diagnostic devices

Abstract

Diagnostic devices, such as lateral flow devices, are described. The devices comprise a microporous phase material and a capture reagent covalently attached to a capture zone of the microporous phase material, the capture reagent comprising a covalent linking moiety, the covalent linking moiety comprising an electromagnetic radiation (EMR)-activated crosslinker having an EMR-activated functional group, wherein the capture reagent is covalently attached to the capture zone via the electromagnetic radiation-activated functional group, and the capture reagent is capable of binding to an analyte of interest. The capture reagent may comprise (i) a first linker moiety, which may be an oligonucleotide such as an aptamer, the covalent linking moiety being located at the 5' or 3' end of the linker moiety, and / or a detection reagent comprising a second linker moiety, the detection reagent being capable of binding to an analyte of interest.

Description

[Technical Field]

[0001] The present invention relates to diagnostic devices, particularly lateral flow devices, methods of making such diagnostic devices, and methods of using such devices. [Background technology]

[0002] A variety of membranes, including nitrocellulose, cellulose, cellulose acetate, polyethersulfone, polyvinylidene fluoride, and nylon, have been used as media for biomolecular analysis (Tonkinson & Stillmann 2002). The requirements for biomolecular analysis media are as follows: I. Having a surface that can immobilize target molecules almost quantitatively. II. Allowing for long-term storage of immobilized molecules. III. Allowing molecules in solution to interact with the immobilized molecules. IV. Do not interfere with signal detection strategies.

[0003] Nitrocellulose has emerged as one of the most widely used membranes due to its large volume-to-surface ratio, high binding capacity, reproducibility, and ease of processing. Applications of nitrocellulose include lateral flow diagnostics, conventional blotting, high-throughput arrays, immunodiagnostics, and mass spectrometry related proteomics applications.

[0004] Lateral flow devices (LFDs) using line deposition are one of the most successful analytical platforms for on-site detection of single biomarkers. In its most common and widely used form, the method is based on the interaction of an analyte / antigen with a selective antibody, forming a complex that can be easily visualized using gold nanoparticles without the need for a reader. LFDs represent a paradigm shift from sample-to-lab to lab-to-sample, aiming to improve decision-making, reduce response times, and reduce the burden on healthcare systems.

[0005] Nitrocellulose has been widely used in the development of lateral flow devices for the immobilization of capture reagents. When proteins are applied to nitrocellulose, they are almost instantly adsorbed through both hydrophobic (van der Waals forces) and hydrophilic (hydrogen bonding) interactions (Buck et al., 2001; EMD Millipore, 2013). However, this passive adsorption strategy is ineffective for small molecules (<1000 Da), such as aptamers, drugs, hormones, small peptides, and nucleic acid polymers, which are washed away when applied to the membrane (Buck et al., 2001). Therefore, alternative approaches for conjugating small molecules to nitrocellulose and other membranes must be found.

[0006] To overcome this challenge, streptavidin has been used to immobilize biotin-functionalized small molecules onto nitrocellulose membranes (Li et al., 2018). However, the use of streptavidin potentially limits the functionality of small molecules due to steric hindrance, potentially reducing the ability to densely pack small molecules onto nitrocellulose. Furthermore, the use of streptavidin potentially negates the benefits of using small molecules such as aptamers. UV crosslinking of nucleic acids functionalized with poly-T termini has also been used to immobilize nucleic acids onto nitrocellulose (Bruno et al., 2014). Unfortunately, the UV light used (254 nm) has the potential to crosslink nucleic acid-based small molecules, thereby reducing their functionalization capabilities.

[0007] Therefore, there is a need to develop approaches that allow for the conjugation of small molecules while retaining the advantages of using small molecules as target recognition elements. Summary of the Invention

[0008] The present invention addresses several problems of the prior art. The inventors have developed a method for manufacturing a lateral flow device that overcomes many of the problems of the prior art while allowing target recognition molecules to be densely packed onto a microporous substrate such as nitrocellulose.

[0009] The inventors have shown that the combination of photoreactive chemistry for covalently attaching capture reagents to microporous phase materials such as those used in lateral flow devices, with capture reagents and / or detection moieties provided with oligonucleotide spacers, can be used to provide directional control of the capture stack and detection moieties, allowing for high-density packing of detection moieties bound to analytes of interest and concentrating the readout signal obtained from ultra-small volume microarray formats.

[0010] According to a first aspect of the present invention, 1. A diagnostic device comprising: a microporous phase material; and a capture reagent covalently bound to a predetermined capture zone of the microporous phase material, the capture reagent comprising a covalently binding moiety, the covalently binding moiety comprising an electromagnetic radiation (EMR) activated crosslinker having an electromagnetic radiation (EMR) activated functional group, the capture reagent being covalently bound to the capture zone via the electromagnetic radiation activated functional group, the capture reagent being capable of binding to a target analyte; (i) the capture reagent further comprises a first linker moiety, and the covalent attachment moiety is located at the 5' or 3' end of the first linker moiety; and / or (ii) the diagnostic device comprises a detection reagent comprising a second linker moiety, the detection reagent being capable of binding to the analyte of interest; Either each linker moiety comprises or consists of either an oligonucleotide moiety, a hydrocarbon chain, or an oligonucleotide moiety linked to a hydrocarbon chain; A diagnostic device is provided.

[0011] According to a second aspect of the present invention there is provided a method of manufacturing a diagnostic device, the method comprising: (a) providing a microporous substrate; (b) contacting a capture reagent with the microporous substrate, the capture reagent comprising a linker moiety and a covalently attached moiety at a terminus of the linker moiety, the covalently attached moiety comprising an electromagnetic radiation (EMR)-activatable functional group, each linker moiety comprising or consisting of either an oligonucleotide moiety, a hydrocarbon chain, or an oligonucleotide moiety linked to a hydrocarbon chain; (c) exposing the microporous substrate to a source of electromagnetic radiation, wherein the exposure covalently binds the capture reagent to the capture zone of the microporous substrate.

[0012] According to a third aspect of the present invention, there is provided a capture reagent comprising a linker moiety and a covalent binding moiety at an end of the linker moiety, the covalent binding moiety comprising an electromagnetic radiation (EMR)-activatable functional group, and the linker moiety comprising or consisting of either an oligonucleotide moiety, a hydrocarbon chain, or an oligonucleotide moiety linked to a hydrocarbon chain.

[0013] Any suitable covalently bonded moiety having an EMR-activatable functional group may be used in the present invention. In one embodiment, the functional group of the covalently bonded moiety is photoactivatable, e.g., activated by visible or ultraviolet (UV) light, depending on the functional group used. However, the inventors have found that deposition efficiency can be significantly improved by using electromagnetic radiation, particularly long wavelengths. As used herein, long wavelengths refer to wavelengths greater than 270 nm. In certain embodiments, wavelengths in the UVB region (280-320 nm) or UVA region (320-400 nm) are used. In certain embodiments, wavelengths in the UVA region (330-365 nm) are used.

[0014] Examples of photoactivatable functional groups and derivatives thereof that can be used in the covalent attachment moiety include anthraquinone, aryl azide, phenyl azide, diazirine, and psoralen groups.

[0015] In certain embodiments of the present invention, each capture reagent comprises an oligonucleotide moiety in addition to a covalent linkage moiety, with the covalent linkage moiety attached to its 5' or 3' end either directly or via a spacer molecule. The oligonucleotide molecule can be of any suitable length. In preferred embodiments, the oligonucleotide molecule comprises 6 to 70 nucleotides, e.g., 6 to 50 nucleotides, 10 to 40 nucleotides, 15 to 30 nucleotides, or 18 to 25 nucleotides. When a spacer molecule is used, any suitable spacer molecule can be used. Typically, the spacer molecule comprises or consists of a hydrocarbon chain, such as a C2 to C50 hydrocarbon chain, a C2 to C20 hydrocarbon chain, a C2 to C12 hydrocarbon chain, a C3 to C9 hydrocarbon chain, etc. In one embodiment, the length of the spacer molecule is in the range of 13 to 55 angstroms. In another embodiment, the length of the spacer molecule is in the range of 18 to 40 angstroms.

[0016] As noted above, linker molecules comprising hydrocarbon chains, such as linear hydrocarbon chains, can be used in place of or in combination with oligonucleotide moieties.

[0017] In certain embodiments of the invention, the oligonucleotide moiety of the capture reagent is capable of binding to the target analyte, hi one embodiment, the oligonucleotide moiety is capable of binding to the target nucleic acid analyte via base-pairing complementarity with the nucleic acid sequence of the target analyte, i.e., hybridization with the analyte sequence.

[0018] In other embodiments, the oligonucleotide moiety can bind to the target analyte by shape or molecular complementarity. In one example of such an embodiment, the oligonucleotide moiety is an aptamer or a biomimetic thereof. The biomimetic aptamer used in the present invention has a designed binding site specific to the target analyte, and binds the analyte to the aptotope recognized by the corresponding aptamer, preferably a biomimetic. In one embodiment, the oligonucleotide moiety is an aptamer. In one example of such an embodiment, the aptamer is a SOMAmer.

[0019] In certain embodiments of the invention, the capture reagent comprises an antibody molecule having binding specificity for an analyte of interest. In embodiments in which the capture reagent comprises an oligonucleotide moiety and an antibody molecule having binding specificity for an analyte of interest, the antibody molecule is attached, preferably to an Fc region, e.g., a CH3 domain, at the 5' or 3' end of the oligonucleotide molecule, with the other end of the oligonucleotide molecule attached to a covalent moiety.

[0020] In embodiments in which the capture reagent comprises an antibody molecule having binding specificity for the analyte of interest but does not comprise an oligonucleotide moiety, the covalent moiety is preferably attached to the Fc region of the antibody molecule, e.g., the CH3 domain. Attachment via the Fc region allows for better directionality and dense packing.

[0021] A particular advantage of embodiments of the capture reagents, diagnostic devices, and methods of the present invention is that by using capture reagents comprising linker molecules, such as oligonucleotide moieties with covalent attachment moieties located at the 5' or 3' end of the oligonucleotide moiety, multiple capture moieties can be deposited in a low volume at high concentrations in a nearly uniform orientation, with the oligonucleotide moiety free to bind to the analyte of interest. This allows for the use of thin lines and small spots of capture reagents in lateral flow devices.

