Lateral flow assay with encrypted diagnostic results

Abstract

This invention relates to a method of manufacturing a membrane for lateral flow assay or rapid diagnostic assay, a device comprising a membrane for detecting an analyte in a sample and the use of the device for detecting an analyte in a sample, wherein a two-dimensional barcode of a plurality of reagent dots comprising bioreceptors is patterned or printed on the membrane, the plurality of reagent dots being arranged in form of a two-dimensional barcode visible after the completion of the test and in which a certain information associated with the analyte is embedded, and to the use of said device.

Description

[0001] Lateral flow assay with encrypted diagnostic results

[0002] Technical Field

[0003] The present invention relates to a method of manufacturing a membrane for a lateral flow assay (LFA) or rapid diagnostic assay (RDA) on which test results are embedded in an encrypted form, a diagnostic or test device for detecting an analyte in a sample comprising a membrane with embedded test results in an encrypted form, and the use thereof for detecting an analyte for monitoring health state and sickness or disease.

[0004] Prior Art and the Problem Underlying the Invention

[0005] The field of diagnosis, particularly self-diagnosis, has entered a new paradigm with the emergence of personalized medicine. Self-testing now appears as a standard tool for the monitoring of health conditions or the detection of viral agents or diseases. Self-diagnosis commonly relies on the use of rapid diagnostic tests, which includes, among others, Lateral Flow Assays (LFA) and strips in a dipstick format. LFAs are simple diagnostic devices used for detecting at least one biological agent and / or target analyte in a sample. Thanks to their low cost and user friendliness, these devices are especially well-suited for point-of-care (POC) testing. They have acquired a dominant position on this market due to their intrinsic qualities: stability, cost effectiveness and clinical performance to analyse various samples without costly equipment at POC, in the laboratory or at home.

[0006] The general functioning of LFAs relies on capillary forces to move a liquid sample along a test strip capable to transport fluids to generate at least a measurable signal often in the form of a continuous line. Typically, a test device comprises a liquid phase and a solid phase including a sample zone, a reaction zone and a detection zone, and an absorbent pad, each zone composed of one or more different pads, such as sample pad, conjugate pad, detection pad. Each pad has a specific function due to the elements contained herein (buffer, affinity molecules, bioreceptors, conjugated antibody) and is composed of different materials enabling fluid transport. When a sample is applied on the sample pad in the sample zone, the sample is put in conditions (pH, ionic strength, purity) to guarantee the functioning of the test and the optimal detection of the target analyte. Through capillary forces, the modified sample reaches the reaction zone including the conjugate pad, wherein the target analyte in the modified sample interacts with a first set of bioreceptor conjugated to often optically signaling particles to form a labeled complex consisting of the analyte bound to the labeled bioreceptor elements. The modified sample comprising the labeled complex and an excess of the first set of bioreceptor elements keeps flowing laterally towards the detection zone comprising the detection pad. On the membrane of the detection pad, two sets of bioreceptor elements or affinity molecules are distributed in the form of two parallel and distant continuous lines perpendicular to the flow direction. The first line near the side of the reaction pad corresponds to the test line comprising a second set of immobilized bioreceptor elements or affinity molecules, named capture bioreceptors, which specifically bind the target analyte in the labeled complex. If the target analyte is present, the labeled complex comprising the analyte interacts with the second set of bioreceptor elements, or capture bioreceptors, and the test line becomes visible. The second line downstream the first line in the flow direction corresponds to the control line which consists of a third set of immobilized bioreceptor elements or molecules, named control bioreceptor, having affinity for the excess of the labeled bioreceptor elements or labeled molecules. When the modified sample continues flowing, the free labeled bioreceptor elements in excess interact with the bioreceptor elements of the control line perpendicular to the flow direction, rendering the control line visible and indicating that the flow test has worked correctly. Finally, the sample then reaches the absorbent pad downstream to the detection pad in the lateral flow direction. The diagnostic or test results are commonly generated in the form of a Boolean response, i.e., in the form of the presence or absence of the continuous lines in the test and control regions of the detection pad, indicating the presence or the absence of the analyte in the sample. This type of results provided by the conventional LFAs can be easily interpreted with naked eye, as well as by dedicated instruments.

[0007] For the diagnosis and monitoring of certain health conditions or diseases, such as stigmatising diseases, sexually transmitted infections (STIs), hepatitis B and C, tuberculosis, malaria, high- risk pregnancies and cancers, the ease with which results can be interpreted by untrained people and non-professional health workers, and the lack of immediate medical advice when interpreting results, can be disadvantages. Further, the subjective interpretation of the test results by the individual can be a source of anxiety, misinterpretation, errors and diagnosis errors, which influence treatment achievement. Moreover, the confidentiality of test results as well as their anonymity cannot be guaranteed. Therefore, some healthcare professionals remain reluctant to use LFAs for self-diagnosis although the POC testing including the patient’s or user's home remains an advantage.

[0008] Providing test results in a form different than a continuous line perpendicular to the sample flow direction or two well-separated continuous lines perpendicular to the sample flow direction, remains a hurdle to the development and generation of results in a complex visual form, such as two dimensional (2D) non-linear patterns, that cannot be interpreted easily or solely by the naked eye. The reproducibility and reliability of test results presented in lines that are not perpendicular to the direction of flow are rendered difficult, as they are affected by the dynamics of flow, the dominant effects of one-dimensional flow, the depletion due to conjugate binding in the strip, interference or cross reactivity from sample components, nonspecific binding, and post zone or prozone effects.

[0009] WO 2012 / 099897 Al has disclosed a manufacturing method of a test device that can produce easily interpreted results in the form of indicia or patterns developed in any orientation relative to the direction of the assay flow. The patterns include standardized symbols or letters easily interpretable by naked eye, a reader, and are representative of the test results by indicating qualitative outcomes, semi -quantitative or quantitative outcomes. To ensure that the results are reproducible, can be multiplexed and less subject to interpretation errors, the patterns are preformed on the matrix of the detection pad by distributing reagent dots, which do not overlap and are sufficiently spaced from one another to avoid affecting the flow of the sample. The exemplified patterns of the test results are very simple geometric forms and remain easily interpretable. In a case of a multiplex LFA based on the aforementioned manufacturing method, the test results are presented in the form of a visible pattern representing letter in Braille, which also remain easily interpretable by naked eye and without a reader (Hofmann et al., ACS Omega, 2021, 6, 22700-22708).

[0010] There is therefore a need for diagnostic devices for self-testing, wherein the test results and the contained information and data can be essentially or only interpreted by trained persons or healthcare professionals, if needed using a dedicated reader and software or computer program, to retain the confidentiality and the anonymity of the tests even during transmission to trained persons or healthcare professionals, and that may include additional information or data.

[0011] The present invention addresses the problems depicted above, such as the issues regarding the lack of confidentiality of the test results, the easiness of the accessibility of the results to misinterpretation or overinterpretation by untrained people, the consequences of misinterpretation on the physical and mental health and treatment of the user, the reproducibility and the reliability of the test results of LFAs presented in a form of 2D pattern, in particular in form of pattern different from continuous line perpendicular to the flow direction, regular and standardized symbol, or alpha and / or numeric features easily interpretable. of the Invention

[0012] To address the problems depicted above, the present invention proposes a method of manufacturing a membrane for lateral flow assay (LFA) or rapid diagnostic assay, on which a two-dimensional barcode of reagent dots including a certain information associated to an analyte in a liquid sample is patterned. The reagent dots are a combination of reagent dots of control reagent and of reagent dots of capture reagents.

[0013] The inventors have found that the as-prepared membrane comprising a two-dimensional barcode of dots of several different reagents enhances the confidentiality and security of access to information relating to the test results, such as whether the test is negative or positive, qualitative or semi-quantitative, as well as further data, by encrypting said information and eliminating the visual interpretation of the test results reading, while maintaining the reproducibility and reliability of the test results. Surprisingly, the combination of dots from different reagents, at least one control reagent and one capture reagent, in a single two- dimensional barcode printed on the porous membrane of a rapid diagnostic test does not impede assay performance or sample flow, or cause depletion of the target analyte and / or free conjugated bioreceptors in the flowing sample.

[0014] Based on a matrix of two-dimensional barcode, in particular on a modified QR (Quick Response) code, fitting the strip structure of the standard rapid diagnostic device and unlike the two-line presentation of LFA results, the two-dimensional barcode also improves the efficiency and accuracy of the test by simplifying data management and interpretation processes through user-friendly software. Presenting the test result in the form of a two- dimensional barcode also enables access to confidential and personal data to be restricted to authorized persons only, avoids misinterpretation of test results and false positives, improves the detection limit of the test through the use of a scanner, and allows medical support and advice to be provided by healthcare professionals when interpreting the test results. Further, the level of encryption of two-dimensional barcode data is increased by combining control and capture reagent dots. Rapid diagnostic assays comprise, but are not limited to, lateral flow assays, dipstick assays and diagnostic or test assays providing a visually readable output signal subject to human interpretation.

[0015] Since the two-dimensional barcode for the LFA or the rapid diagnostic assay can be designed by using a platform that provides standard or off-the-shelf software or computer programs and can be read using a reading device such as mobile phone, there is also a definite economic advantage.

