This strong negative surface charge indicates that the gold nanoparticles were sufficiently stabilized by the citrate coating. When the BSA-biotin-caffeine complex was bound to the gold nanoparticles, the zeta potential increased from -37.2 mV to -31.5 mV. This indicates that some of the negatively charged citrate molecules on the AuNPs were replaced by BSA binding via thiol-gold bonds, confirming the success of the binding. The overall surface charge remains negative. This is because BSA becomes negatively charged at pH levels above its isoelectric point of 4.7.

[0203] d) Aggregation studies using avidin To investigate the binding ability of the AuNP-BSA-biotin-caffeine complex to avidin, the complex was incubated with avidin overnight. Avidin is a tetrameric protein with four biotin-binding sites. Kim et al. (WJ Kim, S.-H. Choi, Y.-S. Rho and D.-J. Yoo, Bull. Journal of the Korean Chemical Society, 2011, 32, 4171-4175) reported that biotinylated AuNPs aggregate due to specific biotin-avidin interactions. This was observed as a gradual color change from red to purple after avidin addition. Aggregation was dependent on the avidin concentration present. Aggregation is observed when avidin bound to a biotin group on a certain AuNP can cross-link with an adjacent AuNP that has an unsaturated biotin site. At low avidin concentrations (0.1 μg / mL), aggregation was not observed because there was insufficient avidin to bind to the biotin groups on the AuNPs. When the avidin concentration was increased to 1 μg / mL, aggregation of AuNPs was observed because each avidin bound to two biotin groups on at least two different AuNPs. After overnight incubation, the absorption peak showed a significant broadening and redshift from 523 nm to 548 nm. The redshift of the absorption peak is due to LSPR binding interactions resulting from the polarization of conduction electron vibrations between adjacent AuNPs linked by avidin molecules (Y. Yang, S. Matsubara, M. Nogami and J. Shi, Mater. Sci. Eng. B, 2007, 140, 172-176). The broadening of the peak is due to variations in binding interactions that are strongly dependent on aggregate size and the orientation of AuNPs within the aggregates (K. Aslan, CC Luhrs and VH Perez-Luna, J. Phys. Chem. B, 2004, 108, 15631-15639).

[0204] Using TEM, we also visualized aggregation of biotin on AuNPs due to avidin molecule binding. This demonstrated that the binding of the BSA-biotin-caffeine complex to AuNPs was not hindered by the binding sites of biotin and avidin. This also supports the finding that treatment of BSA-biotin-caffeine with 2-mercaptoethanol did not affect the binding of biotin to proteins.

[0205] Enzyme-linked immunosorbent assay (ELISA) i) Capture ELISA To measure the binding ability of the BSA-biotin-caffeine complex to anti-caffeine antibodies and avidin, capture enzyme immunosorbent assay (ELISA) was performed. The format of the capture ELISA used is schematically shown in Figure 2.

[0206] The wells were coated with anti-caffeine antibody, and excess binding sites were blocked with BSA. Other blocking agents, such as Tween-20 and commercially available synthetic blockers (ELISA SynBlock), were also tested, but the resulting signal-to-noise ratio was poor due to high nonspecific binding. Milk protein-based blocking agents, such as casein, were avoided because the presence of trace amounts of biotin may affect the specific biotin-avidin interaction in the assay. By using 2% (w / v) BSA as the blocking agent, it was ensured that the obtained signal was due to a BSA-biotin-caffeine conjugate specifically bound to the anti-caffeine antibody, and not due to hydrophobic or electrostatic interactions between the conjugate and the wells.

[0207] Different concentrations of BSA-biotin-caffeine complex were added to each well, followed by HRP-labeled avidin. Subsequently, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate reacted in the presence of horseradish peroxidase (HRP) enzyme to form a chromogenic derivative. The amount of color development was proportional to the amount of bound BSA-biotin-caffeine complex. The absorbance of the solution at 450 nm was recorded and plotted against the concentration of the BSA-biotin-caffeine complex. The presence of a signal confirmed that the synthesized BSA-biotin-caffeine complex could simultaneously bind to both the anti-caffeine antibody and avidin. As the concentration of the BSA-biotin-caffeine complex increased, the amount of HRP-labeled avidin captured by the biotin-avidin interaction increased, and an increase in absorbance at 450 nm was observed. The plot of absorbance against concentration is nonlinear. This is because as the concentration of the BSA-biotin-caffeine complex increases, the saturation of the anti-caffeine antibody binding site increases.

[0208] ii) Competitive ELISA To investigate the competition between the BSA-biotin-caffeine complex and free caffeine for binding to the anti-caffeine antibody coated in the wells, the same ELISA format was used. A fixed amount of BSA-biotin-caffeine complex (20 μg / mL) was used, and the free caffeine concentration was varied. Absorbance at 450 nm was plotted against caffeine concentration on a semi-logarithmic graph (see Figure 5). The data were fitted to a dose-response model. 、 I C 50 The value was calculated to be 1 ng / mL. The detection limit, calculated from the caffeine concentration that elicits 10% inhibition, was 0.03 ng / mL.

[0209] Competitive ELISA was repeated using the AuNP-BSA-biotin-caffeine complex to evaluate whether binding to AuNP affected the binding ability of the BSA-biotin-caffeine complex to anti-caffeine antibodies and avidin. A similar semi-logarithmic graph was created, as shown in Figure 6. The data were fitted to a dose-response model, and the results showed IC50 The value was 7 ng / mL. The limit of detection was 0.4 ng / mL. When using AuNP-BSA-biotin-caffeine, the sensitivity was slightly reduced, which may be due to steric hindrance of the AuNP conjugate. Because the AuNP-BSA-biotin-caffeine complex is quite large, when one AuNP complex binds to an antibody, it may inhibit the binding of other AuNP complexes to adjacent antibodies. However, since the caffeine concentration to be detected in blood is 0.1–10 μg / mL, this assay had sufficient sensitivity for its intended use.

[0210] ELISA results showed that the AuNP-BSA-biotin-caffeine complex maintained its binding ability to both anti-caffeine antibodies and avidin. By utilizing the competition between the AuNP-BSA-biotin-caffeine complex and free caffeine, the caffeine concentration in a sample can be accurately measured with a low detection limit.