[0022] In certain embodiments of the present invention, the diagnostic device further comprises a detection reagent capable of specifically binding to the analyte. The detection reagent used in the invention can be any suitable one. The detection reagent preferably comprises a detectable moiety such as gold nanoparticles. However, any suitable detection reagent can be used. These can include nanoparticles such as quantum dots, nanocellulose, gold nanorods, gold nanoshells, etc. Further examples of suitable detection reagents include europium chelate-based fluorescent nanoparticles (e.g., Cy5, FAM, lanthanide, selenium, cellulose nanobeads).

[0023] In certain embodiments, the detection reagent comprises an antibody molecule having binding specificity for the analyte of interest.

[0024] In some embodiments of the present invention, instead of or in addition to the capture reagent comprising an oligonucleotide portion, the diagnostic device may comprise a detection reagent having an oligonucleotide portion. Similar to the oligonucleotide portion of the capture reagent (herein referred to as the first oligonucleotide), the oligonucleotide contained within the detection portion (herein referred to as the second oligonucleotide) may also contribute to the directionality of analyte detection, allowing for densification and concentration of the readout signal.

[0025] In such embodiments, the second oligonucleotide molecule may have a detectable particle, such as a gold nanoparticle, attached to the 5' or 3' end of the oligonucleotide molecule. In certain embodiments, the detection reagent comprises an antibody molecule having binding specificity for the analyte of interest, and the second oligonucleotide molecule is attached to the CH3 domain of the antibody molecule.

[0026] The second oligonucleotide itself may have binding specificity for the target analyte. In some such embodiments, such as when the analyte is a nucleic acid and the second oligonucleotide has binding specificity for the nucleic acid analyte, the second oligonucleotide may bind via base-pairing complementarity with the nucleic acid sequence of the target analyte, i.e., hybridization with the analyte sequence. In other embodiments of the present invention, the second oligonucleotide portion may be capable of binding to the target analyte, such as a protein, through shape or molecular complementarity. In such embodiments, the second oligonucleotide portion may be an aptamer.

[0027] The second oligonucleotide portion can be of any suitable length. In preferred embodiments, like the first oligonucleotide, the second oligonucleotide molecule can comprise 6 to 70 nucleotides, e.g., 10 to 40, 15 to 30, or 18 to 25 nucleotides.

[0028] As shown in the Examples, unique advantages in analyte detection are demonstrated when a capture reagent and a detection reagent are combined, where one capture reagent and detection reagent comprises an aptamer with binding specificity for the analyte, and the other capture reagent and detection reagent comprises an antibody molecule with binding specificity for the analyte. Examples of embodiments with such hybrid configurations are shown schematically in Schemes 9 and 10 of Figure 2.

[0029] In certain embodiments in which the detection moiety comprises an antibody molecule but does not comprise an oligonucleotide moiety, the detectable moiety, such as a gold nanoparticle, is preferably attached to the Fc region of the antibody molecule, eg, the CH3 domain of the antibody molecule.

[0030] These advantages make the capture and detection reagents particularly suitable for use in multiplexed devices. Thus, in certain embodiments of the present invention, the diagnostic device is a multiplexed device. In one such embodiment, the diagnostic device comprises multiple capture zones, and the capture reagent bound to one or more capture zones is different from the capture reagent bound to one or more other capture zones. In one such embodiment, the diagnostic device comprises multiple capture zones, and the capture reagent bound to each capture zone is different from the capture reagent bound to each of the other capture zones. In such embodiments, the capture reagent in each capture zone may have a different binding specificity for the analyte of interest than the capture reagent in each of the other capture zones. Thus, the multiplexed device may be used to determine the presence of two or more different analytes / biomarkers.

[0031] A particular advantage of the device and method of the present invention is that two or more different types of analytes can be detected in the same device by using two or more different capture reagents. For example, in one embodiment, the capture reagent in at least a first capture zone has binding specificity for a nucleic acid analyte, and the capture reagent in at least a second capture zone has binding specificity for a protein analyte. In such an embodiment, the capture reagent in the first capture zone can bind to the nucleic acid analyte via nucleic acid hybridization, and the capture reagent in the second capture zone can be an aptamer and can bind to the protein analyte via aptamer-protein interaction.

[0032] In the methods of the present invention, the capture moiety can be deposited onto the capture zone using any suitable technique, for example, using a manual or automated dispensing system. In one embodiment, the capture reagent is deposited in a volume sufficient to produce a color signal associated with the detection of the analyte that is visible to the naked eye.

[0033] As described herein, the inventors have shown that the present invention allows for the use of surprisingly small volumes of capture reagent compared to those used in conventional lateral flow devices. This enables multiplexed embodiments of the invention in which multiple different capture reagents are used to detect different analytes within the same device. It also enables embodiments in which multiple samples with different concentrations of the same capture reagent are used to allow quantitation of the amount of analyte in a test sample.

[0034] For example, in one embodiment, the capture reagent is deposited in a volume ranging from 0.5 to 25 nanoliters, e.g., 1.0 to 20.0 nanoliters, e.g., 2.0 to 12.0 nanoliters, or 3.0 to 8.0 nanoliters, in a spot having a diameter of 100 to 500 nm. In one embodiment, the capture reagent is deposited in a volume ranging from 4.0 to 6.0 nanoliters.

[0035] In certain embodiments, for example when the capture reagent comprises an antibody molecule, each spot is filled with a capture reagent having a concentration of 140 μg / μm 2~1400μg / μm 2 , e.g., 300 μg / μm 2 ~1000μg / μm 2 , e.g., 500 μg / μm 2 ~800μg / μm 2 The capture reagent may be provided in the range of

[0036] When the diagnostic device is used with an automated reader that does not rely on the naked eye to determine the signal or color change associated with binding of the analyte to the capture moiety, smaller volumes of capture moiety may be used. In such embodiments, the capture reagent may be deposited in small volumes, such as picoliters, e.g., 5 picoliters, 10 picoliters, 25 picoliters, 50 picoliters, or 100 picoliters. In such embodiments, the capture reagent may be deposited in picoliter volumes ranging from 5 to 800 picoliters, e.g., 10 to 500 picoliters, 25 to 250 picoliters, or 25 to 150 picoliters.

[0037] For example, in certain embodiments, such as when the capture reagent comprises an oligonucleotide, such as an aptamer, the capture reagent may be deposited in picogram amounts, such as in the picogram range, e.g., 8 picograms, 10 picograms, 12 picograms, 25 picograms, or 50 picograms. In such embodiments, the capture reagent may be deposited in picogram amounts ranging from 5 to 10,000 picograms, e.g., 5 to 5,000 picograms, 5 to 1,000 picograms, 5 to 500 picograms, 8 to 250 picograms, 8 to 100 picograms, 8 to 50 picograms, or 8 to 20 picograms.

[0038] In embodiments in which the capture reagent comprises an aptamer, the volume of capture reagent required is typically significantly less than when the capture reagent comprises an antibody molecule.

[0039] In some embodiments, for example when the capture reagent comprises an oligonucleotide, for example when the capture reagent comprises an aptamer, the capture reagent is provided at a concentration of 0.140 μg / μm 2 ~140μg / μm 2The amount of the deposited material can be in the range of 1000 to 10 ... For example, each spot contains 0.140 μg / μm 2 ~140μg / μm 2 , 1 μg / μm 2 ~70μg / μm 2 , 10 μg / μm 2 ~50μg / μm 2 , 20 μg / μm 2 ~40μg / μm 2 , or 28 μg / μm 2 ~35μg / μm 2 The capture reagent may be provided at a concentration ranging from 0.1 to 0.5.

[0040] Given the directional high-concentration / low-volume deposition of capture reagents enabled by the devices and methods of the present invention, multiplexed devices of the present invention may comprise a sufficient number of different capture moieties to allow the detection of more than two different analytes. For example, in one embodiment, up to 5, e.g., up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, or up to 75 different capture moieties may be used in a single multiplexed device to distinguish between different analytes, hi one embodiment, up to 80 different capture moieties may be used in a single multiplexed device to distinguish between up to 80 different analytes.

[0041] Optionally, to ensure that the capture reagent is retained in a predetermined area of ​​the predetermined capture zone, regions of the microporous substrate other than the predetermined capture zone can be masked to prevent activation of the EMR-activated functional groups in such zones other than the capture zone, thereby limiting covalent binding of the capture reagent to the predetermined capture zone. Masking can be achieved using any suitable mask. The mask can be made of any suitable material capable of preventing electromagnetic radiation from reaching the surface of the microporous membrane. For example, materials that can be used to make the mask include, but are not limited to, paper, plastic materials, metal materials, polymeric materials, foil, etc. The mask typically includes one or more sections, such as gratings, through which electromagnetic radiation can pass, allowing for focused irradiation of one or more predetermined capture zones with the electromagnetic radiation.

[0042] To remove capture reagent remaining in zones other than the capture zone, the method of the present invention may include a step of washing the non-covalently bound capture reagent from the microporous substrate. The washing step may be included during the manufacturing process of the diagnostic device or before the diagnostic device is used by a user. For example, the membrane may be washed by either spraying with a buffer or by immersing in a buffer with or without agitation. If included before diagnostic use, a buffer may be applied to the diagnostic device before applying the sample. Any suitable washing buffer may be used. For example, suitable buffers may include phosphate buffer, borate buffer, or Tris buffer. The buffer may contain suitable additives, such as a non-ionic surfactant, e.g., Tween 20, and / or a protein or polymeric substance, e.g., BSA.

[0043] Any suitable type of electromagnetic radiation capable of activating EMR-activatable functional groups may be used in the present invention. In one embodiment, the electromagnetic radiation is visible light. In another embodiment, the electromagnetic radiation is ultraviolet (UV) light. In yet another embodiment, the electromagnetic radiation is electron beam radiation.

[0044] In particularly preferred embodiments of the present invention, radiation having wavelengths in the UVB spectrum (280-320 nm) or UVA spectrum (320-400 nm) is used. In particular embodiments, UVA wavelengths in the range of 330-365 nm are used.