[0016] According to one aspect, the present invention provides a method of manufacturing a membrane for lateral flow assay or rapid diagnostic assay, on which a two-dimensional barcode of reagent dots including a certain information associated to an analyte in a liquid sample is patterned, said method comprising the steps of:

[0017] - generating a negative two-dimensional barcode including a certain information associated to the absence of said analyte in the liquid sample, by way of a computer program;

[0018] - generating a positive two-dimensional barcode including a certain information associated to the presence of said analyte in the liquid sample, by way of said computer program; generating a mask two-dimensional barcode by computing pixel difference between the negative two-dimensional barcode and the positive two-dimensional barcode by way of said computer program;

[0019] - printing a visual representation of the negative two-dimensional barcode on the porous membrane by dispensing a or at least one control reagent;

[0020] - printing a visual representation of the mask two-dimensional barcode by dispensing a or at least one capture reagent over the as-printed visual representation of the negative two- dimensional barcode on said porous membrane.

[0021] In a second aspect, the present invention relates to a test device for detecting an analyte in a liquid sample comprising a membrane on which a plurality of reagent dots are arranged in form of a two-dimensional barcode, wherein said reagent dots are dots of at least one control reagent and dots of at least one capture reagent, said at least one control reagent comprising bioreceptors having affinity for free labelled bioreceptors with affinity for the analyte and / or bioreceptors having affinity for free labelled bioreceptors without affinity for the analyte, and said at least one capture reagent comprising bioreceptors having affinity for the analyte; and wherein the reagent dots of the two-dimensional barcode are visible after completion of the test so as to display the layout of the two-dimensional barcode, in which is embedded a certain information associated with the analyte.

[0022] In a third aspect, the present invention relates to a use of a test device for detecting an analyte in a sample, wherein the analyte is associated with signs and symptoms of an altered physiological state or with health monitoring.

[0023] Further aspects and embodiments of the invention are detailed herein below and in the appended claims. Further features and advantages of the invention will become apparent to the skilled person from the description of the preferred embodiments given below.

[0024] Brief Description of the Drawings

[0025] Figure l is a scheme of a test device for LFA with three zones: a sample zone 1 comprising a sample pad, a reaction zone 2 comprising a conjugate pad and a detection zone 3 comprising a membrane on a back card, and an absorbent pad 4.

[0026] Figure 2 is a scheme of QR code structure relative to rMQR code structure (https: / / www.qrcode.com / en / codes / rmqr.html)

[0027] Figure 3 is a scheme of an embodiment of the method of manufacturing a membrane for lateral flow assay or rapid diagnostic relative to the step of generating negative, positive and positive test two-dimensional barcodes. Figure 3A: generation of the negative two- dimensional barcode by way of off-the-shelf computer program after generating two native two-dimensional barcodes and reworking the first native one so as to match white pixels between both native barcodes. Figure 3B: generation of the positive two-dimensional barcode by way of off-the-shelf computer program after reworking the second native barcode so as to match black pixels between the negative two-dimensional barcode and the second native barcodes. Figure 3C: generation of the positive test two-dimensional barcode by the addition of the pixel layout of the mask two-dimensional barcode to the negative two-dimensional barcode by way of a computer program.

[0028] Figure 4 are schemes of operating principle of a test device of the invention for the detection of a target analyte comprising a membrane with two layouts of two-dimensional barcode as illustrated in Figure 4A or one layout of two-dimensional barcode as illustrated in Figure 4B. At least one layout of the two-dimensional barcode comprises a control two-dimensional barcode pattern printed with at least one control reagent and a mask two-dimensional barcode pattern printed with at least one capture bioreceptors. Capillary action drives the flow upon sample application, resuspending conjugate bioreceptors in the conjugate pad of the reaction zone 2 and enabling the immunoassay reaction.

[0029] Figure 5 is pictures of strip structures with membranes on which rMQR codes are printed and rendered visible after completion of the LFA detecting the analyte hCG (human Chorionic Gonadotropin). The two-dimensional barcodes have been printed as described herein. Figure 5A illustrates negative tests displaying "neg" and Figure 5B illustrates positive tests displaying "pos" after having been scanned by a reader a reader using the off-the- shelf DENSO application.

[0030] Figure 6 illustrates steps for reducing the effects of depletion for effective readability of the two-dimensional barcode printed on a membrane through gradient printing. Figure 6A illustrates blank-corrected absorbance of consecutive "control" points (point encircled of framed in red) along the flow direction for different printed antibody concentrations as shown in the graphic of Figure 6B. The gray curve (no gradient printing) represents a single printed layer across the two-dimensional barcode (rMQR code), while the red curve (gradient printing) corresponds to a gradient printing approach where the antibody solution was printed once across the full QR code and a second time on the last 10 rows. hCG concentration is 0 ng / mL (N = 5 independent assays). Figure 6C represents the hCG-response curve at three test point positions relative to the sample pad: far, middle-distance and close. Mean blank- corrected absorbance values are fitted by a four-parameter logistic (4PL) regression model, yielding R2values of 0.92 (gray curve or bottom curve), 0.97 (red curve or middle curve), 0.98 (blue curve or top curve) (N = 5 independent assays). Figure 6D presents pictures of rMQR code LFIAs run with samples at 0, 100, and 500 ng / mL hCG concentrations. Figure 6E is a schematic representation of the rMQR code system: control rMQR code (blue) printed with control antibodies and mask (green) printed with the capture antibodies (Fabrication parameters: gradient printing, capture antibody concentration = 2.0 mg / mL, conjugate pad optical density = 5.5.)

[0031] Figure 7 illustrates fFN-response curve. Positive scan proportion of rMQR code LFIAs for different fFN concentrations. Values are fitted with a four-parameter logistic regression method, with initial value fixed to 0 and final value to 1, yielding R2 value of 0.99 (N = 5 independent assays per concentration). Fabrication parameters: gradient printing, capture antibody concentration = 2.0 mg / mL, conjugate pad optical density = 6.0. Detailed of the invention

[0032] In the following, embodiments of the invention shall be described in detail with reference to the above-mentioned figures.

[0033] The invention provides a method of manufacturing a membrane for lateral flow assay or rapid diagnostic assay, on which a two-dimensional barcode of reagent dots including a certain information associated to an analyte in a liquid sample is patterned or printed.

[0034] Before printing the two-dimensional barcode of reagent dots on a membrane for detecting an analyte in a liquid sample, one or more two-dimensional barcodes are generated by way of computer or a computer program with respect of one or more specific analytes. The computer program may be specifically designed to generate such two-dimensional barcodes or, where economically advantageous, may be off-the-shelf or already available. Once said one or more two-dimensional barcodes are computed for one or more specific analytes, they are merged, if more than one two-dimensional barcodes, and reworked by way of a computer program so as to print one single two-dimensional barcodes of reagent dots comprising two types of reagent, one type being a control reagent leading to indicate the status of the test functioning of the LFA and the other type being a capture reagent leading to indicate information concerning the one or more analyte, such as its presence, amount, e.g. The reworked two- dimensional barcode, once computed for one or more target analytes, is used as a printing model for manufacturing a membrane for one or more specific analyte.

[0035] Two-dimensional barcodes are selected from QR codes storing information in a matrix pattern of black squares on a white background, namely a matrix pattern of black and white pixels. As matrix-type symbols, the two-dimensional barcodes include functionality patterns such as finder patterns, alignment patterns, timing patterns, quiet zone, for facilitating the reading and a data area for storing information as illustrated in Figure 2. There are various QR codes: original square-shaped QR code, micro QR code, rMQR code (rectangular micro QR code), SQRC (Secret-function equipped QR code) and frame QR code, all with different sizes, capacity of data storage and levels of error correction, and types of data to be stored.

[0036] To fit the standard structure of the detection zone 3 of a test device for LFA in form of a strip, a rectangular pattern for the design of the two-dimensional barcode is preferred, but not limited to this type. The two-dimensional barcodes are selected from rectangular two-dimensional barcodes or square two-dimensional barcodes. The two-dimensional barcode has to include a certain information associated with an analyte in a liquid sample. Said certain information comprises a certain information associated with the presence of the analyte in the sample and / or a certain information associated with the absence of the analyte in the sample. In the present invention, a single two-dimensional barcode comprising reagent dots is patterned on the membrane for LFA and replaces the test results of standard LFA in form of a test line and a control line. The two-dimensional barcode hence includes the information associated with the presence and the absence of the analyte in the sample as well as information associated with the test functioning. The single two-dimensional barcode serves both as a control to confirm that the test is working and as an indication of the presence or absence of the analyte in the sample. Therefore, dots from capture reagent and control reagent have to be printed on the membrane designed for the detection zone 3 of a test device for LFA.

[0037] Without to be bound by theory, an analyte is a chemical species being sought, detected and measured according to analytical protocol. In particular, LFA can accurately detect simultaneously one or more analytes such as proteins, cells, virus, peptides, antibody, DNA, and RNA.

[0038] The certain information associated with the analyte in a sample may include or comprise numeric and alphanumeric data, text, number, URL (Uniform Resource Locator), contact information, batch number, date, data relating to the physiological condition to be tested, and / or data relative to the device identification, in addition to the information associated with the presence or absence of the analyte in the sample. The certain information associated with the absence of the analyte in the sample is different from the certain information associated with the presence of the analyte in the sample. For increased confidentiality and anonymity when reading test results, especially when reading with a standard application, the data to be encrypted or encoded in the two-dimensional barcode is arbitrarily chosen, i.e., without any correlation or particular meaning that could define the analyte.