[0211] Lateral flow assay (LFA) i) Optimization of assay design LFA was developed by utilizing the competition between the AuNP-BSA-biotin-caffeine complex and free caffeine for binding to an anti-caffeine antibody. The initial design of the LFA for caffeine detection is shown in Figure 7. An anti-caffeine antibody was incubated with an excess amount of the AuNP-BSA-biotin-caffeine complex. After centrifugation to remove the excess antibody, the resulting antibody-AuNP-BSA-biotin-caffeine complex was deposited on a conjugate pad. An anti-mouse antibody was coated onto the first test line to capture all of the anti-caffeine antibody. Free caffeine in the sample substituted the AuNP-BSA-biotin-caffeine complex from the anti-caffeine antibody, and the substituted AuNP-BSA-biotin-caffeine complex was captured on the second test line via a specific biotin-avidin interaction.

[0212] Initial testing was conducted using this format, but the signal observed in test line 1 was extremely weak. This suggests insufficient 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 have competed with the antibody-AuNP-BSA-biotin-caffeine complex, saturating the binding site on the anti-mouse antibody. Alternatively, the binding of the AuNP-BSA-biotin-caffeine complex to the anti-caffeine antibody may have caused steric hindrance to the binding of the secondary antibody. The epitope on the caffeine antibody recognized by the anti-mouse antibody may have been shielded by the AuNP label, resulting in a significant decrease in sensitivity. The binding rate in this format was deemed too slow and unsuitable for caffeine detection.

[0213] As shown in Figure 3, an improved competitive LFA method without secondary antibody was proposed. Instead of drying the AuNP-BSA-biotin-caffeine complex on the complex pad, an anti-caffeine antibody was deposited as the first test line. This causes competition between the AuNP-BSA-biotin-caffeine complex and free caffeine in the sample for the caffeine antibody to occur on the test line rather than along the membrane.

[0214] ii) Principle of measurement Figure 8 schematically illustrates the operating principle and expected test results using the LFA design shown in Figure 7. The AuNP-BSA-biotin-caffeine complex is deposited on the conjugate pad. When a sample is applied, the liquid flows along the membrane, moving the gold complex. If caffeine is not present in the sample (Figure 8a), the AuNP-BSA-biotin-caffeine complex is captured by the anti-caffeine antibody in the first test line. If caffeine is present in the sample (Figure 8b), free caffeine competes with the AuNP-BSA-biotin-caffeine complex to bind to the anti-caffeine antibody. The AuNP-BSA-biotin-caffeine complex substituted from the first test line binds to the biotin-binding protein (avidin) in the second test line. The signal intensity in the second test line is proportional to the caffeine concentration in the sample.

[0215] iii) Optimization of LFA conditions To improve assay performance, various conditions were optimized, including the amount of conjugate, antibody and avidin concentrations in the test line, and blocking of nonspecific binding.

[0216] a) Membrane blocking When no blocking agent was used in the sample buffer, a high background signal was observed. The AuNP-BSA-biotin-caffeine complex nonspecifically bound to the negatively charged nitrocellulose membrane, and via hydrophobic and electrostatic interactions, a faint pink background was generated on the rest of the nitrocellulose membrane after each measurement. Since the background signal was not removed by washing the membrane with PBS buffer after the procedure, a blocking agent to reduce nonspecific binding was necessary. When the same blocking buffer used for ELISA (2% (w / v) BSA, 0.05% (v / v) Tween-20 in PBS) was tested with LFA, the background signal was significantly reduced.

[0217] b) Cross flow velocity Capillary flow rate decreases exponentially as the sample passes through the membrane (GA Posthuma-Trumpie, J. Korf and A. van Amerongen, Anal. Bioanal. Chem., 2008, 393, 569-582). Therefore, the further the test line is located from the sample pad, the longer the interaction time between the ligand and receptor. Because the anti-caffeine antibody test line was positioned relatively close to the sample application site, the fast sample flow rate may not have allowed sufficient time for the AuNP-BSA-biotin-caffeine complex to interact with the anti-caffeine antibody. The migration speed of the AuNP-BSA-biotin-caffeine complex can be slowed by introducing an agent that reduces the lateral capillary action velocity of the membrane. For example, sucrose is widely used to reduce flow rate in paper-based assays (B. Lutz, T. Liang, E. Fu, S. Ramachandran, P. Kauffman and P. Yager, in 16th (International Conference on Miniaturized Systems for Chemistry and Life Sciences, Okinawa, Japan, 2012, vol. 20, pp. 788-790). Sucrose is used because it has excellent water solubility and flow-inhibiting properties (WO 2005069007 A1, 2005, 1-25). 30% (w / v) sucrose was dried on a conjugate pad to form a sucrose glaze. This glaze dissolves when a sample is applied and can slow the flow rate along the membrane. A decrease in the intensity of the second test line was observed compared to the first test line, but the decrease in flow rate was still insufficient to capture all of AuNP-BSA-biotin-caffeine into the first test line. Tests at higher concentrations of sucrose (over 50% (w / v)) were not performed because the viscosity was too high and difficult to handle. The sucrose glaze dried on the binding pad significantly hardened the binding pad, potentially hindering the capture and release of the AuNP-BSA-biotin-caffeine complex on the binding pad.

[0218] 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., 2015, 5, 17178) reported that the use of PEG and glycerol affected the signal intensity of the test and control lines of LFA due to the high viscosity of the solution. Therefore, the use of glycerol in optimizing LFA was investigated. Different concentrations of glycerol were tested, as shown in Figure 9. Two lines of anti-caffeine antibody were added to the first test zone, and one line of avidin was added to the second test zone. The second anti-caffeine antibody line was intended to capture the excess AuNP-BSA-biotin-caffeine complex that had passed through the first anti-caffeine antibody line. However, it did not capture all of the AuNP-BSA-biotin-caffeine complex before it reached the avidin test line. Without glycerol addition, three lines were observed on the test strip scanned with buffer alone. The addition of 2.5% (v / v) glycerol reduced the signal intensity in the second test zone, but it was still insufficient to capture all complexes in the first test zone (Figure 9).