[0045] As noted above, a particular advantage of the present invention is the ability to precisely control the deposition of capture moieties into the capture zone, thereby achieving fine capture zones with high concentrations of capture moieties, enabling the fabrication of highly sensitive multiplexed devices.

[0046] In the diagnostic devices of the present invention, the capture zone may be of any suitable shape.

[0047] In one embodiment, the capture zone can be a dot. The dot can be of any shape. In such an embodiment, the area of ​​the dot can correspond to the area of ​​a circular dot having a diameter of 100 to 500 μm, e.g., 200 to 400 μm, e.g., 250 to 300 μm. In one embodiment, the dot is circular. The dot-shaped capture zones can be arranged in an array. In such an array, the kerf value (gap between adjacent spots) can be in the range of 50 to 200 μm. Alternatively or additionally, in such an array, the pitch (center-to-center distance between adjacent dots) can be 2.1 to 3 times the diameter. As an example, the dots in the array can have a diameter of 300 μm, a pitch of 750 μm, and a kerf of 150 μm.

[0048] In one embodiment of the method of the present invention, when the diagnostic device is a multiplexed device, the method comprises: (a) contacting a first capture reagent with the microporous substrate, the first capture reagent comprising an oligonucleotide portion and a covalently attached moiety at a 5' or 3' end of the oligonucleotide portion, the covalently attached moiety comprising an electromagnetic radiation (EMR)-activated functional group; (b) applying a first mask to the microporous substrate, the first mask having a grating corresponding to a first capture zone of the microporous substrate; (c) exposing the first capture zone to a source of electromagnetic radiation, wherein a mask prevents exposure of areas of the microporous substrate other than the first capture zone to the electromagnetic radiation, wherein the exposure covalently bonds the first capture reagent to the first capture zone of the microporous substrate via the EMR-activated functional group; (d) removing the first mask and, if necessary, washing unbound first capture reagent from the microporous substrate; (e) contacting a second capture reagent with the microporous substrate, the second capture reagent comprising an EMR-activated functional group; (f) applying a second mask to the microporous substrate, the second mask having a grating corresponding to a second capture zone of the microporous substrate; (g) exposing the second capture zone to a source of electromagnetic radiation, wherein the mask prevents exposure of areas of the microporous substrate other than the second capture zone of the microporous substrate to the electromagnetic radiation, wherein the exposure covalently bonds the second capture reagent to the second capture zone of the microporous substrate via the EMR-activated functional group of the second capture reagent; (h) removing the second mask and, if necessary, washing any unbound second capture reagent from the microporous substrate; (i) repeating steps (e)-(h) for the third and subsequent capture reagents, if desired, using a specific mask for each capture zone; may include:

[0049] A fourth aspect of the present invention provides a diagnostic device manufactured by the method of the second aspect of the present invention.

[0050] In the present invention, the microporous substrate may be formed from any suitable material through which the test sample can diffuse. In certain embodiments of the present invention, the microporous substrate comprises nitrocellulose.

[0051] In one embodiment, the diagnostic devices of the invention and diagnostic devices manufactured by the methods of the invention are lateral flow devices. In another embodiment, the diagnostic devices are flow-through assay devices. In another embodiment, the diagnostic devices are microfluidic diagnostic devices.

[0052] In one embodiment, the diagnostic device is a lateral flow device comprising at least one capture zone and may additionally comprise, for example, one or more of an absorbent pad downstream of the capture zone, a control zone downstream of the capture zone, and a quenching zone upstream of the capture zone.

[0053] The diagnostic devices of the present invention can be used to detect any suitable analyte, or combination of analytes if the diagnostic device is a multiplexed device. For example, analytes of interest can include antigenic substances, antibodies, proteins, amino acids, nucleic acids, haptens, bacteria or components thereof, viruses, virus particles, hormones, metabolites, or antibodies to any of the above substances.

[0054] The analyte of interest may be indicative of an infectious or non-infectious disease. Thus, the diagnostic device may be used for prognostic or diagnostic purposes. The analyte of interest may be indicative of response to treatment (companion diagnostic), either before or after treatment has begun.

[0055] In certain embodiments of the invention, the capture zone comprises a reagent specific for an analyte of interest indicative of Ebola virus.

[0056] In other particular embodiments of the invention, the capture zone comprises a reagent specific for an analyte of interest indicative of a coronavirus, e.g., the SARS-CoV-2 virus.

[0057] In embodiments of the present invention, the reaction between the reagent and the analyte of interest can result in a detectable color change. In one embodiment, the color change in one or more capture zones can be non-proportional and can indicate only the presence or absence of a particular analyte. However, in some embodiments, the color change in one or more capture zones can be proportional to the analyte concentration in the test sample. Alternatively, the color change in one or more capture zones can be non-proportional and can indicate only the presence or absence of a particular analyte. Indeed, the inventors are the first to demonstrate that a diagnostic device, such as a lateral flow device, can be used to quantitatively determine the amount of an analyte in a particular sample.

[0058] Thus, in a fifth aspect of the present invention, there is provided a diagnostic device comprising a microporous phase material, a capture reagent bound to a capture zone of the microporous material, the capture reagent for binding to an analyte of interest in a test sample, and a calibration zone comprising a plurality of different reference concentrations of a calibration molecule of the analyte of interest, wherein in use the concentration of the analyte of interest in the test sample bound to the capture reagent in the capture zone can be determined by comparing the signal produced by binding of the analyte of interest in the test sample in the capture zone with the signal produced by one or more calibration molecule concentrations in the calibration zone.

[0059] The calibration molecule can simply be a predetermined concentration of the analyte of interest. In other embodiments, the calibration molecule can be, for example, a predetermined concentration of a capture moiety known to bind to a particular concentration of the analyte. For example, the calibration zone may include multiple reference concentrations of capture moieties (e.g., aptamers), and the conjugate pad may be preloaded with a conjugate of a detection molecule and an analyte of interest (e.g., a gold nanoparticle-aptamer-analyte conjugate). In use, when a sample is added to the conjugate pad, the conjugate flows into the calibration zone and binds to the capture moieties in the calibration zone. The concentration of the analyte in the test sample may then be determined by reference to the signal generated in the calibration zone. In one embodiment, signals generated by multiple calibration molecules in the calibration zone may be used to generate a calibration curve, against which a signal generated by an analyte in the test sample bound to a capture reagent in the capture zone may be compared, allowing the concentration of the analyte in the test sample to be determined, if desired.

[0060] The diagnostic device of the fifth aspect of the invention may comprise any of the features of the diagnostic device of the first aspect, and vice versa.

[0061] A sixth aspect of the present invention provides a method for detecting the presence of an analyte in a sample, the method comprising contacting a sample comprising the analyte with a diagnostic device of the present invention or a diagnostic device manufactured by a method of the present invention, and detecting a colour change in the capture zone.

[0062] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0063] [Figure 1A] FIG. 1 shows examples of photoreactive functional groups that can be used to generate capture reagents for binding to microporous phase materials such as nitrocellulose. [Figure 1B] FIG. 1B shows specific examples of compounds having photoreactive functional groups that can be used to generate the capture reagent of the present invention shown in FIG. 1A. [Figure 2] FIG. 1 shows a schematic representation of various combinations of capture reagents, analytes and detection (sensor) molecules described in connection with the present invention. [Figure 3]Figure 1 shows a schematic diagram of an example of click chemistry (strain-promoted alkyne-azide cycloaddition, or SPAAC) used to link dibenzocyclooctyne (DBCO) to an azide-labeled oligonucleotide. In this example, a sensor antibody is first reacted with DBCO-NHS-ester and then coupled to an azide-oligonucleotide. The resulting antibody-oligonucleotide conjugate is then hybridized to gold nanoparticles functionalized with complementary oligonucleotides, enabling the generation of site-specific, directional sensors for LF devices. [Figure 4] FIG. 1 shows a schematic representation of the anthraquinone and SANPAH synthesis chemistry used to obtain 5′-modified oligonucleotide captures, including aptamers, compatible with ultra-low volume deposition and UV-mediated directional immobilization onto porous membranes such as nitrocellulose for LF device fabrication. [Figure 5] Figure 1 shows examples of UV-visible spectra showing SPR (surface plasmon resonance) shifts indicating successful functionalization of 40 nm AuNPs functionalized with appropriate ligands. (A) AuNPs functionalized with an antibody-oligo conjugate for CRP antibody. (B) AuNPs functionalized with oligo18S single-stranded DNA. (C) AuNPs functionalized with the aptamer SOMA16. (D) AuNPs functionalized with IP10 antibody. In each spectrum, a rightward shift in the absorption maximum around 523 nm is observed. In each case, a schematic of the final AuNP sensor is shown to the right of the spectrum. [Figure 6] Figure 1 shows an example of dynamic light scattering (DLS) analysis demonstrating the increase in hydrodynamic diameter of 40 nm AuNPs after functionalization with appropriate ligands. (A) AuNPs functionalized with an antibody-oligo conjugate for CRP antibody. (B) AuNPs functionalized with oligo 18S single-stranded DNA. (C) AuNPs functionalized with an aptamer SOMAmer. (D) AuNPs functionalized with sTREM1 antibody. The increase in diameter is indicated by a rightward shift in DLS signal intensity. In each case, a schematic of the final AuNP sensor is shown to the right of the DLS graph. [Figure 7]Example of LF strip showing titration to determine optimal size deposition for oligonucleotide capture, comparing (A) AQ chemistry and (B) SANPAH chemistry. (C) Schematic of hybridization-based detection using AuNPs functionalized with antisense oligosensors. [Figure 8] (A) Microarray layout showing deposition patterns of SANPAH and AQ-oligo capturers with 12.5 nL volume drops as dark circles, and pitch (no deposition) as dotted white circles. (B) LF strip showing hybridization-based detection results using AuNPs functionalized with antisense oligo sensors. (C) Schematic of the interaction mode. [Figure 9] (A) Microarray layout showing the deposition pattern of SANPAH oligo capture by 6 nL drop volume, as shown in red circles, and pitch (no deposition), as shown in white circles. This configuration allows multiplexing of up to 50 detections, in addition to four reference spots at each corner. (B) LF strip showing hybridization-based detection results using AuNPs functionalized with antisense oligo sensors. (C) Schematic of the interaction mode. [Figure 10] (A) Microarray layout showing the deposition pattern of target biomarker / VLPs dispensed at various concentrations in 6.0 nL volumes at each defined location. (B) Microarray layout showing the deposition pattern of SOMA43-AQ / VLP capture dispensed at various concentrations in 6.0 nL volumes at each defined location. Guide spots / assay validation spots were deposited at positions 1A, 1L, 6A, and 6L. (C) and (D) LF strip results after 20 minutes of reaction. Each interaction mode is shown with a corresponding schematic diagram. [Figure 11](A) Microarray layout showing SOMA-IP10 / SANPAH as capture reagent, dispensed in 6.0 nL volumes at positions 3C, 3E, 3G, and 3I, and recombinant monoclonal antibody-IP10 as capture reagent, deposited at positions 6B, 6D, 6F, and 6H. Guide spots / assay validation spots were deposited at positions 1A, 1J, 10A, and 10J. (B) LF strip results after 20 minutes of reaction. Each interaction mode is illustrated by a corresponding schematic diagram. [Figure 12] (A) Microarray layout showing the deposition pattern of target biomarker / IP10 at various concentrations in triplicate at each defined location in 6.0 nL volumes. Guide spots / assay validation spots were deposited at locations 1A, 1J, 10A, and 10J. (B) LF strip showing titration results between sensor IP10 antibody and target biomarker after 20 minutes of reaction. (C) Linear regression analysis showing dose-response from titration. (D) Schematic of interaction pattern. [Figure 13] Figure 1 shows the LF cassette design and microarray volumes, highlighting features that allow for detection and quantification of signals from calibration standards (STDs) and test samples (TSs). The red line indicates the physical separation between the two channels dedicated to either STDs or TSs, avoiding signal interference between Area A, which is used to read recombinant biomarker standards, and Area B, which is used to read sample signals. The reader acquires the resulting LF images, and custom software generates standard curves for each biomarker from the data in Area A. The software then analyzes the data in Area B by averaging the LF signals from multiple volumes of each capture reagent to calculate the exact concentration of each analyte biomarker under investigation. [Figure 14]Figure 1 shows the experimental design to further demonstrate the quantitative capability and molecular hybridization of multiplexed LFD using nucleic acid-based reagents as the interaction modality with SANPAH-oligo as the capture and AuNP-thiol-antisense oligo as the detector. (A) Microarray deposition of capture oligos at different concentrations for standard curve generation. (B) Microarray deposition showing the simulated test sample analysis design, in this example with known concentrations but requiring experimental validation. (C) and (D) LF strips after assay development. Reference guides were used for technical assay validation and accurate detection and quantification of signals from each LF assay. [Figure 15] Analysis of data from the experiment shown in Figure 14. Mean intensity values ​​from sciREADER LF1 shown in panel (A) were analyzed in GraphPad Prism, and dose-response curves were generated from three LF assay replicates as shown in panel (B). CV was less than 4.0%. Graphs in panels (C) and (D) show linear regression analysis in the low and mid-concentration ranges, with coefficients of determination R-squared of 0.9 and 0.8, respectively. Experimental validation of unknown concentrations is shown overlaid with arrows, as illustrated in panels B and C. DETAILED DESCRIPTION OF THE INVENTION