[0039] The method comprises the steps of generating a negative two-dimensional barcode including a certain information associated to the absence of said analyte in the liquid sample, by way of a computer program, generating a positive two-dimensional barcode including a certain information associated to the presence of said analyte in the liquid sample, by way of said computer program, generating a mask two-dimensional barcode by computing pixel difference between the negative two-dimensional barcode and the positive two-dimensional barcode by way of said computer program. The method further comprises the steps of printing a visual representation of the negative two-dimensional barcode on a porous membrane by dispensing a or at least one control reagent and printing a visual representation of the mask two- dimensional barcode by dispensing a or at least one capture reagent over the as-printed visual representation of the negative two-dimensional barcode.

[0040] According to an embodiment, the step of generating the positive two-dimensional barcode by computer and / or the step of generating the mask two-dimensional barcode by computer are repeated. At least one or two positive two-dimensional barcodes and / or at least one or two mask two-dimensional barcodes are generated. Each computed mask two-dimensional barcode is different one to other and each computed mask two-dimensional barcode includes either information associated with a determined concentration of the same analyte present in a sample or information associated with the presence of several specific analytes of interest. The visual representation of each computed mask two-dimensional barcode being different from one to the other is printed by dispensing at least one capture reagent over the as-printed visual representation of the negative two-dimensional barcode on said porous membrane and / or over the as-printed previous visual representation of a mask two-dimensional barcode on the porous membrane.

[0041] Negative and positive two-dimensional barcodes can be generated by way of a computer program specifically designed to generate such two-dimensional barcodes. The negative and positive two-dimensional barcodes have different layouts in pixel or patterns but have a certain similarity between them such as the functionality patterns and the level of error correction. This ensures efficient scanning by a reading device and that the difference in pixels between them is not high to impede the accuracy and the readout of the two-dimensional barcode of reagent dots providing test results relating to the test functioning and to the analyte in a single two-dimensional barcode printed on the membrane.

[0042] Standard computer programs or computer programs already-available can be used for the generation of two-dimensional bar codes. Generating a negative and / or positive two- dimensional barcode by way of a standard code generator program involves the generation of one, two or more native two-dimensional barcodes. In this embodiment, the method step of generating a negative two-dimensional barcode comprises the steps of generating a first native two-dimensional barcode including a certain information associated to the absence of said analyte in the liquid sample, by way of a computer program, and generating a second native two-dimensional barcode pattern including a certain information associated to the presence of said analyte in the liquid sample by way of said computer program. This latter step is followed by a step of reworking the first native two-dimensional barcode so as to match white pixels between the second native two-dimensional barcode and the first native two-dimensional barcode, as illustrated in Figure 3A. The method further comprises a step of generating a positive two-dimensional barcode, said step comprises the step of reworking the second native two-dimensional barcode so as to match the black pixels between the negative two- dimensional barcode and the second native two-dimensional barcode, as illustrated in Figure 3B.

[0043] The two-dimensional barcodes are selected from QR codes, e.g., square-shaped, micro QR codes or rMQR codes, preferably rMQR codes. They can be native or modified and / or reworked. Even when modified, the two-dimensional barcodes comply with QR code-like patterns, in which the functionality patterns such as finder pattern, timing pattern and alignment pattern are preserved for ensuring efficient scanning by a reader. Taking advantage of the level of error correction, the first and second native two-dimensional barcodes are generated so as to include data providing patterns of black and white pixels having a medium to high level of similarity between the two patterns, but not the same pattern. In the case of generating a rectangular-shaped two-dimensional barcode, the level or error correction is in the range from 1% to 30%, preferably 30%. The level of similarity between the two patterns or layouts of two-dimensional barcode, one associated with the control of the test working and the other associated with the information relating to the analyte ensures that there are only a small number of pixels to modify between the two patterns. Each native two-dimensional barcode is reworked by way of a computer program so as that the level of error correction and the own embedded information in each barcode are kept and that the difference in pixels between the layouts of the two reworked two-dimensional barcodes is maintained small as represented in Figure 2.

[0044] After the steps of generating negative and positive two-dimensional barcodes directly through a dedicated computer program or through reworking native negative and positive two- dimensional barcodes, the two layouts or patterns in pixels are merged, combined and / or compared by way of the computer program and the difference between the layouts of white and black pixels of the negative two-dimensional barcode and the positive two-dimensional barcode is computed so that a mask two-dimensional barcode is generated by way of computer. The mask two-dimensional barcode does not comprise the functionality patterns of the negative two-dimensional barcode. By adding the pixel layout of the mask two- dimensional barcode, a positive test two-dimensional barcode is generated by the computer program. The method further comprises a step of generating a positive test two-dimensional barcode by computing the pixel addition from the mask two-dimensional barcode to the negative two-dimensional barcodes by way of a computer program, as illustrated in Figure 3C. The manufacturing method further comprises a step of storing the negative two- dimensional barcode and the positive test two-dimensional barcode in a database for subsequent verification of test results by way of controlling computer program. Said positive test two-dimensional barcode and negative two-dimensional barcode stored in a database or an electronic health record system may serve for the analysis of test results embedded in the two-dimensional barcode of reagent dots, which is printed on the membrane of the LFA device, by way of a connecting mobile device communicating with a secure database manager by way of a computer. The positive test two-dimensional barcode has a layout different than the positive two-dimensional barcode, but the information associated with the presence of the analyte in the sample and embedded in said positive test two-dimensional barcode is the same as the information embedded in the positive two-dimensional barcode.

[0045] The patterns of the negative two-dimensional barcode and the mask two-dimensional barcode being visual representations of two-dimensional barcode can be directly used for printing the reagent dots in a layout of two-dimensional barcode, provided that the size of the patterns fits with the width and length of the membrane of the detection zone 3 of the test device.

[0046] Alternatively, the method of manufacturing comprises a step of converting the negative two- dimensional barcode and the mask two-dimensional barcode into a printable visual representation of full and empty squares or white and black squares by way of computer prior to the printing. Each full or black square on the visual representations is processed in the printing as a reagent dot to be printed. The negative and the mask two-dimensional barcodes are converted into a matrix, in particular a binary matrix, which is then converted into a visual representation which can be printed by dispensing the reagents. The conversion of the two- dimensional barcodes into printable visual representations is performed by an off-the-shelf computer program using, but not limited to, Python code.

[0047] The printable visual representation of the negative two-dimensional barcode is printed by dispensing the control reagent in form of dots on the membrane. The visual representation of the mask two-dimensional barcode is printed over the as-printed visual representation of the negative two-dimensional barcode by dispensing a capture reagent on the membrane. This step is performed so as to prevent dispensing capture reagent dots on as-printed control reagent dots on the membrane.

[0048] To test two or more different target analytes on the same membrane, the two-dimensional barcode comprises, in addition to the dots of control reagent, reagent dots of two or more capture reagents, each capture reagent being specific to a particular or specific target analyte. The step of generating a positive two-dimensional barcode is hence repeated as many times as the number of analytes to be analyzed. One positive two-dimensional barcode per target analyte is generated by way of computer on the basis of the visual representation of one or a single negative two-dimensional barcode and the visual representation of one mask two- dimensional barcode per specific analyte being generated by way of computer as described herein. Subsequently to the step of printing the visual representation of the negative two- dimensional barcode, the visual representation of each mask two-dimensional barcode associated to a specific target analyte is printed by dispensing different capture reagents, each capture reagent being specific to one target analyte, over the as-printed visual representation of the negative two-dimensional barcode. This step is performed so as to prevent dispensing first, second or further specific capture reagent dots on as-printed control reagent dots and / or second or further specific capture reagent dots. The visual representations of each mask two- dimensional barcode can be printed sequentially or simultaneously.

[0049] For semi-quantitative or multiplexed readouts in the case of the analysis of one specific target analyte, at least two or more visual representations of mask two-dimensional barcodes for the same analyte can be generated to enable the scanning of one or more mask two-dimensional barcodes as a function of the concentration of the analyte. The visual representations of mask two-dimensional barcodes differ from one another. One positive two-dimensional barcode for the specific target analyte is generated by way of computer on the basis of the visual representation of one or a single negative two-dimensional barcode and at least two or more different mask two-dimensional barcodes per a specific concentration of the same analyte being generated by way of computer as described herein. The visual representation of the negative two-dimensional barcode is printed by dispensing control reagent dots. The visual representation of each different mask two-dimensional barcode associated to the presence of the same analyte in different concentrations is printed by dispensing capture reagent dots, each visual representation of each mask two-dimensional barcode being printed by dispensing dots of capture reagents comprising a determined concentration of capture bioreceptors or with a specific concentration of capture bioreceptors, over the as-printed visual representation of the negative two-dimensional barcode and / or the as-printed visual representation of a mask two- dimensional barcode printed by using a capture reagent with a different concentration of capture bioreceptors. This step is performed so as to prevent dispensing capture reagent dots of different concentrations on as-printed control reagent dots and / or as-printed capture reagent dots of a different concentration. The visual representation of each mask two-dimensional barcode can be printed sequentially or simultaneously.