[0219] The addition of 10% (v / v) glycerol confirmed that all AuNP-BSA biotin-caffeine complexes could be captured in the first test line. However, in tests using a sample containing 100 μg / mL caffeine, no positive signal was obtained in the second test line (Figure 9). The presence of glycerol excessively slowed the movement of the AuNP-BSA-biotin-caffeine complex, and competition for anti-caffeine antibodies by free caffeine and the complex did not occur in the first test line. Since free caffeine molecules in the sample have a much lower molecular weight than the AuNP-BSA-biotin-caffeine complex, they cross the membrane much faster than the complex, even in the presence of glycerol. Kim et al. (YA Kim, EH Lee, KO Kim, YT Lee, BD Hammock and HS Lee, Anal. Chim. Acta, 2011, 693, 106-113) previously reported that the relative migration speed between two competing molecules is important for the sensitivity of competitive LFAs. The analyte in the sample should migrate before the conjugate, but in order to obtain sufficient overlap between the two competing molecules on the film, the migration speed of the analyte needs to be only slightly faster than that of the conjugate.

[0220] 5% (v / v) glycerol was found to be optimal for reducing the migration rate of the AuNP-BSA biotin-caffeine conjugate without adversely affecting competition between the competing antibody and free caffeine (Figure 9). Most of the AuNP-BSA-biotin-caffeine conjugate was captured by the anti-caffeine antibody in the first test zone. Scanning a sample containing 100 μg / mL of caffeine achieved competition between the migrating AuNP-BSA-biotin-caffeine conjugate and free caffeine, resulting in a strong signal in the second test zone.

[0221] iv) Sensitivity test To determine the operating range and sensitivity of caffeine detection, the effects of applying different concentrations of caffeine to LFA were investigated. LFA test strips were prepared based on the assay design shown in Figure 3 and scanned with different caffeine concentrations ranging from 1 ng / mL to 100 μg / mL. The optimized scanning buffer contained glycerol as a capillary action rate modifier, and BSA and Tween-20 as blocking agents to reduce nonspecific binding. The test strip results are shown in Figure 10.

[0222] As caffeine concentration increased, the intensity of test line 1 decreased accordingly, as expected in a competitive assay. When the caffeine concentration exceeded 10 μg / mL, no color development occurred in test line 1. This is the visual detection limit for caffeine presence in the sample without a calibration curve. This is an inherent limitation of conventional competitive assays, because a signal will always appear on the test line unless the analyte is present at a concentration high enough to completely saturate the test line. Without a standard curve to calibrate signal intensity against the concentration of the target analyte, this assay provides qualitative results only for caffeine concentrations above 10 μg / mL. This does not meet the necessary working range for detecting caffeine in blood. The assay sensitivity can be improved by amplifying the signal using silver (R.-H. Shyu, H.-F. Shyu, H.-W. Liu and S.-S. Tang, Toxicon, 2002, 40, 255-258) or gold enhancement (J. Kaur, KV Singh, R. Boro, KR Thampi, M. Raje, GC Varshney and CR Suri, Environ. Sci. Technol., 2007, 41, 5028-5036). However, such methods are undesirable because they negate the convenience of a one-step LFA.

[0223] This limitation was overcome by providing a second test line that captures the AuNP-BSA-biotin-caffeine complex displaced from the binding to the anti-caffeine antibody by free caffeine in the sample. As the caffeine concentration in the sample increased, the signal intensity of test line 2 also increased correspondingly (Figure 10). With the second test line, the visual detection limit was improved to 10 ng / mL. As a result, the sensitivity of the caffeine detection assay was improved by 1000-fold. Since test line 2 shows a positive signal at caffeine concentrations above 10 ng / mL, qualitative analysis of the caffeine content in the sample has become easier compared to the negative determination associated with conventional competitive assays. Due to the improved sensitivity, this assay is suitable for examining blood samples with caffeine concentrations ranging from 0.1 μg / mL to 10 μg / mL.

[0224] In addition to visual inspection, the signal intensity of the test lines was analyzed using ImageJ software. The signal intensities of test lines 1 and 2 at different caffeine concentrations were plotted on a semi-logarithmic graph as shown in Figure 11 (results are from three measurements). The IC50 values for test lines 1 and 2 are 5 ng / mL and 8 ng / mL, respectively. These IC50 values are equivalent to those obtained by competitive ELISA. As shown in Table 1, semi-quantitative analysis of the caffeine content in the sample is possible by comparing the signal intensity ratios of the two test lines.

[0225] Semi-quantitative analysis of caffeine concentration based on the signal intensity ratio of test lines 1 and 2

[0226]

Table 1

[0227] v) Stability of the test strip Since LFA test strips are typically batch-produced and stored for a certain period before use, shelf-life stability is crucial. We verified the stability of the reagents printed on the nitrocellulose membrane and the AuNP-BSA-biotin-caffeine complex deposited on the conjugate pad. Test strips were prepared and stored in a desiccator at room temperature for 7 days. Tests were performed at different caffeine concentrations, and the results are shown in Figure 12. The visual detection limit for caffeine concentration in test line 2 remained at 10 ng / mL.

[0228] The results of plotting the signal intensities of test lines 1 and 2 at different caffeine concentrations on a semi-logarithmic graph showed the IC values ​​of test lines 1 and 2. 50 The values ​​were 3 ng / mL and 11 ng / mL, respectively. No significant change in assay sensitivity was observed after one week of storage, indicating that the dried reagent on the membrane maintained good stability and reactivity. Conventional competitive and sandwich-type LFAs incorporate a positive control line to guarantee reagent reactivity and confirm the validity of the test results. While this format does not include a control line, the two test lines function as cross-validators, and the validity of the test is indicated if at least one line is detected. If the reagent on the strip is degraded, the reactivity of both the anti-caffeine antibody and avidin should decrease, and neither line should develop color.