[0064] (definition) Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0065] Throughout this specification, unless the context otherwise requires, terms such as "comprises" or "including", or variations such as "comprises", "comprising", "includes", or "including", should be understood to imply the inclusion of the stated element or elements but not the exclusion of other elements or elements.

[0066] As used herein, terms such as "a," "an," and "the" include both the singular and the plural unless the context clearly requires otherwise.

[0067] As used herein, the term "consisting essentially of" means that the invention necessarily includes the recited items and permits the inclusion of unrecited items that do not materially affect the basic and novel characteristics of the invention.

[0068] In one aspect, the present invention provides a diagnostic device comprising: a microporous phase material; and a capture reagent covalently attached to a predetermined capture zone of the microporous phase material, the capture reagent comprising a covalently attached moiety, the covalently attached moiety comprising an electromagnetic radiation (EMR) activated crosslinker having an electromagnetic radiation (EMR) activated functional group, the capture reagent being covalently attached to the capture zone via the electromagnetic radiation activated functional group, the capture reagent being capable of binding to a target analyte; (i) the capture reagent further comprises a first linker moiety, and the covalent attachment moiety is located at the 5' or 3' end of the first linker moiety; and / or (ii) the diagnostic device comprises a detection reagent comprising a second linker moiety, the detection reagent being capable of binding to the analyte of interest, each linker moiety comprising or consisting of either an oligonucleotide moiety or a hydrocarbon chain (e.g., a linear hydrocarbon chain), or an oligonucleotide moiety linked to a hydrocarbon chain (e.g., a linear hydrocarbon chain).

[0069] The device may be used in any suitable field, for example, human health, veterinary fields, food science, environmental science, etc. Capture of target recognition molecules may be achieved by conjugating functionalized target recognition molecules to capture reagents bound to the microporous phase material.

[0070] The use of diagnostic devices of the present invention and diagnostic devices manufactured according to the present invention allows for improved diagnostic performance and stability. Covalent attachment to the microporous substrate improves the stability of the capture reagent attachment to the device, improving stability under humidity fluctuations and long-term storage compared to prior art devices. Covalent attachment of the capture reagent to the microporous phase material using electromagnetic radiation allows for precise control of the direction, volume, and area of ​​deposition of the capture moiety on the microporous phase material, allowing for improved diagnostic performance.

[0071] (Microporous base material) In the present invention, the microporous substrate can be formed from any suitable material through which the test sample can be diffused. In a specific embodiment of the present invention, the microporous substrate is a nitrocellulose-based material. The nitrocellulose-based material can comprise nitrocellulose alone or a mixture of nitric acid and other acids, such as mixed esters of aliphatic C1-C7 carboxylic acids. Other materials suitable for use in lateral flow devices can also be used in the present invention. Further examples of materials that can be used to form the microporous substrate include, but are not limited to, cellulose acetate, paper, polymers such as vinyl chloride, vinyl chloride-propylene copolymer, vinyl chloride-vinyl acetate copolymer, polyacrylamide film, polyester sulfone material, nylon, etc. The choice depends on the means for attaching the capture reagent to the microporous substrate.

[0072] (Capture Reagent) The capture reagents of and for use in the present invention comprise a covalent moiety that, upon activation by electromagnetic radiation, covalently binds to a capture zone of a diagnostic device via an electromagnetic radiation (EMR)-activatable functional group.

[0073] In certain embodiments of the present invention, each capture reagent comprises, in addition to the covalent linking moiety, a linker moiety that comprises or consists of an oligonucleotide moiety, a hydrocarbon chain, or an oligonucleotide moiety linked to a hydrocarbon chain. When the linker moiety comprises a hydrocarbon chain, the chain can typically comprise or consist of a C2-C50 hydrocarbon chain, e.g., a C2-C20 hydrocarbon chain, e.g., a C2-C12 hydrocarbon chain, or a C3-C9 hydrocarbon chain. In one embodiment, the hydrocarbon chain is a linear, unbranched hydrocarbon chain. In one embodiment, carbons in the hydrocarbon chain can be substituted with oxygen or nitrogen atoms. In one embodiment, the hydrocarbon chain is unsubstituted. In one embodiment, such a hydrocarbon chain can be composed of or consist of ethylene glycol monomers, i.e., PEG monomers. Other suitable linkers can be based on, for example, enzyme-cleavable linkers (peptide-like linkers, pyrophosphate, carbohydrates), acid-cleavable linkers (PEGylated or hydrazone linkers), etc.

[0074] In one embodiment of the invention, the length of the linker ranges from 8 to 70 angstroms, such as from 10 to 55 angstroms, for example from 15 to 40 angstroms.

[0075] In a particularly preferred embodiment of the invention, the linker moiety is an oligonucleotide moiety.

[0076] Thus, in certain embodiments of the invention, each capture reagent comprises an oligonucleotide moiety in addition to a covalent binding moiety, which is attached to the 5' or 3' end of the oligonucleotide moiety either directly or via a linker (spacer) molecule.

[0077] In such embodiments, an oligonucleotide moiety capable of specifically binding to the target analyte can be used in the capture reagent. Suitable oligonucleotide molecules that can be used include, but are not limited to, single-stranded nucleic acids, double-stranded nucleic acids, aptamers, or SOMAmers®. SOMAmers® are single-stranded deoxyoligonucleotides in which the dU residues are uniformly functionalized with a moiety (e.g., benzyl, 2-naphthyl, 3-indolylcarboxamide) at the 5-position. SOMAmers® are subjected to in vitro selection from large random libraries, similar to aptamers (Kraemer et al. 2011).

[0078] In one embodiment, the binding of the oligonucleotide to the analyte of interest is by base pairing with the nucleic acid sequence of the analyte of interest, i.e., hybridization with the analyte sequence. In other embodiments, the oligonucleotide moiety can bind to the analyte of interest through shape or molecular complementarity. In such embodiments, the oligonucleotide moiety can be an aptamer or a biomimetic thereof. In one embodiment, the oligonucleotide moiety is an aptamer. In one embodiment, the aptamer is a SOMAmer.

[0079] Any suitable covalent linking moiety having an EMR-activatable functional group can be used in the present invention. In one embodiment, the functional group of the covalent linking moiety is photoactivatable and is activated by visible or ultraviolet (UV) light, depending on the functional group used. In certain embodiments, the functional group of the covalent linking moiety is photoactivatable and is photoactivated by light greater than 270 nm, e.g., light in the UVA or UVB spectrum. Examples of photoactivatable functional groups and their derivatives that can be used in the covalent linking moiety include anthraquinone, aryl azide, phenyl azide, diazirine, psoralen groups, and the like. Examples of usable photoactivatable functional groups are shown in Figure 1.

[0080] In one embodiment, the EMR-activated functional group comprises a photoactivatable anthraquinone moiety. In one embodiment, the covalent binding moiety comprises anthraquinone C2-dT.

[0081] In another embodiment of the invention, the EMR-activated functional group comprises a 6-(4'-azido-2'-nitrophenylamino)hexanoate group. In one embodiment, the capture reagent can be generated by reaction of the N-hydroxysuccinimide ester moiety of the Sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate) reagent with an amino group of a linker group attached to an oligonucleotide.