[0050] The control reagent comprises control bioreceptors having affinity for free labeled bioreceptors with affinity for the analyte and / or without affinity for the analyte and the capture reagent comprises capture bioreceptors having affinity for the analyte. A bioreceptor is a biological element (e.g., antibody, biomolecule, enzyme, DNA, RNA) which is sensitive to recognizing the analyte and / or, presently in an LFA, another bioreceptor sensitive to recognizing the analyte. The control bioreceptors are selected from antibody, biomolecule, enzyme, nucleic acids (DNA, RNA), peptides or proteins, which bind free labeled or unlabeled bioreceptors with affinity for the analyte and / or labeled bioreceptors without affinity for the analyte, said labeled or unlabeled bioreceptors being named conjugate bioreceptors. Conjugate bioreceptors are comprised in the conjugate pad of the reaction zone 2 of the test device, and may form a complex with the analyte or be captured or bound by the control bioreceptors immobilized on the membrane, when they are free and / or in excess in the flowing sample. The capture bioreceptors are selected from antibody, biomolecule, enzyme, nucleic acids (DNA, RNA), peptides or proteins, which bind the analyte, in particular the analyte conjugated or bound to the conjugate bioreceptor. When labeled, the conjugate bioreceptor comprises a labeling moiety or molecule selected from, but not limited to, standard labelling molecules for LFA, or gold particles, biotin, latex particles, fluorescent labels or molecules, enzymes, colloidal metals, or magnetic particles. The control reagent and the capture reagent are liquid compositions and optionally comprise a blocking agent to prevent non-specific affinity or binding, said blocking agent being selected from usual blocking agents for LFA, including but not limited to as BSA (Bovine Serum Albumin), gelatin, generic proteins (albumin, sperm), detergents and the like. Another way to prevent non-specific affinity or binding and blurred printed patterned is to apply or dispense blocking agent on the membrane.

[0051] To obtain a clear and readable signal from the two-dimensional barcodes of reagent dots on the membrane after completion of the test is essential and involves an accurate printing of the visual representations of two-dimensional barcodes. The size of the two-dimensional barcode has to be sufficiently small to be integrated or embedded in the membrane of LFA device, a strip or a dipstick of a rapid diagnostic assay, while maintaining the resolution of the two- dimensional barcode for ensuring the successful scanning and reading of the data included or embedded in the two-dimensional barcode.

[0052] The main types to accurately apply the reagents or the bioreceptors on the membrane include manual pipetting beings suitable for low-throughput applications but can be labour-intensive and variable, and automated pipetting systems, which use machines for higher precision and reproducibility. Microarray printers, employing technologies like pin-based, inkjet, syringe pumps or piezoelectric dispensers are designed to print small volumes of bioreceptors reagents in precise patterns. The choice of contact and non-contact dispensers depends on factors as volume and precision requirements. Non-contact dispensing method including application by spray or by jetting, typically requires less volume to dispense but can result in greater run-to- run variability. On the other hand, contact dispensing systems have relatively low run-to-run variability, but require additional volume to dispense the same amount of material, which can be costly.

[0053] The relevant parameters for printing the two-dimensional barcode with a spray-coating method on a printing machine selected form piezo-based spray coater are the Stroke of the piezo actuator (S) in percentage [%], the Stroke Velocity (SV) in [pm / ms], the Drop Pitch (P) measured in [mm], and X, Y and Z velocities measured in [mm / s]. The Stroke of the piezo actuator (S) in percentage [%] is the piezo deflection distance which correlates with the dispensed volume. A larger stroke results in a larger droplet. The effective S in pm can be sr%i calculated according to S[pm] = - . The preferred values are in the range from 15 % to 100

[0054] %, from 10 % to 40 %, from 20% to 80%, or from 80 to 90%. Stroke Velocity (SV) in [pm / ms] is velocity of the piston during displacement. Increasing SV results in faster droplets. SV values are in the range from 60-100 pm / ms for water-based samples, 50-80 pm / ms for solvents-based sample and > 150 pm / ms for viscous sample. Preferably SV values for printing reagents for rapid diagnostic assay are in the range from 60-100 pm / ms, more preferably 70- 90 pm / ms. Drop Pitch (P) measured in [mm] is distance between pixels or printed dots. For example, P of 0.45mm for a ’’short” rMQR code corresponds to a real size of 12.15x4.95 mm2. Preferably P values are in the range from 0.35mm to 1 mm, more preferably from 0.4 to 0.45 mm. X, Y and Z velocities measured in [mm / s] is velocity of the axis movement. If the axis velocity is too fast compared to the ratio of P and actuation cycle duration, drops may be omitted during operation.

[0055] In contrast to a standard rapid diagnostic assay with test and control lines, the sample in a test device of the invention has to flow through the complex pattern of the two-dimensional barcode of reagent dots embedded in the membrane. The presence of bioreceptors, namely control and capture bioreceptors over a long distance has a much more significant impact on the gradient of the species (analyte, conjugate bioreceptors) flowing in the test membrane than the effect of the flow rate decreasing with the distance.

[0056] This implies sequential exposure of the control bioreceptors and the capture bioreceptors with the flowing analyte and flowing free labelled bioreceptors or conjugate bioreceptors. The control bioreceptors and capture bioreceptors immobilized on the membrane and located close to the zone of reaction 2 bind or capture more species in the flowing sample, which causes a depletion, for both analyte and flowing free labelled bioreceptors, in the sample moving towards the absorbent pad 4. This gradient is an issue, in particular in the visibility or the appearance of elements in the two-dimensional barcode necessary to obtain an accurate reading and functional scanning of the two-dimensional barcode pattern. This issue leading to an indistinct two-dimensional barcode cannot be corrected by the error correction level of the two-dimensional barcode design, since it has already been used. To prevent this depletion effect, the visual representations of the negative two-dimensional barcode and the mask two- dimensional barcode are printed so as to form a concentration gradient of the control bioreceptors and the capture bioreceptors in the printed two-dimensional barcode. As the reagent dots of the two-dimensional barcode are closer to reaction zone 2, the concentration of bioreceptors in the reagent dots is lower. Thus, one or more steps of repeating printing the visual representation of the negative two-dimensional barcode and / or one or more steps of repeating printing the visual representation of the mask two-dimensional barcode is introduced so as to form a concentration gradient of the at least one control reagent and / or of the at least one capture reagent in the two-dimensional barcode formed by the reagent dots being printed. The repeated printing of the visual representations of the negative and / or the mask two- dimensional barcodes may be complete or partial in order to obtain a linear, sequential or incremental concentration gradient. This step allows to create a concentration gradient of the control and / or capture reagents in the printed dots and decrease the probability that the control bioreceptors trap the conjugate bioreceptor before the distal end of the two-dimensional barcode close to the absorbent pad. Alternatively to form the concentration gradient of reagents in the dots forming the printed two-dimensional barcode, the visual representations of the control and / or the mask two-dimensional barcodes are printed by using at least two control reagents of different concentrations and / or at least two capture reagents of different concentrations. This step of printing the visual representations of the negative two- dimensional barcode and / or the mask two-dimensional barcode using two or more control reagents, each comprising control bioreceptors at different concentrations, and / or two or more capture reagents, each comprising capture bioreceptors at different concentrations, can be repeated.

[0057] Mitigating the depletion effect with gradient printing and tuning the optical density of the conjugate pad and the concentration of the capture antibody enables maintaining both the diagnostic performance and the scannability of the two-dimensional barcode while adjusting the assay sensitivity and dynamic range.

[0058] To reduce competition between control and capture bioreceptors, which contributes to the depletion effect, the conjugate pad in reaction zone 2 may comprise one or more types of conjugate bioreceptors, each type of conjugate bioreceptors being different from the others. For example, but not limited to, the conjugate pad may comprise two types of conjugate bioreceptors, each type of conjugate bioreceptors being selected from two groups of detection antibodies, each group comprising labeled antibodies from a host animal species different from the other group (e.g., goat versus mouse) with or without affinity for the analyte. This enables selective binding by the control bioreceptors and / or the capture bioreceptors. The visual representation of the negative two-dimensional barcode can be printed with a control reagent comprising control bioreceptors having affinity for free labeled bioreceptors, said free labeled bioreceptors having or not having affinity for the analyte. The negative two- dimensional barcode can also be printed by dispensing one, two or more control reagents having different control bioreceptors, e.g. as described above, and / or of different concentrations of control bioreceptors.

[0059] The invention further provides a test device for detecting an analyte in a liquid sample comprising a membrane on which a plurality of reagent dots are arranged in form of a two- dimensional barcode, wherein said reagent dots are dots of control reagent and dots of capture reagent, said control reagent comprising bioreceptors having affinity for free labelled bioreceptors with or without affinity for the analyte and said capture reagent comprising bioreceptors having affinity for the analyte; and wherein the reagent dots of the two- dimensional barcode are visible after completion of the test so as to display the layout of the two-dimensional barcode, in which is embedded a certain information associated with the analyte.

[0060] The test device of the invention is used for assays or diagnostic tests for detecting an analyte in a sample selected from rapid diagnostic assays including, but not limited to, lateral flow tests, lateral flow immunoassay, dipstick, test strip. In particular, when the test device is used for assays enabling lateral flow, it further comprises a structure with a plurality of zones comprising a sample zone 1 for applying the liquid sample, a reaction zone 2 comprising labelled bioreceptors with affinity for the analyte, a detection zone 3 comprising the membrane and an absorbent pad 4, the zones being configured to enable lateral flow of the liquid sample from the sample zone to the absorbent pad being at the distal end of the structure. The sample being liquid or rendered liquid by solubilization in a standard liquid, solution or buffer is a sample derived from, but not limited to, body fluids, cells, or biological tissues. Body fluids may include, but not limited to, blood, urine, sweat, vaginal secretions, saliva, lymph, spinal fluid, and bile. The sample can be provided from any living being, from animals or from human being. The liquid sample is applied on a sample pad comprised in the sample zone 1. The reaction zone 2 comprises the conjugate pad further comprising conjugate bioreceptors, which may be labelled bioreceptors with or without affinity for the analyte as described herein. The detection zone 3 comprises the membrane on which the control and capture reagents dots are dispensed in form of a two-dimensional barcode. The membrane comprises or is made of a material which is porous and selected from nitrocellulose, cellulose acetate, polyvinylidene fluoride, charge-modified nylon, polyether sulfone, glass, fibreglass, sintered glass, or ceramic. Finally an absorbent pad is located at the end of the detection zone 3 in the flow direction. The conjugate pad, the membrane and the absorbent pad are assembled on a backing card.