[0229] conclusion We successfully developed a competitive LFA for colorimetric detection of caffeine. The signal intensity is proportional to the caffeine concentration in the sample. The binding ability of the AuNP-BSA-biotin-caffeine complex to avidin and anti-caffeine antibodies was demonstrated by ELISA. By optimizing the design, blocking, and flow rate of an improved competitive LFA format, we developed an LFA suitable for caffeine concentration detection. The main advantages of this LFA format are its high sensitivity and clear positive result. By incorporating a second test line, sensitivity was increased 1000-fold, and the visual detection limit for caffeine became 10 ng / mL. This makes the assay suitable for detecting caffeine concentration in blood samples. Meanwhile, commercially available LFAs for caffeine detection... 74 It lacks the necessary sensitivity. This test is also characterized by its speed, and qualitative results can be obtained by visual inspection of the test strip 5 minutes after sample addition. It does not require special equipment or additional washing steps and can be performed with only a small amount of sample ( ), making it easy to perform as a fingertip blood test.

[0230] This embodiment also serves as a proof-of-concept example of a competitive assay that enables the detection of trace amounts of target analytes with a positive result. Conventional competitive assays suffer from limitations such as low sensitivity and a narrow detection range, but these limitations are overcome. By changing the hapten bound to BSA and the antibody immobilized on the first test line, this competitive assay format can be applied to the detection of other small molecule compounds. The use of two test lines allows for cross-referencing of results, and semi-quantitative data can be provided using the signal intensity ratio of both test lines. The one-step assay requires no washing, sensitization, or calibration steps, making it relatively low-cost and highly convenient as a point-of-care test.

[0231] Example 2 - Detection of NHPA The objective of this study is to develop an in vitro diagnostic test for measuring urinary NHPA, specifically by employing an improved competitive lateral flow immunoassay and demonstrating its performance (sensitivity, detection limit, and linearity) using laboratory (non-clinical) samples.

[0232] This study focused on developing a point-of-care (POC) test to detect NHPA in solution using a 3-nitrotyrosine (3-NTyr) antibody and a lateral flow assay (LFA).

[0233] Although no NHPA-specific antibodies have been reported, it has been reported that the BSA conjugate of NHPA binds to monoclonal antibodies against 3-nitrotyrosine (3-NTyr), and it is expected that these antibodies will also bind to NHPA, similar to 3-NTyr. While 3-NTyr is present in urine, its concentration is several hundredths of that of NHPA, and interference with this assay is not expected. The following work was performed to evaluate the feasibility of developing a 3-NTyr antibody for detecting urinary NHPA in a modified competitive assay format.

[0234] A - Synthesis of compound i) Binding of biotin and BSA 1% BSA (20 mg, 0.3 μmol) was dissolved in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2 (2 mL) and added to NHS-LC-biotin (1.12 mg, 2.46 μmol) in dimethyl sulfoxide (DMSO) (40 μL). The reaction was left on ice for 2 hours. Subsequently, the sample was purified by gel filtration using 20 mM borax buffer pH 8.0 as the elution buffer. The fraction containing biotinylated BSA was identified and bound by absorbance measurement at 280 nm.

[0235] To quantify the amount of biotinylated protein, a solution containing biotinylated protein is added to a mixture of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) and avidin. Because biotin has a high affinity for avidin, it substitutes for HABA, and the absorbance at 500 nm decreases proportionally.

[0236] The amount of biotin 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 formula as in Example 1 above.

[0237] The biotinylation reaction was successful, and calculations showed that the synthesized product contained an average of 3.3 molecules of biotin per molecule of BSA.

[0238] ii) Coupling of NHPA and biotinylated BSA A solution of 3-nitro-4-hydroxyphenylacetic acid (NHPA) (1.43 μmol, 5.64 μL, 50 mg / mL in ethanol) was added to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride (6.85 mg) in DI water (3000 μL, pH 4.5). The biotinylated BSA solution prepared in Step 1 (0.24 μmol, 3000 μL, 5.28 mg / mL in 20 mM borax buffer (pH 8.0)) was added. The reaction mixture was allowed to react on a rotator at room temperature for 2 hours, then overnight at 4°C. The sample was purified by gel filtration using 2 mM borax buffer (pH 8.0) as the elution buffer. The fractions containing the highest protein concentration were identified by recording the absorbance at 280 nm and then conjugated.

[0239] To quantitatively evaluate the success of the reaction, spectroscopic analysis was performed using a calibration curve. Based on calculations, the synthesized product contained, on average, 3.6 molecules of NHPA per molecule of BSA.

[0240] iii) Binding of NHPA-biotin-BSA to gold nanoparticles Spectroscopic analysis of PBS-stabilized gold nanoparticles revealed a distinct peak at 523 nm, which is consistent with the properties expected of 20 nm nanoparticles.

[0241] Gold nanoparticles (GNP) (1 mL, 1 OD) were first centrifuged at 14,000 rpm for 5 minutes. The solution became clear, and a precipitate of nanoparticles formed at the bottom of the Eppendorf tube. The supernatant was carefully removed without 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 rotated at room temperature for 4 hours.

[0242] Subsequently, the conjugate was centrifuged at 8500 rpm for 15 minutes, and the supernatant was removed. Finally, the GNPs were resuspended in 1 mL of buffer containing 20 mM Tris, 2% BSA, 4% sucrose, 5% glycerol, 0.05% Tween 20, pH 7.2.

[0243] B - Urine Strip Test In this section, the performance of the conventional LFA for NHPA detection under actual background conditions was evaluated.

[0244] i) Assembly of the Lateral Flow Immunoassay The LFA was assembled by attaching a nitrocellulose membrane to a plastic support card (Figure 13). The anti-3-nitrotyrosine antibody line was printed at a concentration of 1 mg / mL and consisted of 20 lines with a width of 25 μm each, using 0.1 μL / cm / line of the antibody solution. The streptavidin line was printed at a concentration of 1 mg / mL and consisted of 10 lines (each 50 μm wide), using 0.1 μL of the streptavidin solution / cm / line. The membrane was sealed in an aluminum bag together with a desiccant silica gel packet and stored in a refrigerator until use.

[0245] On the day of the test, a blotting pad (5 × 17 mm) was attached to the membrane (with a 2 mm overlap), and the plastic card was cut into strips (5 mm wide). The glass fiber conjugate pad (5 × 7 mm) was immersed in the AuNP-BSA-biotin-NHPA conjugate (0.5 OD) and dried in a desiccator for 2 hours. Then, the conjugate pad was attached to the membrane (with a 2 mm overlap). The sample pad (5 × 17 mm) was immersed in the 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 so as to overlap the conjugate pad by 2 mm.