[0082] The reaction of an N-hydroxysuccinimide ester moiety with an amino group of a linker group attached to an oligonucleotide can also be used to generate alternative capture moieties with other functional groups. Thus, in one embodiment, a capture reagent of the invention can be generated by reacting an N-hydroxysuccinimide ester moiety of an EMR-activated functional group-containing reagent with an amino group of a linker group attached to an oligonucleotide.

[0083] (EMR activation) As noted above, in embodiments of the present invention, the capture reagent is attached to the microporous substrate via a functional group that is activated by electromagnetic radiation.

[0084] Examples of photoactivatable functional groups that can be used for covalent attachment include anthraquinone, aryl azide, phenyl azide (including orthohydroxyphenyl azide, metahydroxyphenyl azide, tetrafluorophenyl azide, orthonitrophenyl azide, and metanitrophenyl azide), diazirine, psoralen group, etc. Examples of these functional groups are shown in Figures 1A and 1B. Methods for photoactivation of such reagents are well known to those skilled in the art; see, for example, Koch et al., Bioconjugate Chem. 2000, 11, 474-483.

[0085] In one embodiment, the functional group comprises a photoactivatable anthraquinone moiety.

[0086] In one such embodiment, the covalently bonded moiety comprises anthraquinone-2-sulfonic acid.

[0087] In another such embodiment, the covalently attached moiety comprises anthraquinone C2-dT. A schematic diagram of a capture reagent comprising anthraquinone C2-dT groups is shown in Figure 4. For reference only, the anthraquinone C2-dT groups within the circles are shown attached to the 5' end of a first oligonucleotide and the 3' end of a second oligonucleotide. In the diagnostic devices of the present invention, the capture reagent comprises only a single oligonucleotide, and the covalently attached moiety is located at either the 5' or 3' end of the oligonucleotide.

[0088] FIG. 1B shows non-limiting examples of compounds having the photoreactive functional groups shown in FIG. 1A that can be used to generate capture reagents of the present invention.

[0089] Figure 1C illustrates the use of one of these compounds, Sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate), to prepare a capture reagent of the invention. The figure shows that a capture reagent can be generated by reaction of the N-hydroxysuccinimide ester moiety of the Sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate) reagent with an amino group of a linker group attached to an oligonucleotide.

[0090] (electromagnetic radiation) In the present invention, any suitable type of electromagnetic radiation and, if necessary, its wavelength that enables activation of the EMR-activatable functional group can be used. In one embodiment, the electromagnetic radiation is visible light. In another embodiment, the electromagnetic radiation is ultraviolet (UV) light. In yet another embodiment, the electromagnetic radiation is electron beam irradiation. Other electromagnetic radiation that can be used includes radioisotopes such as X-rays, laser light, neutron beams, positively charged beams, alpha rays, beta rays, and gamma rays. Those skilled in the art can select the appropriate type of electromagnetic radiation and wavelength depending on the properties of the EMR-activatable functional group.

[0091] For example, simple phenyl azides generally require short-wavelength UV light (e.g., 254 nm, 265-275 nm) to be efficiently activated, whereas long-wavelength UV light (e.g., 300-460 nm) is sufficient for nitrophenyl azides. Because short-wavelength UV light can damage other molecules, nitrophenyl azides are usually preferred for crosslinking experiments. Diazirines are a new class of photoactivatable chemical groups used in crosslinking and labeling reagents. The diazirine (azipentanoate) moiety has higher photostability than phenyl azide groups and is more easily and efficiently activated with long-wavelength UV light (330-370 nm).

[0092] The inventors have found that particularly good results are achieved in binding of the capture reagent to a microporous phase material such as nitrocellulose when the capture reagent comprises or consists of an oligonucleotide, such as when the capture reagent comprises an aptamer.

[0093] In some embodiments of the invention, the electromagnetic radiation has a wavelength greater than 280 nm. In some such embodiments, the electromagnetic radiation is ultraviolet B (UVB) or ultraviolet A (UVA). In particular embodiments of the invention, the electromagnetic radiation has a wavelength of 330-370 nm, e.g., 350-370 nm, e.g., 365 nm. The inventors have found that this wavelength range minimizes damage to the oligonucleotides and aptamers in the capture reagent.

[0094] (mask) In certain embodiments of the present invention, deposition of capture reagents and / or EMR activation of covalently attached capture moieties on the microporous phase material may be performed using masking to focus the covalent attachment to specific capture zones.

[0095] The mask used in the present invention can be made of any suitable material capable of preventing electromagnetic radiation from reaching the surface of the microporous membrane. For example, materials that can be used to make the mask include, but are not limited to, paper, plastic materials, metal materials, polymer materials, foil, etc. The mask usually has one or more sections, such as gratings, through which electromagnetic radiation can pass, thereby enabling focused irradiation of one or more predetermined capture zones with the electromagnetic radiation. A mask with an appropriate number and shape of gratings is applied between the nitrocellulose membrane and the electromagnetic radiation source. The type of mask selected can depend on the number of test and control lines that the final diagnostic device should have.

[0096] (Lateral flow device) The diagnostic devices of the present invention and diagnostic devices manufactured using the present invention may be lateral flow devices, an example of which is shown in FIG. 13. In such diagnostic devices, a microporous substrate may define a detection zone, within which one or more capture zones are included, and capture moieties are covalently attached to the surface of the microporous substrate so that capture reagents do not diffuse through the microporous substrate outside of the capture zones. Each detection zone may include two or more distinct capture zones (e.g., lines, dots, etc.). The capture zones may be any shape. However, typically, the capture zones are line-shaped, which may be substantially perpendicular to the flow of the test sample through the assay device. In other embodiments, the capture zones may have other shapes, such as dot-shaped, or may be line-shaped parallel to the flow of material through the microporous substrate.

[0097] The test sample is typically applied to the sample pad of a lateral flow type diagnostic device, from which it diffuses through the microporous substrate to the detection zone and capture zone therein, where the analyte of interest can bind to the capture moiety. To aid in the flow of the test sample, the diagnostic device may include an absorbent pad downstream of the detection zone.

[0098] Optionally, the device may further comprise a conjugate pad between the sample pad and the detection zone. Various reagents specific to the analyte of interest may be present on the conjugate pad, which bind to the analyte of interest as it migrates through the conjugate pad, either by causing a chemical or structural change in the analyte that enables it to bind to or react with a capture moiety in the capture zone, or by conjugating the analyte with a reagent that can specifically bind to the capture moiety in the capture zone. In an alternative embodiment, instead of separate sample and conjugate pads, the sample pad may comprise one or more reagents specific to the analyte of interest, which bind to the analyte of interest as it migrates through the sample pad, either by causing a chemical or structural change in the analyte that enables it to bind to or react with a capture moiety in the capture zone, or by conjugating the analyte with a reagent that can specifically bind to the capture moiety in the capture zone.

[0099] The device of the present invention may further include a control zone. Preferably, the control zone is located downstream of the detection zone. The control zone may include one or more reagents that react with components of the test sample or components from the conjugate pad to generate a detectable signal, thereby indicating that a sufficient volume of the test sample has passed through the lateral flow device or that the test has been completed. Reagents suitable for use in the control zone will be apparent to those skilled in the art and will depend on factors such as the nature of the test sample, and may include, but are not limited to, antigens, antibodies, aptamers, single- or double-stranded nucleic acids, peptides, haptens, proteins, etc.

[0100] The device of the present invention may further comprise a quenching zone. The quenching zone may comprise a quencher capable of removing contaminants from the test sample that may interfere with the accuracy of detection in the detection zone. The quencher used will depend on the type of sample and the target antigen. These may include buffers such as phosphate buffer, borate buffer, or carbonate buffer. Additives such as non-ionic surfactants or polymers may also be added to the quenching zone. If present, the quenching zone is preferably located upstream of the detection zone, and may be located upstream of the conjugate pad, if present.

[0101] The diagnostic device of the present invention may optionally include an analyte concentration calibration zone. Such a zone may include multiple predetermined concentrations of calibration molecules, and the signals of these may be compared to the signal generated by the analyte of interest to determine the concentration of the analyte of interest. If the diagnostic device is a multiplexed device detecting multiple analytes, the calibration zone may include multiple concentrations of calibration molecules, each different for each analyte of interest. In one embodiment, the multiple predetermined concentrations of calibration molecules are multiple predetermined concentrations of the analyte of interest. In another embodiment, the multiple predetermined concentrations of calibration molecules are multiple predetermined concentrations of aptamers flowing from a conjugate pad that, when reacted with an indicator (e.g., AUNP)-aptamer-antigen complex, generate reference signals indicative of different standard concentrations of the analyte of interest. In one embodiment, the calibration zone is located adjacent to or parallel to the detection zone for the analyte of interest. A buffer pad may be provided for applying a sample buffer that flows through the microporous substrate into the calibration zone, and is optionally positioned adjacent to the sample pad. In one embodiment, during use, a test sample is applied to the sample pad and allowed to diffuse to the detection zone, and a sample buffer (without the test sample) is applied to the buffer pad and allowed to diffuse to the calibration zone, allowing the calibration molecule, e.g., a reference analyte, to be detected. For a particular analyte, the concentration of the analyte in the test sample can be estimated or determined by comparing the signal of the reference concentration of the calibration molecule to the signal for that analyte at the detection zone.

[0102] In one embodiment, the concentration can be assessed visually. Alternatively, a lateral flow device reader or a smartphone equipped with appropriate software can be used to assess the concentration of the analyte of interest by comparison with the signal of the calibration zone. For example, such a reader or smartphone can be programmed to generate a calibration curve from the calibration molecule signals of the calibration zone, thereby assessing the concentration of the analyte of interest at the detection zone.