[0061] In the test device of the invention, the certain information associated to the analyte comprises information associated with the presence or absence of the analyte in the sample and / or information associated with the identification of the test device and / or the test. After the completion of the test, the layout of a two-dimensional barcode is rendered visible and can be scanned or read by a reader device. After scanning said two-dimensional barcode, the readout provides a certain information depending on the layout of the visible two-dimensional barcode, said information being embedded in the layout or pattern of the two-dimensional barcode. When the analyte is absent or is not detected, the control reagent dots of the two- dimensional barcode are visible after completion of the test so as to display the layout of a negative two-dimensional barcode. In this case, the capture reagent dots of the two- dimensional barcode are not rendered visible after completion of the test. After scanning the visible two-dimensional barcodes by a reader device communicating with a computer comprising a data managing and controlling system, the readout of said two-dimensional barcode provides the information associated with the absence of the analyte in the sample. Information associated with identity or identification of the test device and / or the test can be also embedded in the layout of the negative two-dimensional barcode, which is rendered visible by the reaction between control bioreceptors and conjugate bioreceptors not forming a complex with the analyte. Information associated with identity or identification of the test device and / or the test include, but are not limited to, information relating to batch, date, gender, age, and further data useful for monitoring a health condition. When the analyte is present or is detected, the control reagent dots and the capture reagent dots of the two-dimensional barcode are visible after completion of the test so as to display the layout of a positive test two-dimensional barcode. In this case, both control and capture reagent dots of the two- dimensional barcode are rendered visible after completion of the test. After scanning the visible two-dimensional barcodes by a reader device communicating with a computer comprising a data managing and controlling system, the readout of said two-dimensional barcode provides the information associated with the presence of the analyte in the sample, which can be quantitative, semi -quantitative and / or qualitative. Since the positive test two- dimensional barcode is based on the layout of the negative two-dimensional barcode, information associated with identity or identification of the test device and / or the test can be also embedded in the layout positive test two-dimensional barcode. The two-dimensional barcode of reagent dots comprises a concentration gradient of the control reagent and / or the capture reagent. The concentration gradient is linear, sequential or incremental, preferably sequential. As described herein, to avoid depletion effect, the two- dimensional barcode of reagent dots dispensed on the membrane includes or comprises a concentration gradient of control and capture bioreceptors from the lowest to the highest concentration in the flow direction. The membrane is positioned in the test device based on the flow of the sample so that the lowest concentration of the control reagent and / or the capture reagent in the two-dimensional barcode of reagent dots is on the side of the reaction zone 2 and the highest concentration of reagent is towards the absorbent pad 4. The concentration of the bioreceptors in the control reagent and / or in the capture reagent are in the range from 103mM to 10'9mM, 1 mM to 10'6mM.

[0062] The test device further comprises a membrane of the present invention, produced or manufactured by the method of the present invention. The two-dimensional barcode may be printed on the membrane enabling the flow of the sample according to the method described herein or a method suitable to dispense biological molecules in form of a complex and nonlinear pattern or layout similar to a two-dimensional barcode or QR-code and the like.

[0063] According to a further embodiment, the reagent dots arranged in form of a two-dimensional barcode printed on the membrane are dots of one, two or more control reagents and / or dots of two or more capture reagents. Each control reagent comprises control bioreceptors or one type of control bioreceptors having affinity for a specific free labelled bioreceptor with or without affinity for the analyte and each capture reagent comprises capture bioreceptors or one type of capture bioreceptors having affinity for a specific target analyte or a specific target on the analyte. The test device, in particular a LFA device, comprising such a membrane, onto which a single two-dimensional barcode comprising dots of one, two or more control reagents anddots of one, two or more capture reagents is printed, allows the detection of several different analytes, at least two analytes with a single test device.

[0064] The test device further comprises a membrane on which a second or more two-dimensional barcodes of reagent dots is patterned or dispensed, wherein the reagent dots are selected from control reagent dots. The second or more two-dimensional barcodes are patterned or printed on the same membranes as the membrane on which the first two-dimensional barcode is printed, said first two-dimensional barcode comprising control and capture reagent dots, or on one or more additional membranes. The second or more two-dimensional barcodes are located upstream of the first two-dimensional barcodes in the flow direction or are located on the side of the first two-dimensional barcode pattern. The information embedded in the second or more two-dimensional barcodes relates to different data associated with the absence or presence or the quantification of the analyte.

[0065] In a third aspect, the invention provides a use of a test device for detecting an analyte in a sample, wherein the analyte is associated with signs and symptoms of an altered physiological state or with health monitoring. The sample is selected from a sample selected from body fluids. Accordingly the sampled of body fluid is selected from, but not limited to, blood, urine, sweat, vaginal secretions, saliva, lymph, spinal fluid, and bile. The impaired physiological state is a disease selected from, but not limited to, sexually -transmitted diseases, viral diseases, cancers, breast cancer, colorectal cancer, lung cancer, cervical cancer, endometrial cancer, ovarian or testicular cancer, skin cancers, prostate cancer, thrombosis, cardiovascular disease. In particular, the analyte is associated with the monitoring of pregnancy.

[0066] The present invention will now be illustrated by way of examples. These examples do not limit the scope of this invention, which is defined by the appended claims.

[0067] Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other modifications and variations can be made without departing from the scope of the invention as hereinafter claimed

[0068] Example 1 : Development of the method

[0069] A) QR Code design

[0070] A single rMQR code is designed to serve as both controls and tests within the LFIA system. Only the squares forming the ’’Control” rMQR code appear when no analytes are present. Upon scanning, it displays a negative result (or any other indication chosen by the creator of the rMQR code). Conversely, when analytes are detected, the squares of the ’’control” rMQR code appear, along with the squares forming the ’’mask”, which corresponds to the test line in a traditional LFA. The assembly of these squares forms a single and complete rMQR code as illustrated by Figure 2. The advantages and disadvantages to use a specific type a type of QR code for LFA device among QR code, micro QR code, rMQR code, SQRC and frame QR code have been analyzed. The type rMQR code was selected for its good balance between size and data capacity, as well as its rectangular shape. Increasing the size of the rectangular QR Code allows us to maintain a constant LFA width while also increasing the error correction rate and data capacity. This provides a reliable solution for our LFA application without compromising on error correction or expenses. However, the resources available regarding how a rMQR code is encoded are limited, particularly the position of redundancy bytes. These bytes can be modified while still allowing the rMQR code to be scanned. Only the patterns present on all rMQR codes, i.e., finder pattern, timing pattern and alignment pattern are referenced in the literature. These patterns also ensure efficient scanning.

[0071] To create the rMQR code to print on a LFA membrane by way of computer, we have considered the example of a rMQR code which scans ”2” for the ’’negative” QR Code, in which the embedded information is associated with the absence of the analyte, and, a rMQR code which scans ”n” for the ’’positive” QR Code, in which the embedded information is associated with the presence of the analyte. These two rMQR codes, also named native two- dimensional barcodes, were chosen because they have a close structure. In other words, by changing only a small number of squares, the two rMQR codes can be obtained.

[0072] The ”n” rMQR code has more black squares than the ”2” so it was chosen as the positive pattern. These two native two-dimensional barcodes were generated by way of an off-the-shelf computer program to generate rMQR codes, e.g., online rMQR code generator: https: / / rmqr.oudon.xyz as illustrated in Figures 3A-C. Then, to ensure that white squares match between the native rMQR code "n" (second native two-dimensional barcode) and the native rMQR code "2" (first native second two-dimensional barcode), the native rMQR code ”2” was modified to generate a negative rMQR code. White squares were added to the native rMQR code ”2” where there are white pixels in the native rMQR code ”n.” After each addition of white pixel, the embedded information in the modified rMQR code displays "2" correctly, when said modified rMQR code is scanned. The rMQR code ”2” resulting from the modification represents the negative rMQR code, or control rMQR code, as illustrated in Figure 3 A. The native rMQR code ”n” (second native two-dimensional barcode) was modified by way of computer program to ensure black squares match between the negative and the second native rMQR codes to generate a positive rMQR code as illustrated in Figure 3B. Black squares were added to the rMQR code ”n” where there are black pixels in the modified rMQR Code ”2”, namely the negative two-dimensional barcode. After each addition of black pixel, the embedded information in the modified rMQR code displays "n" correctly, when said modified rMQR code is scanned. The modified rMQR code ”n” is the positive rMQR code or the positive two-dimensional barcode as illustrated in Figure 3B.

[0073] A mask two-dimensional barcode is created from the merging of the negative and positive two-dimensional barcodes to determine pixel differences and reworking of these differences so as to obtain a two-dimensional barcode that corresponds to the test line in a standard LFA. During the printing of the visual representation of the two-dimensional barcodes, there is one pattern printed with CTRL cAbs (control bioreceptors or control capture antibodies or control antibodies) in form of the negative rMQR code and another one printed above the pattern printed with CTRL cAbs with the test cAbs (capture bioreceptors or test capture antibodies) in form of the mask two-dimensional barcode.