[0246] ii) Testing of the LFA Strip Samples with varying NHPA concentrations in synthetic urine (200 μL) were prepared in each well of a low-adsorption 96-well microplate. Test strips were inserted vertically into each well and left until the liquid level reached the edge of the absorbent pad, then removed and imaged. Images of the test strips (Figure 14) were analyzed using ImageJ software. ImageJ analysis revealed two peaks in the streptavidin line and the antibody line (Figure 15a). The gray level of the pixels in each test line was measured, and the intensity and NHPA concentration dependence of each line were calculated by subtracting the background (Figure 15b).

[0247] As NHPA concentration increased, the amount of AuNP-BSA-biotin-NHPA complex captured by anti-3-nitrotyrosine antibody decreased. This resulted in a linear decrease in signal intensity in the antibody test line (Figure 15b). When the NHPA concentration in the urine sample was 250 ng / ml, the antibody line intensity decreased by more than 90%.

[0248] The reproducibility of this assay was evaluated using three strips for each NHPA concentration (Figure 16). These strips were then compared with strips prepared from two other batches of AuNP. The results are summarized in Figure 17, which is an Image J analysis of the antibody lines, with each data point representing the average value of five strips. The test line analysis of all strips showed good reproducibility (Figure 17).

[0249] Figure 17 shows the linear response at NHPA concentrations of 0–150 ng / ml, where the response levels off above this concentration. This indicates that NHPA in the solution occupies all antibody binding sites.

[0250] Conclusion: These results clearly demonstrate the establishment of a practical assay method for detecting NHPA in urine. The detection limit is 170–330 ng / ml (250 ± 80 ng / ml). The proposed protocol is suitable for distinguishing samples above the detection limit as positive and samples below as negative. Figure 18 shows that, as expected, a visible line appears only on the streptavidin test line in positive samples, demonstrating the good reproducibility of the assay.

[0251] Example 3 - Detection of creatinine Using the same method as in Example 1, the LFT design shown in Figure 3 was applied to creatinine detection.

[0252] method Gold nanoparticle synthesis Gold(III) chloride hydrate (30 mg, 0.0762 mmol) was added to Milli-Q water (250 ml) and heated under reflux for 30 minutes. Sodium citrate solution (500 mg, 1.94 mmol) was added. After heating for another 30 minutes, the solution was cooled and filtered through a 0.2 micron PTFE membrane to obtain a red solution. Characterization was performed using UV-Vis spectrophotometry and NTA. This is an improved version of the Frens protocol.

[0253] Synthesis of creatinine derivatives Ethyl 4-bromobutyrate (26.6 ml, 100 mmol) was added to creatinine (9 g, 80 mmol) in DMF (50 ml) and heated at 85°C for 4 hours. After cooling the solution, ethyl acetate (500 ml) was added to produce a precipitate (12.71 g, 52% yield). This was filtered. The resulting precipitate (3.1 g, 10 mmol) was added to KOH (10 mmol) in methanol (25 ml). Methanol was evaporated and ethanol was added. The precipitated salt was removed by vacuum filtration, and the filtrate was heated to evaporate the ethanol. The solution was added to NaOH (0.5 M, 10 ml) and heated under reflux for 6 hours. Ethanol was heated and removed, and concentrated hydrochloric acid was added until the pH was 3. Water was evaporated to produce a yellow oily substance and NaCl, which were removed by centrifugation. The product was identified by NMR, mass spectrometry, and IR.

[0254] Biotin binding to BSA 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 of buffer (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2). The mixture was kept at room temperature for 5 hours and then gel filtered using the same buffer. UV-Vis spectroscopy at 286 nm was performed on different fractions, and the fractions showing the highest absorbance were combined.

[0255] HABA assay 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. This mixture was added to the biotinylated sample, and the absorbance at 500 nm was recorded.

[0256] Creatinine binding to BSA Creatinine derivative (20 μL) was added to EDC (20 mg) in phosphate buffer (80 μmol, pH 7.4). After holding at room temperature for 4 hours, the solution was purified by gel filtration using the same phosphate buffer. The maximum protein concentration was detected using a UV-Vis spectrophotometer.

[0257] TNBS assay BSA conjugate (200 μg / ml) was added to 2,4,6-trinitrobenzenesulfonic acid (TNBS) (500 μL in 0.12 M NaHCO3) and incubated at 37°C for 3 hours. Absorbance at 335 nm was recorded, and the amino group concentration was calculated.

[0258] Capture ELISA Anti-creatinine antibody (50 μL, 8 μg / mL) was added to a 96-well ELISA plate and incubated for 1 hour. Then, 2% BSA in PBS (10 μL) was added. Different concentrations of BSA-biotinylated creatinine (50 μL) were added. HRP-labeled avidin (50 μL) was added, and the plate was left in the dark for 1 hour. After that, the plate was washed four times with PBS buffer, and TMB (50 μL) was added (resulting in blue color development). The color changed to yellow upon addition of H2SO4 (50 μL, 1M). The absorbance at 450 nm was then recorded.

[0259] Competitive ELISA The same method as described above was used. However, the amount of BSA conjugate was kept constant at 25 μL, and creatinine of different concentrations was added to bring the total volume to 50 μL.

[0260] Gold and BSA conjugate combination The reduction method described in Example 1 was used. BSA conjugate was added to 2-mercaptoethanol (0.50 μL, 25 mM) and kept at room temperature for 6 hours. Then, gel filtration was performed using phosphate buffer. 1000 μL of the sample was added to the filtered gold particle solution (1 ml) and left 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 spectrophotometric methods.

[0261] Elman's Law Elman's reagent was prepared by adding 5'5'-dithiobis-2-nitrobenzoic acid (DTNB) (0.42 mg, 1.22 μmol) to sodium phosphate buffer (0.1 M, pH 8, 1 mM EDTA, 5 mL). A reduced BSA sample (20 μL) was added to Elman's reagent (180 μL), and the absorbance at 412 nm was recorded using a UV-VIS spectrophotometer.