[0103] Furthermore, while such embodiments may be particularly useful for diagnostic device embodiments of the present invention in which the capture reagent comprises an oligonucleotide moiety and a covalently attached moiety at the 5' or 3' end of the oligonucleotide moiety, such calibration zones may also be used in other types of lateral flow devices in which the capture reagent is an antibody. [Example]

[0104] Example 1 (material and method)

[0105] (reagent) Custom oligonucleotides containing aptamers were synthesized by a third party using solid-phase synthesis with the phosphoramidite method. The oligonucleotides were specified by the supplier to contain either anthraquinone-C2-dT or sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate) at the 5' end. The oligonucleotides were purified by analytical RP-HPLC. Prior to deposition, the oligonucleotides were redissolved in 0.2 M MgCl2 and 0.2 M NaCl to a final concentration of 100 μM. For aptamer-specific applications, DNA was used as supplied at concentrations ranging from 100 to 300 μM. The redissolved oligonucleotides / aptamers were stored at -20°C.

[0106] Custom SOMAmer (Slow Off-Rate Modified Aptamer) reagents were synthesized by SomaLogic with specified 5'-end modifications using photoreactive anthraquinone or SANPAH for capture reagents and 5'-thiol modifications for gold nanoparticle sensor generation. SOMAmers were supplied with a certificate of analysis and were reconstituted and stored according to the supplier's recommendations. DBCO-PEG4-NHS ester (BP-22288) was purchased from BroadPharma. Azide-free recombinant antibodies against BSA and CRP (ab270347), Mx1 (ab290698), sTREM1 (ab253319), and IP10 (ab253846) were purchased from Abcam. A gold conjugate kit (40 nm, 20 OD, ab154873) was purchased from Abcam. The recombinant protein CRP used as the analyte was obtained from Sigma (SRP6267-100UG). Recombinant MX1 (ATGP2826) and sTREM1 (ATGP1771) were obtained from NKMAXBIO. Recombinant IP10 (10768-HNAE) was purchased from Sino Biological. Recombinant Ebola virus-like particles (EBOLA VLPs) were purchased from IBT BIOSERVICES (0550-001). Molecular Probes™ DMSO, Anhydrous (13406448) was purchased from Fisher Scientific. Tris(2-carboxyethyl)phosphine hydrochloride, TCEP (51805-45-9) was purchased from MERCK. HEPES (1M) buffer solution, Gibco (15630-080) was purchased from ThermoFisher Scientific. Bovine serum albumin, BSA (9048-46-8) was purchased from Sigma. 20X PBS buffer, Thermo Scientific (28348) was obtained from ThermoFisher Scientific. Tween 20, Ultrapure, Thermo Scientific (J20605.AP) was purchased from ThermoFisher Scientific. 1 M MgCl2 (AM9530G) was obtained from ThermoFisher.2 M KCl (AM9640G) was obtained from ThermoFisher. Nitrocellulose membranes, Whatman FF120 HP (10547001), conjugate pad Fusion 5 or ST 17 membranes, Whatman CF7 dipstick pads and papers (8117-2250), and back cards were obtained from Cytiva Life Science.

[0107] (Deposition onto nitrocellulose membrane) Ultra-microvolume dispensing, 50–1.0 nanoliters, was performed using, for example, the sciFLEXARRAYER S3 system from SCIENION, UK, or the AD1520 Aspirate / Dispense system from BioDOT, UK. The deposition protocol followed the in-house approved SOP 007.01, "Start-up Procedure for the BioDot AD1520 Dispensing System," SOP 008.01, "Software Setup Procedure for the BioDot AD1520 Aspirate Dispense System," and SOP 009.01, "Cleaning and Shutdown Procedure at the End of Dispense for the BioDot Dispensing System."

[0108] (UV irradiation) After deposition, the nitrocellulose membrane was placed in a UVILink CL 508 Crosslinker (UVITec, UK) and irradiated with UV light at 365 nm for 10 minutes. The membrane was then wrapped in aluminum foil and stored in a vacuum chamber with a moisture absorbent at room temperature under controlled humidity of 11–14%.

[0109] (Functionalization of thiol oligos / aptamers or antibodies onto gold nanoparticles (AuNPs)) Aptamer / oligonucleotide labeling to AuNPs (40 nm diameter) was performed using a microwave-assisted heating method (Mengqi Huang et al., Nat Com. https: / / doi.org / 10.1038 / s41467-022-28627-8) according to our in-house approved SOP 001.01 (Reduction of thiol-modified oligonucleotides with TCEP) and SOP 002.01 (Gold nanoparticle labeling with thiol-oligoaptamers via microwave heating), with modifications. Conjugation of recombinant antibodies to oligonucleotides using DBCO-azide ligation was performed as described by Gong et al. (Bioconjugate Chem. 2016, 27, 217-225).

[0110] Alternatively, we used the protocols and reagents associated with Abcam's 5' Feature Barcode antibody conjugate kit, Lightning-Link®, oligos 1-10, ab270703. For sensor fabrication, after successful antibody-oligo synthesis, we performed hybridization to link AuNPs pre-functionalized with the corresponding complementary oligos. The AuNP-oligo-antibody sensors were further purified by sequential centrifugal washes. For direct conjugation of recombinant antibodies to AuNPs, we used the protocols and reagents associated with Abcam's gold conjugate kit (40 nm, 20 OD) ab154873. In-process quality control of AuNP labeling was performed by UV-visible spectral analysis using an NP80 IMPEN NanoPhotometer® spectrophotometer and dynamic light scattering measurement of the hydrated diameter of AuNPs using a DynaPro® NanoStar®, Wyatt / Watters Technology. Prior to loading onto the conjugate pad, the functionalized AuNPs were prepared in binding buffer (50 mM HEPES buffer pH 7.0, 150 mM NaCl, 0.25% Tween-20, 10% sucrose, 2.5% BSA, 5 mM MgCl2, 5 mM KCl) and dried at 37 °C for 1.5 h before being incorporated into the LFD strips.

[0111] Preparation of Lateral Flow Device (LFD) Strips The test strips consisted of a conjugate pad, a nitrocellulose (NC) membrane (FF120 HP) with deposited capture reagents (oligonucleotides / aptamers, antibodies, and target recombinant biomarkers), an absorbent pad, and an adhesive pad. The conjugate pad (Whatman Fusion 5) was first immersed in 20 mM HEPES buffer pH 7.0, 150 mM NaCl, and 0.05% Tween-20, then dried at 37 °C for 1.5 h before being loaded with functionalized AuNPs as sensors at a predetermined OD525 nm concentration. The LFD strips, consisting of the NC membrane, conjugate pad, and absorbent pad, were attached to an adhesive card with a 3 mm overlap. Finally, the LFD strips were cut to the appropriate size, wrapped in aluminum foil to block light, and stored at room temperature in a vacuum chamber under controlled humidity of 11–14%.

[0112] (Lateral flow (LF) assay) All LF assays were performed at room temperature. For sandwich-format-based detection, the running buffer consisted of 50 mM HEPES buffer pH 7.0, 150 mM NaCl, 0.1% Tween-20, 5 mM MgCl, 5 mM KCl, and the target biomarker / analyte present at the indicated concentration. For direct interactions, the LF assay was performed in the absence of the target biomarker / analyte already present as a deposited capture, followed by a 15-minute wash step with 200 μL of 1× PBS, 0.05% Tween-20.

[0113] (result) Example 1 (In-process quality control of AuNP sensors) We have employed various approaches to generate AuNP sensors with different ligands. For the directed and site-specific conjugation of AuNPs to single-stranded DNA oligos, including aptamers, we utilized the high-affinity interaction between the thiol (SH) groups present in the 5'-oligos to mediate and functionalize the AuNPs using a microwave-assisted method (Figures 3 and 4).

[0114] To conjugate the detection antibody to the oligo, we first reacted the antibody with DBCO-NHS-ester, and then linked the antibody-DBCO to an azide-labeled oligo via DBCO (Figure 3). The resulting antibody-oligo could be hybridized at room temperature in an approximately equimolar ratio with AuNPs already functionalized with complementary thiol-oligos (Figure 3). Non-directed functionalization of AuNPs was achieved by linking the antibody to carboxylated AuNPs via lysines using NHS / EDC chemistry.

[0115] After functionalization of the AuNPs, we used two independent methodologies to monitor the successful generation of AuNP sensors. First, we used UV-visible spectroscopy, which allows monitoring of the SPR (surface plasmon resonance) shift, indicating that the AuNPs were successfully and stably functionalized with the appropriate ligands. As shown in the spectra in Figure 5, a rightward shift in the absorption maximum around 523 nm can be clearly seen in all spectra when compared to the supplied control AuNPs.

[0116] Second, we analyzed the size distribution of the functionalized AuNPs using dynamic light scattering (DLS). Figure 6 shows an example of a DLS measurement comparing the diameter (nm) of each sensor with that of the as-received (i.e., control) 40 nm-AuNPs. DLS analysis clearly demonstrated an increase in the diameter of the AuNPs after functionalization with all ligands.

[0117] In this section, we used two independent approaches to monitor some of the physical properties of AuNPs and showed that sensors with different conjugate configurations met predetermined quality control criteria, such as maintaining colloidal properties, exhibiting an SPR shift under UV-visible testing, and exhibiting a relative increase in diameter compared to the supplied control AuNPs. Therefore, these sensors are ready for functional analysis in the LF assay.

[0118] Example 2 (Titration to determine optimal droplet size deposition on nitrocellulose membrane) We performed deposition titrations using volumes of 100, 50, 25, 12.5, 6.25, 3.0, and 1.25 nL on a SCIENION sciFLEXARRAYER S3 system or, alternatively, a BioDot BioDot AD1520 to compare the deposition of SANPAH-oligos and AQ-oligos after UV irradiation and LF strip development using hybridization-based detection as the interaction mode (Figure 7). Prior to deposition, the nucleic acid capture reagent stock was diluted 1:1 with 0.2 μm-filtered 0.2 M MgCl2, 0.2 M NaCl solution to avoid potential clogging of the Piezo Dispensing Capillaries (PDC).

[0119] The results in Figure 7 demonstrate that a clear visible signal can be obtained with both AQ and SANPAH capture oligos even with a deposition volume as small as 1.25 nL (equivalent to 0.15 pmol of oligo). Visual evaluation of the LFD strips indicated that the SANPAH chemistry appeared to perform slightly better than the AQ chemistry. This may be explained by the difference in the length of the spacer arm separating the photoreactive moiety from the AQ or SANPAH: approximately 8.0 Å and 18 Å, respectively. The longer spacer arm in SANPAH may be more suitable for avoiding potential steric hindrance, thus allowing for directional immobilization of the capturer more freely available for interaction with the antisense-functionalized AuNP sensor. Based on this visual evaluation, we chose to use either a 12.5 or 6 nL deposition volume for further experiments, including the simulation of multiplexed detection in a microarray format with up to 50 spot volumes.