[0074] The differences between the negative and positive rMQR codes were computed pixel by pixel. If there is a black square in the positive rMQR code and a white pixel in the negative rMQR code at the same position, the mask code displays a black pixel. If there is a black square in the negative rMQR code and a white square at the same position in the positive rMQR code, a white pixel is displayed by the mask code. Finally, if the squares are black or white at the same position in both negative and positive rMQR codes, the mask code displays a white pixel. Then the negative rMQR and the mask two-dimensional barcode are superimposed or added to create the positive test rMQR code or the positive test two-dimensional barcode as illustrated in Figure 3C.

[0075] To simplify the manipulation of black and white squares on rMQR codes, image files, namely bmp files or printable visual representations, were generated by way of computer using Python code. These printable visual representations of the negative and mask two-dimensional barcodes are useful for printing with BiofluidiX printer. The code utilizes the Pillow library for image manipulation, Matplotlib for visualization, and NumPy for numerical operations. At first the rMQR codes were converted in a matrix. By this way of computer program, the two- dimensional codes are resized to fit the size and dimension of the membrane. The matrices were converted in a binary format matrix followed by the creation of images or printable visual representations (bmp files) of the rMQR codes. The generation of images from rMQR codes may be performed after generating the various rMQR codes. The reworking pixel by pixel of said rMQR codes may be performed on the images generated from rMQR codes through the conversion and resizing of said rMQR codes into matrices converted into image format files.

[0076] Developing a rMQR code integrated into the LFA membrane involves creating patterns printed with control and capture bioreceptors. These patterns are designed to accurately indicate the presence or absence of target analytes by scanning the rMQR code that has appeared on the LFA membrane 20 minutes after the beginning of the assay. In the absence of analytes, the negative rMQR code has appeared, which corresponds to the pattern printed with CTRL cAbs. On the other hand, if analytes are present, the positive test rMQR code has appeared as a result of the superposition of the mask printed with test cAbs (capture bioreceptors) and the pattern printed with CTRL cAbs (control bioreceptors), as illustrated in Figures 5A-B.

[0077] This method of QR code design leverages the 30% error correction level to modify existing rMQR codes and includes developing a Python script to facilitate the conversion of images to matrices and vice versa, aiding in the manipulation and analysis of rMQR codes. After analyzing the advantages and disadvantages of QR code types for LFA applications, the rMQR code type is the suitable type due to its balance between size and data capacity, as well as its rectangular shape. Despite these advantages of these rectangular two-dimensional barcodes, wider but shorter rMQR codes are designed to enhance LFA sensitivity and improve rMQR code scanning.

[0078] B) Two-dimensional barcode printing on LFA membrane.

[0079] B. l. rMQR Code printing with dyes on LFA membrane

[0080] To print a rMQR code on LFA membranes, it was essential to understand the parameters of the BiofluidiX Biospot Arc printer. This printer provides various printing options and settings that significantly impact the quality and accuracy of printed patterns and are relevant for the printing of biological reagents on a membrane. These are the following:

[0081] • Stroke of the piezo actuator (S) in [%]: piezo deflection distance which correlates with the dispensed volume. A larger stroke results in a larger droplet. The effective S in pm can be calculated according t

[0082] • Stroke Velocity (SV) in [pm / ms]: velocity of the piston during displacement. Increasing SV will result in faster droplets. If it is too low, no droplet will tear off, if it is too high, satellite droplets may appear. Typical values range from 60-100pm / ms for water-based samples, 50- 80 pm / ms for solvents and > 150 pm / ms for viscous media.

[0083] • Drop Pitch (P) [mm]: distance between pixels as shown in Figure 6.1. P of 0,45mm for a ’’short” rMQR code corresponds to a real size of 12,15x4, 95mm2.

[0084] • X, Y and Z velocities [mm / s]: velocity of the axis movement. If the axis velocity is too fast compared to the ratio of P and actuation cycle duration, drops may be omitted during operation. Therefore, a series of experiments was conducted to test and understand these parameters. For the settings, dyes were chosen to print on the membrane.

[0085] Experimental Section

[0086] Instruments and Reagents: Nitrocellulose FF170HP membrane, 25><300mm was purchased from GE Healthcare (Parramatta, NSW, Australia) and backing card from Merck Millipore (Buchs, Switzerland). Black dyes PATIS-colour Lebensmittelfarbe are diluted with MilliQ water. The dye was printed on the membrane using the BioSpot Arc from BiofluidiX, tips 200S. QRQR app from DENSO permits to scan the printed rMQR code.

[0087] Fabrication: Three rMQR codes were printed with dyes: ”2”, ”13” modified corresponding to the ’’negative” pattern and ”16” modified corresponding to the ’’positive” pattern. The bmp files containing the rMQR code were generated thanks to the Python code. The tested parameter values are:

[0088] • P = 1, 0.5, 0.45, 0.43, 0.4, 0.38 and 0.35mm

[0089] • S = 15, 20, 40, 80 and 100%

[0090] • SV = 70, 80 and 90pm / ms

[0091] The constant parameters used are Z = 40mm, n = 1, f = 1Hz, X, Y and Z velocities = 25mm / s. Results and Discussion

[0092] Primarily, the ’’Print BMP Pattern” command aligns perfectly with the desired application. With only one command, the tips dispense droplets at the precise position where the black pixels are in the bmp file. Next, as S increases, the volume of dispensed droplets also increases. This observation is crucial as it directly impacts the clarity and legibility of the printed rMQR codes. When the S exceeds 70%, we encountered diffusion problems, resulting in a blurred rMQR code that is difficult to discern. On the other hand, when the S is between 10 and 25%, the dispensed volume is moderate, allowing for precise deposition without excessive spreading, solution Regarding SV, when it is too low (< 60 pm / ms), no droplets tear off, leading to incomplete printing. We did not observe significant differences in droplet formation between 80 and 90 pm / ms. However, at 80 pm / ms with a low S of 10%, the rMQR code is not printed straight. In the end, a SV of 90 pm / ms consistently produced the most reproducible results in experiments. Finally, when P is too large (20,7mm), the droplets / squares of the rMQR code are too far apart, and the reader is no longer able to scan the rMQR code. On the other hand, diffusion issues start to appear if P is too small (<0,35mm). Droplets are too close and merged, leading to an unreadable rMQR code. S must be reduced to avoid diffusion problems. Moreover, these results are influenced by the viscosity of the solution. Higher viscosity tends to mitigate diffusion problems or, at the very least, allows for smaller pitches or larger dispensed volumes, which can help maintain resolution and readability. For instance, when attempting to print with a much more diluted dye, diffusion problems occurred even at a relatively high P of 0.5mm and a S of 50%. This indicates that the lower viscosity of the dye solution caused the droplets to spread out more. After several trials, optimal parameters were found to ensure the rMQR code is scannable by the reader and that it is not too wide or long to be easily integrated onto an LFA:

[0093] 1. SV = 90pm / ms

[0094] 2. S = 20-10% 3.

[0095] P = 0.4-0.45 mm 49

[0096] B.2. rMQR codes printing with antibodies on LFA membrane

[0097] Experimental Section

[0098] Instruments and Reagents: hCG (human chorionic gonadotropin) active protein, mouse anti-a hCG monoclonal dAbs (detection antibodies), mouse anti-P hCG monoclonal cAbs (capture antibodies), and mouse anti-mouse IgG Abs (control antibodies), were all supplied from MyBioSource (California, USA). 40nm Au NPs (gold nanoparticles) were purchased from nanoComposix (California, USA). Phosphate buffer saline (PBS) (0.01 M, pH 7.4), sucrose, tween-20, tris, boric acid, hydroxide de sodium (NaOH) and bovine serum albumin (BSA), were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Water was treated with a Millipore (Bedford, USA) Milli-Q water purification system and was used throughout. Nitrocellulose Whatman FF80HP, FF170HP, and FF120HP membranes, 25><300mm, and sample pad, 22^300 mm, were purchased from GE Healthcare (Parramatta, NSW, Australia). Glass fiber membrane, 20x300mm, and absorbent pad, 20x300mm, and backing card were purchased from Merck Millipore (Buchs, Switzerland).

[0099] Fabrication of the LFA.

[0100] Membrane preparation: The anti-mouse IgG and the anti-P hCG monoclonal cAbs (capture antibodies or cAbs) are immobilized on the membrane and dried at 37°C for two hours using the BioSpot Arc from BiofluidiX (Freiburg, Germany).

[0101] Conjugate pad (CP) preparation: P-hCG dAbs (conjugate antibodies) are diluted with PBS and 20 mM borate buffer (pH = 8.0). Au NPs solution (10 O.D.) is added and mixed. BSA is included to obtain a 0.1% BSA solution, followed by centrifugation and removal of the supernatant. The solution is then resuspended in 1 mL of 20 mM borate buffer with 1% BSA (pH = 8.0). the conjugate antibody is resuspended in loading buffer (2 mM borate buffer at pH 7.0 with 5% sucrose) to achieve an O.D. of 1.85. 1 mL and is dispensed onto the conjugate pad and incubated in an oven for an hour at 38°C.