[0262] LFA strip creation The sample pad was attached to a support card. A nitrocellulose membrane was attached, and anti-creatinine antibody and avidin were immobilized in two linear fashions (one for the antibody, the other for the avidin). 1% BSA solution was added to the membrane and allowed to dry. A strip was prepared, and a wick was attached to the opposite end. The conjugate pad was immersed in a 30% sucrose solution, the AuNP-BSA-biotin-creatinine complex was added twice, and it was allowed to dry for 2 hours. The pad was then attached to the strip.

[0263] Testing of LFA strips Creatinine solutions of different concentrations were prepared and added to the wells of a 96-well plate. Strips were placed in the wells, and the sample pads were immersed in the solution for 10 minutes. After drying, images were taken with a camera and analyzed using ImageJ software. The obtained values ​​were plotted on a semi-logarithmic graph (Figure 20).

[0264] Results and Discussion Biotinylation Similar to Example 1, biotin bound to BSA was quantified by the HABA assay. The ratio of biotin to BSA was determined to be 2.2 molecules of biotin per molecule of BSA.

[0265] Synthesis of creatinine derivatives Since creatinine itself does not contain a carboxylic acid group, derivatives having an acid group capable of forming an amide bond with BSA were synthesized. The synthesis procedure for the creatinine derivatives is as follows, as shown in Figure 1. Step 1: Creatinine was reacted with ethyl 4-bromobutyrate to produce a yellow solid intermediate, ethyl 4-(2-imino-3-methyl-5-oxoimidazolidine-1-yl)butyrate hydrobromide. Step 2: The hydrated chloride salt was neutralized with a KOH base. Step 3: Ethyl 4-(2-imino-3-methyl-5-oxoimidazolidine-1-yl)butyrate was refluxed in the presence of a base to remove the ester group. Step 4: The sodium carboxylate salt was converted to 4-(2-imino-3-methyl-5-oxoimidazolidine-1-yl)butyric acid, a creatinine derivative, using a strong acid. NaCl precipitate was formed at the bottom.

[0266] [ka]

[0267] Subsequently, creatinine derivatives were identified by NMR, IR, and mass spectrometry.

[0268] i) Infrared spectroscopy (IR) The carboxylic acid group bound to the creatinine molecule was identified using IR. The broad peak is on the far left. ( 3375 cm-1) This confirmed the OH extension oscillation of the COOH group. Sharp peak ( )は 1697 cm- 1 The carbon (C=O) in COOH was confirmed. These values ​​were within the range of literature values, as shown in Table 2.

[0269] IR value

[0270] [Table 2]

[0271] ii) NMR 1 HNMR and 13¹¹C NMR was performed. To compare the structures of the creatinine derivatives, literature values ​​for creatinine and 4-aminobutyric acid (H2N-CH2-CH2-CH2-COOH) were used. The creatinine portion of the product was within the literature range for creatinine. The carboxylic acid portion of the product was also within the literature range for 4-aminobutyric acid. However, one CH2 group of the carboxylic acid was outside the literature range - the measured range for ¹¹H NMR should have been 3.25-3.75 ppm, but the observed value was 4.36 ppm. This difference is reasonable because the literature value pertains to the CH2 group bonded to NH2, while in the creatinine derivative, the CH2 is bonded to the nitrogen atom, which is part of the five-membered ring. Resonance within the ring weakens the electron shielding effect of the atom bonded to it. Increased deshielding leads to a shift to the left of the chemical shift. A similar phenomenon was observed in ¹¹C NMR. All values ​​were within the range, but only the same CH2 group discussed in ¹¹H NMR showed a value of 70.61 ppm. This high chemical shift is due to electron deshielding due to resonance in the ring structure.

[0272] The OH group of the COOH group was not detected in the HNMR spectrum. In HNMR, D2O is used as the solvent, and H and D in solution exchange with each other to form COOD and HOD. Deuterium has NMR activity, but its signal has a different energy and does not appear in the NMR spectrum. Therefore, the OH signal was not observed in the HNMR spectrum.

[0273] Carbonyl carbon 13 The 13C NMR spectrum showed no signal. However, the presence of a carboxylic acid group in the creatinine derivative has already been confirmed by IR spectroscopy.

[0274] iii) Mass spectrometry The creatinine derivative was identified using electro-negative ionization. The derivative had a molecular weight of 199, and a large peak was observed around 200, confirming the presence of the product.

[0275] Therefore, we succeeded in synthesizing creatinine derivatives containing a carboxylic acid group linked to BSA via an amide bond.

[0276] Binding of creatinine derivatives This creatinine derivative, possessing a carboxylic acid group, can be bound to BSA. EDC was added to activate the derivative. The carboxylic acid group of the derivative reacted with EDC to form an intermediate. This intermediate formed an amide bond with the lysine residue of BSA. The mixture was purified by gel filtration to remove excess creatinine derivative, EDC, and any resulting by-products.

[0277] Since the first three fractions contained the largest amount of BSA conjugates, these three fractions were combined and used in the next step.

[0278] 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to measure the amount of bound creatinine, and the results are shown in Table 3 below.

[0279] Determination of amino group substitution by TNBS assay

[0280] [Table 3]

[0281] The absorbance of the BSA-biotin-creatinine mixture was lower than that of the BSA-biotin mixture, and even lower than that of BSA alone. This is because BSA linked biotin and creatinine via amide bonds, reducing the number of available primary amines. As a result, the amount of TNBS that could bind to BSA decreased, leading to reduced formation of the orange product and a decrease in color intensity.

[0282] Amine concentration values ​​were obtained from standard curves for lysine and glutamate.

[0283] On the surface of the BSA molecule, 30–35 of the 59 total lysine residues are available for binding. This is consistent with data measuring 32.6 primary amines per BSA molecule. Biotin binding reduced this to 30.35 molecules, a net reduction of 2.4 amines. This is consistent with the HABA assay value of 2.1 biotins per BSA molecule. Approximately 2 biotin molecules were bound to each BSA molecule. Further creatinine binding reduced the amount to 26.53 amines per BSA molecule, a net reduction of 3.82 molecules. Therefore, on average, approximately 4 creatinine molecules were present per BSA molecule.