[0120] Example 3 (Microarray deposition simulating LFD multiplexing) We performed 12.5 nL depositions in a microarray format using a SCIENION sciFLEXARRAYER S3 system, as shown in Figure 8A. We determined that the spot size was approximately 300 nm in diameter. We also determined that a pitch size of 750 nm was optimal to achieve optimal separation between spots. This avoided interference and bleeding between spots during development of the LF strips. As shown in Figure 8A, one strip contained 20 spots corresponding to SANPAH-oligo capturers, a second strip contained 20 spots of AQ-oligo capturers, and a third strip containing a combination of both oligos contained 10 spots of AQ-oligo capturers in the first four columns and 10 spots of SANPAH-oligo capturers in columns 5–8 following columns 1–4.

[0121] The LF strip results in Figure 8B demonstrate successful hybridization-based detection between capture and sensor oligos. Signal onset for this microarray multiplex detection was achieved within the first 2 minutes after applying the running buffer to the conjugate pad. Based on visual evaluation of the spot color intensity, both AQ and SANPAH chemistries performed equally well. The results were recorded in a time-lapse video, available upon request. This demonstrates that LFD assays can be achieved very quickly when combining optimal directional deposition, spot size deposition, and staggered spot placement within the microarray.

[0122] The precision afforded by ultrasmall volume and directional deposition of capture oligos, along with the flexibility to configure them into various shapes and forms of microarray layouts, allowed us to deposit the capture oligos in a manner that could reveal meaningful signal readouts, such as capture or analyte names or symbols. The results of this experiment are shown in Figure 8B, displaying the initials for anthraquinone (A), SANPAH (S), and the three-letter abbreviation (MRF) within the LF strip.

[0123] These results demonstrate that, once deployed within the LFD, deposition is possible that can provide direct information about the nature of the investigated biomarker, such as the use of initials or symbols or emojis. Interpretation of the LFD strips becomes much easier for users if the caption code is displayed on the side of the LFD cassette. Through connectivity, one of the World Health Organization's REASSURED criteria for field diagnostics, test results can be interpreted using a standard smartphone or a fully validated app with a certified reader.

[0124] The multiplexing capability of our LF platform has also been demonstrated, as shown in Figure 9, where accurate readout of the region of interest is successfully achieved using 50 depositions in addition to four reference guide spots.

[0125] Example 4 Microarray deposition for LFD multiplexing with nucleic acid-based reagents / aptamers Having demonstrated that both SANPAH and AQ chemistries function as photoreactive moieties in the directional immobilization of hybridization-based capture oligos as a mode of interaction in LFD strips, we decided to extend this technology to LFD multiplexing using aptamer sandwich detection. We used pairs of aptamers (also known as SOMAmers™) capable of binding with high affinity to EBOLA VLPs. These aptamers were SOMA16 and SOMA43. For convenience and to account for the novel photoreactive chemistry added to these aptamers during synthesis, we refer to them in this section as SOMA-S16 (SANPAH-SOMA-16), SOMA-A16 (AQ-SOMA-16), SOMA-S43 (SANPAH-SOMA-43), and SOMA-A43 (AQ-SOMA-43). The aptamer pairs were tested in both direct and sandwich interactions, as shown in Figure 10C-D. All combinations and chemistries were tested and confirmed to be functional. Therefore, SOMA-16 was selected as a representative sensor and SOMA-A43 as a representative capture aptamer (Figure 10). The microarray layout of the deposition pattern is shown in Figure 10A for the direct interaction and Figure 10B for the sandwich interaction. The LF strip results, shown in Figure 10D, demonstrate that the aptamer sandwich assay functions as expected. The aptamer used as the sensor (SOMA16) was able to recognize the VLP as the analyte in the running buffer (which simulates the test sample), and the entire complex (VLP-SOMA16-AuNPs) was recognized by the immobilized capture aptamer (SOMA-A43), resulting in a clear signal shown as a dark spot in the LF strip. Immobilized EBOLA VLP, used as a positive control for direct interaction, was also detected, demonstrating dose-dependent intensity, indicating that the sensor aptamer (SOMA-16) can be used as a calibration standard and for quantification. The presence of reference guide spots in the LF strip allows a reading device or a suitably programmed smartphone to accurately locate and measure the intensity of spots within the region of interest as required.They also serve as technical validation of the LF assay.

[0126] Example 5 Microarray deposition for LFD multiplexed testing of antibody / aptamer hybrid interactions We wanted to test whether their LF platform could accommodate interaction modes that integrated both protein-based (antibody) and nucleic acid-based (aptamer) interactions. We considered that there might be situations in which a homogenous detection and capture reagent pair is not always available for our LF platform. To explore whether hybrid modalities could be used in these or other situations, we used IP10 (C-X-C motif chemokine 10), a 10 kDa interferon-γ-inducible protein, UniProt ID# P02778, a recombinant antibody pair from Abcam (cat# ab253846), and the single aptamer SOMAmer IP10 from SomaLogic, SeqID# SANPAH-4141-79_1, as examples. Figure 11A shows a microarray pattern in which both IP10 antibody and SOMA-IP10 / SANPAH were deposited as capture reagents. The sensor pad contains an IP10 antibody-AuNP-sensor. The sandwich LF interaction was performed in the presence of IP10 as analyte in the running buffer, and the results are shown in Figure 11B.

[0127] Simple visual assessment of signal intensity surprisingly confirmed the superior performance of the sandwich hybrid interaction compared to the whole antibody sandwich interaction, across all repeats (Figure 11B).

[0128] To demonstrate that the weak LF signal detection from the whole-antibody sandwich interaction was not due to the performance of the sensor-IP10 antibody itself, we performed the experiment shown in Figure 12. Results from the direct interaction of the AuNP-IP10 antibody sensor with the immobilized IP10 biomarker (Figure 12B) show a clear dose-response effect in all titration iterations (Figure 12C). These data sets suggest that combining hybrid interaction modes, such as those between antibodies and aptamers, can be highly advantageous in combining the strengths of each reagent, and that our LF platform is fully capable of accommodating these interactions.

[0129] In summary, this example demonstrates that combining different interaction modes can result in surprising performance improvements. Without being bound by any particular theory, we believe the improvement can be explained as follows: For small protein biomarkers, the number of epitopes available for antibody recognition may be very limited or overlapping, leading to antibody clones competing for identical or nearby binding sites. Similarly, aptatopes (aptamer binding sites) may be subject to similar constraints, albeit to a lesser extent due to their smaller size relative to antibodies. In contrast, because antibodies and aptamers typically bind to different portions of the analyte, the use of a hybrid configuration in this example reduces steric hindrance between antibody or aptamer pairs. Thus, this example demonstrates steric hindrance between antibody pairs when used as sensors and detectors, potentially competing for identical or nearby epitopes on the antigen (IP10). However, by using SANPAH-SOMAmers as capturers, this competitive interaction is avoided or reduced because the capture SOMAmers target different binding sites on the analyte / biomarker IP10. Another advantage of combining a hybrid interaction mode with aptamers and antibodies is that aptamers can be easily chemically modified during synthesis to incorporate photoreactive moieties for directed deposition of capture reagents.

[0130] Example 6 Microarray deposition to test the quantitative capabilities of LFD The quantitative capabilities of our LFD multiplexing are demonstrated by the data presented in the Examples, such as Figures 10A, 10C, 12A, B, and C. To further address the quantification challenges within multiplexed LFDs, we employed the dual-channel cassette design shown in Figure 13 and implemented the experimental design shown in Figures 14A-B. The results are shown in Figures 14C-D.

[0131] Signal intensities from dose-response titrations (Figure 14C-D) were acquired with the sciREADER LF1 reader shown in Figure 13. Mean intensity data were analyzed using GraphPad Prism 9.5.0, and the results are shown in Figure 15B, C, and D. In this interaction mode, the lower limit of detection for our platform appears to be approximately 8 picograms (equivalent to 1 femtomole). Mean intensity values ​​were analyzed, and dose-response curves were generated from three LF assay replicates, as shown in Figure 15B. The coefficient of variation (CV) was determined to be less than 4.0%. The graphs in Figure 15C and D show linear regression analysis at the low and mid-concentration ranges, with R-squared values ​​of 0.9 and 0.8, respectively. Experimental validation of unknown concentrations is overlaid in red, and values ​​fall within the linear range of the standard curve, as shown in the examples shown in panels B and C of Figure 15, demonstrating the quantitative capability of our LFD platform.

[0132] Example 7 (Lateral flow device manufacturing) Lateral flow device strips are fabricated using stacked pads on a back card. The various pads include a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. Each pad is pretreated appropriately depending on the application of the lateral flow device, and an appropriate buffer is selected for the sample and / or analyte being determined. For example, if the sample is a blood sample, the sample pad can be pretreated with an appropriate surfactant. Next, the conjugate pad is treated with a target recognition element-conjugate complex. Different capture moieties are dispensed onto the nitrocellulose membrane using a lateral flow dispenser such as a BioDot dispenser. A mask, e.g., with a grating 1 mm wide and spaced 1.5 mm apart, is placed between the electromagnetic radiation source and the nitrocellulose membrane. The nitrocellulose membrane is then exposed to electromagnetic radiation, e.g., using a UV crosslinker, to activate the photoactivatable functional groups of the capture moieties exposed to the radiation. The mask is then removed. The membrane is washed with an appropriate buffer as needed to remove unbound capture moieties. Next, target recognition elements are dispensed onto the nitrocellulose and bind to the areas previously activated by electromagnetic radiation. Control lines or spots for different test samples are then dispensed using a lateral flow dispenser such as the BioDot dispenser. These can be dispensed either after or before exposing the nitrocellulose to electromagnetic radiation. The nitrocellulose membrane is then dried at 37°C for 1 hour. Next, the nitrocellulose membrane is blocked with 0.1% BSA solution to prevent nonspecific binding and dried at 37°C for 1 hour. Finally, the strips are assembled, cut to 5 mm widths, and stored at room temperature and below 20% RH.