[0102] Assembly test assay: strips are laminated onto the backing card with 2mm intervals between each component (sample pad, CP, membrane and absorbent pad). hCG Solutions hCG solutions were prepared in running buffer (lx tris-HCl, 1,5% Tween20, 1% BSA) at ten hCG concentrations: 0, 0.2, 1, 5, 10, 20, 50, 100, 500, lOOOng / mL. 120pL of each solution were loaded onto the sample pad of each strip. Two replicates of each concentration were run.

[0103] Quantification: After 20 minutes, the colorimetric signal of the two lines on the strip was measured using a reading device or reader which utilizes high-precision camera optics for imaging the entire test strip. The measured signals are normalized by subtracting the mean background signal obtained next to the line. Due to the limitations of the reader in quantifying results for patterns and the variability in printing, each rMQR code was analyzed semi- quantitatively. The rMQR codes were scanned using the QR code scanning app of DENSO, and their resolution and readability were assessed visually. In the initial experiments, the modified ”13” rMQR code was printed with a CTRL cAbs concentration of Img / mL and an O.D. of 1,85 of the CP. Only the ’’negative” pattern was printed due to its cost-effectiveness, speed, and simplicity. This approach allows us to use only one channel with a single tip during printing, avoiding the need for test cAbs and enabling us to run the test with only buffer. Once the rMQR code was successfully scanned, the mask printed with test cAbs was added. Figures 5 and 6 illustrates a successful example including strips with rMQR codes for detecting hCG. Results and Discussion

[0104] During the initial tests, three primary challenges emerged:

[0105] - colour gradient of the signal: uneven distribution of colour across the printed rMQR code. The presence of cAbs over a long distance has a much more significant impact on the colour gradient compare to the decrease effect due to the flow rate as in standard LFA (as the capture line is placed further from the origin, the flow rate at which the analyte passes the capture reagent line is slower, resulting in a higher effective concentration of the analyte in the sample and thus an increased signal).

[0106] - weak colour intensity of the signal: poor contrast between the rMQR code and the background.

[0107] - lack of consistency: variability in the quality and readability of the printed codes between batches.

[0108] To address these challenges, we studied and optimized key parameters related to fabrication, run conditions, and scanning processes. For each experiment, one or two parameters were modified at a time to observe their impact on the identified issues and determine their dependencies. The constant parameters are presented in Table 1 For all experiments : Z = 40, n = 1, f = 1Hz, 200pm. The bmp files containing the rMQR code were generated by using Python code

[0109] Table 1 : constant parameters during rMQR code integration experiments

[0110] B.3. Reducing the signal color gradient of the rMQR codes

[0111] By decreasing the concentration and the volume of the dispensed antibodies on the membrane, printing or repeating the printing of the distal end of the rMQR code several times, reducing the length of the rMQR to be printed and using a "faster" membrane allowing less time for antigens (analytes) to bind at the beginning of the rMQR code, we have significantly reduced the signal colour gradient in the rMQR code for LFA. First, reducing the length of the rMQR code decreased the width of the cAb barriers, allowing dAbs to reach the end of the rMQR code more effectively by not being trapped at the beginning. The same concept is implemented by decreasing the amount of printed control and capture Abs. Second, printing the end of the rMQR code pattern twice or more with control and capture cAbs enhanced the cAb-dAb binding, thereby increasing signal intensity. Third, using a faster membrane facilitates the faster migration of analytes and conjugate antibodies, reducing the likelihood of said conjugate antibodies and analytes being trapped at the beginning of the rMQR code.

[0112] B.4. Improving the signal intensity and contrast of the rMQR codes

[0113] To successfully scan a two-dimensional barcode of type QR code, the contrast between its dark and light squares (features) is essential. A high contrast ratio ensures that these features stand out clearly and can be accurately interpreted by scanners. Factors such as ambient light, print quality and surface reflections can affect this contrast. Enhancing this contrast can improve scanning reliability and speed.

[0114] Using a slower membrane enhances the antigen-antibody interactions by providing more time for these reactions to occur, which leads to higher sensitivity across the rMQR code. Employing a camera flash during the scanning process ensures uniform illumination, which reduces shadows and enhances the contrast between the dark and light features of the rMQR code. Additionally, applying a larger volume of running buffer in multiple steps permits that all the conjugate antibodies move through the membrane and participate to the signal. Increasing the concentration of conjugate antibodies and control and capture antibodies further contributes to better signal contrast by amplifying the binding events. All these optimizations significantly increase the accuracy of the scanning and decoding processes.

[0115] B.5. Printing consistency between printings and batches

[0116] In order to scan the rMQR code effectively, several critical factors must be ensured. Firstly, there must be a strong contrast between the dark points and the background. Moreover, the printing process must be precise and aligned. The printed code should maintain consistent concentration of control and capture antibodies or bioreceptors for each point to avoid any variations that could affect the readability of the code. The printing must be performed accurately, without any omissions, ensuring that no points of the rMQR code are missing. This precision in printing is paramount to maintain the integrity and functionality of the rMQR code. Additionally, another challenge is to position the ’’mask” pattern on the ’’negative” patterned of dispensed control antibodies without having a mismatch so as to form the ’’positive” rMQR code. Optimizing printing parameters, using high-quality materials, and conducting regular calibration of equipment enhance the consistency of results.

[0117] To improve the consistency of the printing process, several key adjustments were implemented. Firstly, the SV (Stroke Velocity) was calibrated to match the viscosity of the fluid being used. This adjustment ensures a smoother and more controlled flow of the fluid, which is critical for precise printing. Secondly, the distance between the dispensing tip and the membrane (Z-axis) was decreased. This reduction minimizes the potential for fluid splash and position offset. Additionally, the axis velocities (X and Y axis) were reduced to ensure no points were omitted during printing. Lastly, the tip size was adapted to correspond with the volume aimed to print, ensuring that each printed spot was uniform and consistent.

[0118] Example 2 : rMQR code Lateral flow Assays detecting hCG

[0119] After the optimization of all the relevant parameters as described in Example 1, functioning LFAs was successfully produced. These tests consistently displayed ”Neg” when the sample was negative and ”Pos” when the sample had a concentration of 600 ng / mL. Figures 5A-B illustrates one of the exemplified LFAs comprising a membrane on which a two-dimensional barcode is printed. The two-dimensional barcodes have been printed according to the following parameters: Drop Pitch = 0,45mm, Stroke Velocity = 90pm / ms, Tips being : 125S, X, Y Velocities = 20mm / s and Z Coordinate = 40, and Gradient Printing Stroke = 80%. The concentration of the capture bioreceptors with affinity for alpha unit of hCG is l,5mg / mL and the concentration of control bioreceptors with affinity for the labelled bioreceptors is 0,3mg / mL. The concentration of the labelled bioreceptors with affinity for the beta unit of hCG or conjugate bioreceptors, labelled with gold nanoparticles and measured in optical density (O.D) is 6.0.

[0120] Summary of the optimal parameters: control rMQR code “Neg” (short) + Mask = positive rMQR code ”pos”

[0121] Tips 125S (One tip for all LFAs in this experiment)

[0122] S CTRL rMQR code = 80-90%

[0123] S Mask = 90-100%

[0124] SV = 90pm / ms

[0125] P = 0,45mm

[0126] FF80HP nitrocellulose membrane

[0127] Conjugate Pad: 5,5 - 6 O.D. 40nm Au NPs-P hCG

[0128] CTRL pattern Cone. = 0,25 mg / mL anti-mouse IgG

[0129] Mask pattern Cone. = 2 mg / mL a-hCG

[0130] Example 3 : rMQR code Lateral flow Assays detecting detecting fetal fibronectin: reducing anxiety in preterm birth monitoring with automated result interpretation and integrated medical support

[0131] Preterm birth, defined as delivery before 37 weeks of gestation, remains the leading cause of death for children under 5, with nearly 1 million deaths annually. Its etiology is multifactorial, involving maternal age, chronic conditions, infections, lifestyle factors, and pregnancy complications such as premature rupture of membranes. Premature delivery is associated with adverse outcomes, including cerebral palsy, sensory disabilities, and learning deficits, underscoring the urgent need for accurate and scalable diagnostic tools to mitigate its impact. Fetal fibronectin (fFN) is a glycoprotein normally present in high concentrations until 22 weeks of gestation, elevated fFN levels beyond this period are associated with increased preterm birth risk. The LFIA's limit of detection is established at an fFN concentration of 50 ng / mL (see Figure 7). The integration of QR codes into LFIAs for fFN detection offers advancements over traditional diagnostic methods. First, self-tests are non-invasive, involving painless collection of vaginal secretions, enabling frequent monitoring without hospital visits. They deliver rapid, cost-effective results, essential for timely preterm birth intervention, particularly in resource-limited settings. QR code LFIA approach addresses maternal anxiety by providing clear, immediate results and streamlined communication with healthcare providers, a key consideration during pregnancy. In our study, developing the rMQR code LFIAs detecting fFN also permits to validate our approach with another clinically relevant protein showing its versatility and flexibility. The probit curve showing the proportion of positive scans according to the fFN concentrations is shown in Figure 7, with a calculated C50 value of 6.73 ng / mL. As with QR code LFIAs detecting hCG, the C50 value in QR code-based assays could be tuned by adjusting fabrication parameters, although this was not demonstrated in this study.

[0132] Example 4 : QR Code LEIA performance with human samples

[0133] To further validate the robustness and verify its practical applicability, we conducted experiments using urine samples from pregnant women and vaginal secretions positive for fFN. These biological matrices introduce additional complexities, such as non-specific interactions and physical property variations, which are critical to assess for real-world deployment of the technology.