[0284] Synthesis of gold nanoparticles and their binding with BSA In Example 1 above, the adsorption method for binding BSA to gold nanoparticles was shown to be unsuitable for passive adsorption by incubation and instead caused aggregation. In particular, a black, insoluble solid precipitated. The electrostatic interaction between the gold nanoparticles and BSA was not strong enough to induce efficient gold-BSA binding. Therefore, a reduction method was used to bind creatinine-biotin-BSA to gold nanoparticles.

[0285] The sulfhydryl groups in BSA were quantified by absorbance measurement at 412 nm using Elman's reagent containing DTNB. The reduced BSA conjugate showed increased absorbance compared to the unreduced BSA conjugate. The concentration of sulfhydryl groups was calculated from the standard L-cysteine ​​curve. The number of sulfhydryl groups increased significantly from 0.81 to 2.51, with a net increase of 1.7. This result confirms the reduction of the BSA conjugate.

[0286] Next, a sample of gold nanoparticles (AuNP) was added to the reduced BSA conjugate. This caused the sulfhydryl groups to form bonds with the gold nanoparticles. UV-Vis spectrophotometric measurements were performed to characterize the AuNP and AuNP-BSA conjugate. The absorption peak of AuNP was at 520 nm. After binding to the BSA-biotin-creatinine conjugate, a new absorption maximum occurred at 526 nm.

[0287] Size characterization was performed using NTA. NaCl-Na3PO4 buffer was used for measurement, and a peak was observed around 600 nm, confirming aggregation. No aggregation was observed in the samples when MilliQ water was used for the AuNP NTA and Tris buffer was used for the AuNP-BSA-biotin-creatinine complex NTA. Aggregation was mainly due to Na, which can bind to AuNP and form larger complex molecules. + Ions and Cl - The issue was caused by a buffer solution containing ions. These ions were not present in the other buffer solutions used.

[0288] A sharp peak at 80 nm was observed in the NTA of AuNP, confirming the size of the gold nanoparticles. An additional peak at 97 nm was observed in the NTA of the AuNP-BSA-creatinine complex, confirming the size of the AuNP-BSA complex molecule.

[0289] Capture ELISA ELISA was performed to confirm that the binding of creatinine antibody and biotin-avidin antibody followed a concentration-proportional relationship. As shown in Figure 27, decreasing the BSA concentration resulted in a decrease in absorbance at 450 nm. Significant changes in color intensity and absorbance were observed in the concentration range from 25 μg / ml to 0.625 μg / ml.

[0290] BSA concentrations exceeding 50 μg / ml were also tested. A 150 μg / ml solution was used and diluted to 125, 100, 75, and 50 μg / ml. No significant change in absorbance was observed with decreasing concentration in this concentration range. This is because, at concentrations above 25 μg / ml, all antibodies reached saturation with BSA. As a result, in the concentration range of 50–150 μg / ml, no significant change in the absorbance curve was observed because the amount of chromogenic agent TMB produced was the same.

[0291] Competitive ELISA This was a reproduction experiment of a capture ELISA, but in this case, the BSA conjugate was kept constant, and different concentrations of free creatinine were added. A semi-logarithmic graph was created to generate a dose-response curve (Figure 28). As the concentration of free creatinine increased, it bound to the antibody, and the absorbance decreased because the number of antibody sites that could bind to the BSA-biotin-creatinine conjugate decreased.

[0292] Lateral flow assay Different concentrations of creatinine were used in each liquid sample, and their effect on the signal intensity of test lines 1 and 2 was examined. Creatinine concentrations ranging from 0 ng / ml to 100 μg / ml were tested. The resulting LFT strips are shown in Figure 19. Figures 19 and 20 demonstrate that the improved LFT design performed as expected in creatinine detection. In particular: At creatinine concentrations of -0 ng / ml and 1 mg / ml, a strong signal was generated in the antibody test line (test line 1), while no signal was generated in the avidin test line (test line 2). This suggests that the AuNP-BSA-biotin-creatinine complex was not substituted in test line 1, and that the entire complex was captured by the antibody. At creatinine concentrations of -5 ng / ml and 10 ng / ml, the signal in test line 1 was slightly reduced, while the signal in test line 2 was slightly increased. This suggests that some AuNP-BSA-biotin-creatinine complexes were substituted and captured by test line 2. - The signal intensity of test line 1 decreased significantly, while the signal intensity of test line 2 increased significantly. This occurred when the creatinine concentration was increased to 100 ng / ml and 1 μg / ml. This suggests that there was intense competition between creatinine and the AuNP-BSA-biotin-creatinine complex, resulting in more AuNP-BSA-biotin-creatinine complex being substituted from test line 1 and captured in test line 2. - The signal intensity of test line 1 was very weak at 10 μg / ml creatinine, and no signal was observed at 100 μg / ml creatinine. The signal intensity of test line 2 was high at both 10 μg / ml and 100 μg / ml creatinine. This suggests that the AuNP-BSA-biotin-creatinine complex did not bind to test line 1, but instead bound to test line 2.

[0293] Figure 20 shows that the signal intensity of test line 2 increases with increasing creatinine concentration, indicating a proportional relationship. The signal intensity of test line 1 decreases with increasing creatinine concentration, indicating an inverse proportional relationship.

Claims

1. A transverse flow testing apparatus comprising a solid support structure having a sample receiving region, a conjugate pad, a first test region, and a second test region, wherein the solid support structure is configured such that liquid flows sequentially from the sample receiving region through the conjugate pad to the first test region, and then to the second test region. i) The binding pad includes a mobile binding analyte containing one or more analytical molecules bound to a detectable label; ii) a) The first test area includes an immobilized analyte-binding molecule that defines the first binding site, b) The binding pad includes a movable analyte binding molecule, and the first test area includes an immobilization capture molecule that immobilizes the analyte binding molecule, the immobilization capture molecule defining the first binding site, or c) The binding pad includes a movable analyte binding molecule, and the first test area includes an immobilized analyte binding molecule defining the first binding site, iii) The second test area contains an immobilized bound analyte molecule that binds the bound analyte but does not bind the unbound analyte molecule, and defines the second binding site. Furthermore, the number of molecules of the bonded analyte is less than or equal to the number of the second bonding sites.