[0134] For hCG experiments, five replicates per urine sample were run, with a total of six positive and two negative samples tested. The results demonstrated consistent performance of the QR code LFIAs, with no degradation in QR code scannability compared to buffer conditions. For negative samples, the unspecific binding signal seems less present compared to buffer conditions. The tests were scanned with a sensitivity of 100 % in less than 5 minutes (Table 2).

[0135] Table 2: Summary of scanned results of rMQR code LFIAs detecting hCG in urine samples. Table 2 presents the number of tests that were scanned as positive or negative for each urine sample (N = 5 independent assays per sample). Fabrication parameters: gradient printing, capture antibody concentration = 2.0 mg / mL, conjugate pad optical density = 5.5

[0136] Regarding fFN, one or two replicates per vaginal secretion sample were run, with a total of two positive and three negative samples tested. As for hCG, the results were consistent with a sensitivity of 100 % (Table 3).

[0137] Table 3: Summary of scanned results of rMQR code LFIAs detecting fFN in vaginal secretions. Table 3 presents the number of tests that were scanned as positive or negative for each vaginal secretion sample which are diluted in a running buffer (N = 1 or 2 independent assays per sample). Fabrication parameters: gradient printing, capture antibody concentration = 2.0 mg / mL, conjugate pad optical density = 6.0.

Claims

Claims1. A method of manufacturing a membrane for lateral flow assay or rapid diagnostic assay, on which a two-dimensional barcode of reagent dots including a certain information associated to an analyte in a liquid sample is patterned, said method comprising the steps of:- generating a negative two-dimensional barcode including a certain information associated to the absence of said analyte in the liquid sample, by way of a computer program;- generating a positive two-dimensional barcode including a certain information associated to the presence of said analyte in the liquid sample, by way of said computer program;- generating a mask two-dimensional barcode by computing pixel difference between the negative two-dimensional barcode and the positive two-dimensional barcode by way of said computer program;- printing a visual representation of the negative two-dimensional barcode on the porous membrane by dispensing at least one control reagent;- printing a visual representation of the mask two-dimensional barcode by dispensing at least one capture reagent over the as-printed visual representation of the negative two- dimensional barcode on said porous membrane.

2. The method according to claim 1, wherein the step of generating a positive two- dimensional barcode by computer and / or the step of generating a mask two-dimensional barcode by computer is repeated.

3. The method according to claim 2, wherein the visual representation of each computed mask two-dimensional barcode being different from one to the other is printed by dispensing at least one capture reagent over the as-printed visual representation of the negative two- dimensional barcode on said porous membrane and / or over the as-printed previous visual representation of a mask two-dimensional barcode on the porous membrane.

4. The method according to to any one of the preceding claims, wherein the step of generating a negative two-dimensional barcode comprises the steps of:- generating a first native two-dimensional barcode including a certain information associated to the absence of said analyte in the liquid sample, by way of a computer program;- generating a second native two-dimensional barcode pattern including a certain information associated to the presence of said analyte in the liquid sample by way of said computer program;- reworking the first native two-dimensional barcode so as to match white pixels between the second native two-dimensional barcode and the first native two-dimensional barcode.

5. The method according to any one of the preceding claims, wherein the step of generating a positive two-dimensional barcode comprises the step of:- reworking the second native two-dimensional barcode so as to match the black pixels between the negative two-dimensional barcode and the second native two-dimensional barcode by way of the computer program.

6. The method according to any one of the preceding claims further comprising a step of generating a positive test two-dimensional barcode by computing the pixel addition from the mask two-dimensional barcode or each different mask two-dimensional barcode to the negative two-dimensional barcode by way of a computer program.

7. The method according to any one of the preceding claims, further comprising a step of storing the negative two-dimensional barcode and the positive test two-dimensional barcode in a database for subsequent verification of test results by way of controlling computer program.

8. The method according to any one of the preceding claims, further comprising, a step of converting the negative two-dimensional barcode and the mask two-dimensional barcode or each different mask two-dimensional barcode into a printable visual representation of full and empty squares or white and black squares by way of computer prior to the printing.

9. The method according to claim 8, wherein each full or black square on the visual representations is processed in the printing as a reagent dot to be printed.

10. The method according to any one of the preceding claims, wherein the step of printing a visual representation of the mask two-dimensional barcode over the as-printed visual representation of the negative two-dimensional barcode and / or over the as-printed previousvisual representation of a mask two-dimensional barcode by dispensing the at least one capture reagent is performed so as to prevent dispensing capture reagent dots on the as-printed control reagent dots on the membrane and / or the as-printed previous capture reagent dots on the membrane.

11. The method according to any one of the preceding claims, wherein the visual representations of the negative two-dimensional barcode and the mask two-dimensional barcode are printed so as to form a concentration gradient of the control bioreceptors and the capture bioreceptors in the printed two-dimensional barcode.

12. The method according to any one of the preceding claims, further comprising one or more steps of repeating printing the visual representation of the negative two-dimensional barcode and / or one or more steps of repeating printing the visual representation of the mask two-dimensional barcode so as to form a concentration gradient of the at least one control reagent and / or of the at least one capture reagent in the two-dimensional barcode formed by the reagent dots being printed.

13. The method according to any one of the preceding claims, wherein the visual representations of the control and / or the mask two-dimensional barcodes are printed by using at least two control reagents having different concentrations of control bioreceptors and / or at least two capture reagents having different concentrations of capture bioreceptors.

14. The method according to any one of the preceding claims, wherein the two- dimensional barcodes are selected from rectangular two-dimensional barcodes or square two- dimensional barcodes.

15. The method according to any one of the preceding claims, wherein the two- dimensional barcodes are selected from QR codes, micro QR codes or rMQR codes.

16. The method according to any one of the preceding claims, wherein the control reagent comprises control bioreceptors having affinity for free labelled bioreceptors with affinity for the analyte and / or control bioreceptors having affinity for free labelled bioreceptors without affinity for the analyte, and the capture reagent comprises capture bioreceptors having affinity for the analyte.

17. A test device for detecting an analyte in a liquid sample comprising a membrane manufactured by a method according to any one of claims 1 to 16 on which a plurality of reagent dots are arranged in form of a two-dimensional barcode, wherein said reagent dots are dots of at least one control reagent and dots of at least one capture reagent, said at least one control reagent comprising bioreceptors having affinity for free labelled bioreceptors with affinity for the analyte and / or bioreceptors having affinity for free labelled bioreceptors without affinity for the analyte, and said at least one capture reagent comprising bioreceptors having affinity for the analyte; and wherein the reagent dots of the two-dimensional barcode are visible after completion of the test so as to display the layout of the two-dimensional barcode, in which a certain information associated with the analyte is embedded.

18. The test device according to claim 17, wherein the certain information associated to the analyte comprises information associated with the presence or absence of the analyte in the sample and / or information associated with the identification of the test device and / or the test.

19. The test device according to any one of claims 17-18, characterized in that, when the analyte is absent or is not detected, the control reagent dots of the two-dimensional barcode are visible after completion of the test so as to display the layout of a negative two-dimensional barcode20. The test device according to any one of claims 17-19, characterized in that, when the analyte is present or is detected, the control reagent dots and the capture reagent dots of the two-dimensional barcode are visible after completion of the test so as to display the layout of a positive test two-dimensional barcode.

21. The test device according to any one of claims 17-20, further comprising a structure with a plurality of zones comprising a sample zone (1) for applying the liquid sample, a reaction zone (2) comprising labelled bioreceptors with affinity for the analyte, a detection zone (3) comprising the membrane and an absorbent pad (4), the zones being configured to enable lateral flow of the liquid sample from the sample zone to the absorbent pad being at the distal end of the structure.

22. The test device according to any one of claims 17-21, wherein the two-dimensional barcode of reagent dots comprises a concentration gradient of the at least one control reagent and / or the at least one capture reagent.

23. The test device according to claim 22, wherein the membrane is positioned so that the lowest concentration of the at least one control reagent and / or the at least one capture reagent in the two-dimensional barcode of reagent dots is on the side of the reaction zone (2) and the highest concentration of reagent is towards the absorbent pad (4).

24. The test device according to any one of claims 22-23, wherein the concentration gradient of the at least one control reagent and / or the at least one capture reagent is sequential.

25. The test device according to any one of claims 17-24, wherein the reagent dots arranged in form of a two-dimensional barcode printed on the membrane are dots of one, two or more control reagents and / or dots of two or more capture reagents.

26. The test device according to any one of claims 17-25, comprising a second or more two-dimensional barcodes of reagent dots patterned or dispensed on the membrane, wherein the reagent dots are selected from control reagent dots.

27. Use of a test device according to any one of claims 17-26 for detecting an analyte in a sample, wherein the analyte is associated with signs and symptoms of an altered physiological state or with health monitoring.

28. The use according to claim 27, wherein the sample is selected from a sample selected from body fluids.

29. The use according to any one of claims 27-28, wherein the impaired physiological state is a disease selected from sexually-transmitted diseases, viral diseases, cancers, breast cancer, colorectal cancer, lung cancer, cervical cancer, endometrial cancer, ovarian or testicular cancer, skin cancers, prostate cancer, thrombosis, cardiovascular disease.

30. The use according to any one of claims 27-29, wherein the analyte is associated with the monitoring of pregnancy.

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