2. In the lateral flow testing apparatus according to claim 1, the number of molecules of the bonded analyte is less than the number of second bonding sites on the second test area.

3. A transverse flow inspection apparatus according to claim 1 or 2, wherein the number of first binding sites is equal to the number of molecules to be analyzed to which the binding site is located.

4. A crossflow inspection device according to claim 1 or 4, i) the bound analyte, ii) the immobilized analyte-bound molecule, and iii) the bound analyte-bound molecule are present in a 1:1:1 ratio; or i) the bound analyte, ii)b) the mobile analyte-bound molecule, ii)b) the capture molecule, and iii) the bound analyte-bound molecule are present in a ratio of 1:1:1:

1.

5. In the transverse flow testing apparatus according to any one of claims 1 to 4, the detectable label is a nanoparticle, preferably a gold nanoparticle.

6. In the transverse flow testing apparatus of any one of claims 1 to 5, each analyte molecule of the combined analyte is bound to a detectable label via a linking molecule, preferably the linking molecule is biotin-BSA.

7. The transverse flow inspection apparatus according to claim 6, wherein the bound analyte binding molecule specifically binds to the linking molecule, and preferably the bound analyte binding molecule is avidin, streptavidin, or polystreptavidin.

8. In the transverse flow detection apparatus according to any one of claims 1 to 7, either the analyte or the analyte-binding molecule is an antibody specific to the other analyte or analyte-binding molecule.

9. In the transverse flow inspection apparatus according to any one of claims 1 to 8, the analyte-binding molecule is an anti-analyte antibody.

10. A method for detecting the presence of an analyte molecule in a test sample, the method comprising: i) A step of preparing a crossflow inspection device according to any one of claims 1 to 9, ii) Apply the test sample to the sample receiving area of ​​the lateral flow inspection device, allowing the test sample to move to the conjugate pad and mix with the conjugated analyte and optionally movable analyte-binding molecules. iii) The test sample, the conjugate analyte, and optionally the movable analyte conjugate molecule are sequentially moved to the first and second test areas, and brought into contact with the immobilized molecules in each of the first and second test areas. iv) Detect a signal in both the first and second test areas. Here, a change in signal intensity in both the first and second test areas indicates the presence of the analyte molecule in the test sample.

11. In the method according to claim 10, the signal is an optical signal.

12. The method according to claim 10 or claim 11 further includes: v) The concentration of the analyte molecule in the test sample is quantified based on the ratio of the signal intensity in the first test area to the signal intensity in the second test area.

13. In the method according to any one of claims 10 to 12, in step v), a decrease in signal intensity in the first test area and an increase in signal intensity in the second test area indicate the presence of the analyte molecule in the test sample.

14. A method according to any one of claims 10 to 13, wherein the signal intensity is measured as a percentage of the maximum signal intensity, and step 4) includes measuring the signal intensity in either the first or second test area and predicting the signal intensity in the other first or second test area based on the fact that the sum of the signal intensity in the first and second test areas equals 100% of the maximum signal intensity.

15. In the method according to claim 14, step IV) includes: a) Measure the signal intensity in the first test area, predict the signal intensity in the second test area based on the fact that the sum of the signal intensities of the first and second test areas equals 100% of the maximum signal intensity, and calculate the ratio of the first measured value to the predicted value of signal intensity. b) Measure the signal intensity in the second test area, predict the signal intensity in the first test area based on the fact that the sum of the signal intensities of the first and second test areas equals 100% of the maximum signal intensity, and calculate the ratio of the second measured value to the predicted signal intensity. c) The average signal intensity in the first and second test areas is calculated based on the first and second measured value:predicted value signal intensity ratio, and the average first:second test area signal intensity ratio is determined. d) Calculate the ratio of the measured signal intensity of the first test area to the second test area. e) Compare the average signal intensity ratio of the first and second test regions with the measured signal intensity ratio, and f) If the average signal intensity ratio of the first to second test areas matches the measured signal intensity ratio, the concentration of the analyte molecule in the test sample is quantified based on the average signal intensity ratio of the first to second test areas. Here, steps a) and b) can be performed in any order.

16. In the method according to claim 15, The signal intensity in the first test area and the signal intensity in the second test area are scaled using a calibration coefficient during measurement; and Step iv) further includes: g) If the average first:second test domain signal intensity ratio does not match the ratio of measured signal in step d), adjust the calibration coefficient and repeat steps a) through f) using the adjusted calibration coefficient.

17. In the method of claim 16, the adjustment of the calibration coefficient includes: Determine whether the sum of the measured signal intensities of the first and second test regions exceeds the maximum signal intensity; The calibration coefficient is reduced in response to the sum of the measured signal intensities of the first and second test regions exceeding the maximum signal intensity; and The calibration coefficient is increased in response to the fact that the sum of the measured signal intensities of the first and second test areas does not exceed the maximum signal intensity.

18. A method according to any one of claims 10 to 17, wherein at least step a) of step IV is carried out using an optical signal reader, preferably a smartphone.

19. A method for diagnosing a disease or condition, comprising carrying out a method defined in any one of claims 10 to 18, characterized in that the test sample is obtained from a human, animal, or plant subject.

20. A computer-implemented method for detecting the presence of an analyte molecule in a test sample, the method comprising: After applying the test sample to the transverse flow inspection device, signal intensity measurements are obtained in the first and second test areas of the transverse flow inspection device according to any one of claims 1 to 9; Determine whether there was a change in signal intensity in both the first and second test areas; If a change in signal intensity is detected in both the first and second test areas, an instruction indicating the presence of the analyte molecule in the test sample is output.

21. The method according to claim 20, the step of determining whether or not there has been a change in signal intensity in both the first test area and the second test area includes the following: Determining whether a change has occurred in one of the test areas, based on whether the signal intensity in either the first or second test area is less than the maximum signal intensity; and When the signal intensity of the other of the first and second test regions exceeds the minimum signal intensity, the change in the other of the first and second test regions is determined.

22. In the method according to claim 20 or claim 21, the signal intensity measurement is obtained from one or more optical measurements for each of the first and second test areas, preferably from one or more images showing the first and second test areas of the transverse flow test apparatus.

23. A kit comprising a transverse flow test apparatus as defined in any one of claims 1 to 9.

Images