[0133] (Conclusion) This study demonstrates that the inventors have developed a strategy using photoreactive chemistry for the directional immobilization of nucleic acid ligands on microporous membranes, such as nitrocellulose membranes, in a manner compatible with the implementation of LF assays. The photoreactive chemistry is compatible with existing DNA synthesis chemistry platforms. The photoreactive chemistry can be used to achieve directional deposition in ultra-small volume microarray formats, allowing for multiplexing of multiple different analytes, e.g., using up to 50 spots, e.g., up to 60 spots, up to 70 spots, up to 80 spots, up to 90 spots, or up to 100 spots, thus enabling the detection of up to 100 different analytes in each sample, or the detection of multiple different analytes together with their multiple calibration concentrations, allowing for quantification of the amount of each analyte in a test sample. Thus, for example, with a device capable of depositing up to 100 spots, the device could be used to test 20 different test analytes, each with four calibration concentrations of the analyte, or 10 different analytes, each with nine calibration concentrations of the analyte. Through multiplexed detection, such LFDs can not only report on the quality of sample preparation (internal control) but also leverage the multifactorial power of multi-omics approaches in biomarker discovery, enabling simultaneous omics detection and differential diagnosis. Photoreactive chemistry can be used to immobilize nucleic acid ligands with distinct binding specificities for target analytes based on either base pair complementarity (nucleic acid hybridization) or specific steric and molecular complementarity (aptamer-protein interactions), enabling the design of "hybrid" diagnostic LFD assays based on both proteomic and transcriptomic biomarkers.

[0134] Although the present invention has been particularly shown and described with reference to specific embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention. [Prior art documents] [Non-patent literature]

[0135] [Non-Patent Document 1] Bruno, J. (2014). Application of DNA Aptamers and Quantum Dots to Lateral Flow Test Strips for Detection of Foodborne Pathogens with Improved Sensitivity versus Colloidal Gold. Pathogens, [online] 3(2), pp. 341-355. Available at: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4243449 / [Accessed November 11, 2019]. [Non-patent document 2] Buck, R.L., Wang, H., Hyatt, T.P. and Mueggler, P.A. (2001). Covalent bonding of molecules to an activated solid phase material. [Online] Available at: https: / / patents.google.com / patent / US6306665B1 / en [Accessed December 12, 2019]. [Non-patent document 3] Holstein, C.A., Chevalier, A., Bennett, S., Anderson, C.E., Keniston, K., Olsen, C., Li, B., Bales, B., Moore, D.R., Fu, E., Baker, D., and Yeager, P. (2016). Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers. Analytical and Bioanalytical Chemistry, [online] 408(5), pp. 1335-1346. Available at: https: / / pubmed.ncbi.nlm.nih.gov / 26427504 / [Accessed July 9, 2020]. [Non-patent document 4] Kannoujia, DK, Ali, S. and Nahar, P. (2010). Single-step covalent immobilization of oligonucleotides onto solid surface. Analytical Methods, [online] 2(3), pp. 212-216. Available at: https: / / pubs.rsc.org / en / Content / ArticleLanding / AY / 2010 / C001661F# [Accessed July 9, 2020]. [Non-Patent Document 5] Kraemer S, Vaught JD, Bock C, Gold L, Katilius E, Keeney TR, et al. (2011) From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications:A SOMAmer-Based, Streamlined Multiplex Proteomic Assay.PLoS ONE 6(10):e26332.https: / / doi.org / 10.1371 / journal.pone.0026332 [Non-patent document 6] Lauritzen, E., Masson, M., Rubin, I. and Holm, A. (1990).Dot immunobinding and immunoblotting of picogram and nanogram quantities of small peptides on activated nitrocellulose.Journal of Immunological Methods, 131(2), pp.257-267. [Non-Patent Document 7] Li, J., Jing, L., Song, Y., Zhang, J., Chen, Q., Wang, B., Xia, X. and Han, Q. (2018). Rapid Detection of Rongalite via a Sandwich Lateral Flow Strip Assay Using a Pair of Aptamers. Nanoscale Research Letters, [Online] 13. Available at: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6153204 / [Accessed July 9, 2020]. [Non-patent document 8] Ma'sson, M., Lauritzen, E. and Holm, A. (1993).Chemical activation of nitrocellulose membranes for peptide antigen-antibody binding studies:Direct substitution of the nitrate group with diaminoalkane.Electrophoresis, 14(1), pp.860-865. [Non-Patent Document 9] Merck Millipore. (2013). Rapid Lateral Flow Test Strips: Considerations for Product Development. [Online] Available at http: / / www.merckmillipore.com / INTERSHOP / web / WFS / Merck-RU-Site / ru_RU / - / USD / ShowDocument-Pronet?id=201306.15671 [Non-Patent Document 10] Tonkinson, JL and Stillman, BA (2002). Nitrocellulose: a tried and true polymer finds utility as a post-genomic substrate. Frontiers in Bioscience: A Journal and Virtual Library, [online] 7, pp. c1-12. Available at: https: / / pubmed.ncbi.nlm.nih.gov / 11779718 /

Claims

1. 1. A diagnostic device comprising: a microporous phase material; and a capture reagent covalently bound to a predetermined capture zone of the microporous phase material, the capture reagent comprising a covalent binding moiety, the covalent binding moiety comprising a long wavelength electromagnetic radiation (EMR) activatable crosslinker having a long wavelength electromagnetic radiation (EMR) activatable functional group, the capture reagent being covalently bound to the capture zone via the electromagnetic radiation activated functional group, the capture reagent being capable of binding to an analyte of interest; (i) the capture reagent further comprises a first linker moiety, and the covalent attachment moiety is located at the 5' or 3' end of the first linker moiety; and / or (ii) the diagnostic device comprises a detection reagent comprising a second linker moiety, the detection reagent being capable of binding to the analyte of interest; Either each linker moiety comprises or consists of either an oligonucleotide moiety, a hydrocarbon chain, or an oligonucleotide moiety linked to a hydrocarbon chain, optionally wherein the hydrocarbon chain is a linear hydrocarbon chain; Diagnostic devices.

2. The diagnostic device of claim 1, wherein the capture reagent comprises the first linker moiety, the first linker moiety being a first oligonucleotide moiety, and the covalent bond moiety is located at the 5' or 3' end of the first oligonucleotide moiety.

3. The diagnostic device of claim 1 or 2, wherein the capture reagent comprises an antibody molecule having binding specificity for the analyte of interest.

4. The diagnostic device of claim 3 , wherein the covalent binding moiety is attached to the Fc region of the antibody molecule.

5. The diagnostic device of claim 2 , wherein the capture reagent comprises an antibody molecule having binding specificity for the target analyte, the antibody molecule being attached to the other of the 5′ end or the 3′ end of the first oligonucleotide at a CH3 domain.

6. The diagnostic device of claim 2 , wherein the first oligonucleotide molecule comprises an aptamer or a biomimetic thereof, optionally wherein the first oligonucleotide molecule comprises an aptamer.

7. 7. The diagnostic device of claim 1, wherein the microporous phase material is nitrocellulose.

8. The diagnostic device of any one of claims 1 to 7, wherein the covalently attached moiety comprises an N-hydroxysuccinimide ester, an aryl azide, a phenyl azide, a diazirine, a psoralen group, or an anthraquinone.

9. 9. The diagnostic device of claim 8, wherein the covalently binding moiety comprises an anthraquinone C2-dT or the covalently binding moiety comprises a 6-(4'-azido-2'-nitrophenylamino)hexanoate group.

10. 10. The diagnostic device of claim 1, wherein the covalently attached moiety is covalently attached to the microporous phase material via photoactivation of a photoreactive functional group.

11. 11. The diagnostic device of claim 1, wherein the long wavelength electromagnetic radiation (EMR)-activatable functional group is activated at wavelengths greater than 270 nm.

12. 12. The diagnostic device of claim 11, wherein the long wavelength electromagnetic radiation (EMR)-activatable functional group is activated at wavelengths in the UVA or UVB range.

13. 13. The diagnostic device of any one of claims 1 to 12, wherein the covalently attached moiety comprises a photoactivatable anthraquinone moiety or a photoactivatable nitrophenyl azide.

14. The diagnostic device of claim 1 , wherein the diagnostic device comprises a detection reagent capable of binding to the analyte of interest.

15. 15. The diagnostic device of claim 14, wherein the diagnostic device comprises a detection reagent comprising a second linker moiety, the second linker moiety comprising a second oligonucleotide moiety, the detection reagent being capable of binding to an analyte of interest.

16. 16. The diagnostic device of claim 15, wherein the detection reagent comprises an antibody molecule having binding specificity for the analyte of interest, and the second oligonucleotide molecule is attached to a CH3 domain of the antibody molecule.

17. 17. The diagnostic device of claim 16, wherein the second oligonucleotide molecule is attached to the CH3 domain of the antibody molecule at either the 5'-end or the 3'-end of the second oligonucleotide molecule and attached to a detectable particle such as a gold nanoparticle at the other 5'-end or the 3'-end of the second oligonucleotide molecule.

18. 16. The diagnostic device of claim 15, wherein the second oligonucleotide has binding specificity for the analyte of interest.

19. 16. The diagnostic device of claim 15, wherein the second oligonucleotide comprises a detectable particle, such as a gold nanoparticle.

20. 20. The diagnostic device of claim 15, 18 or 19, wherein the second oligonucleotide molecule comprises an aptamer or a biomimetic thereof, and optionally the first oligonucleotide molecule comprises an aptamer.

21. 21. The diagnostic device of any one of claims 14 to 20, wherein (i) either the capture reagent or the detection reagent comprises an antibody molecule having binding specificity for the target analyte, and (ii) the other of the capture reagent and the detection reagent comprises an aptamer having binding specificity for the target analyte.

22. 22. The diagnostic device of claim 21, wherein the capture reagent comprises an antibody molecule having binding specificity for the analyte of interest and the detection reagent comprises an aptamer having binding specificity for the analyte of interest.

23. 22. The diagnostic device of claim 21, wherein the detection reagent comprises an antibody molecule having binding specificity for the analyte of interest and the capture reagent comprises an aptamer having binding specificity for the analyte of interest.

24. A diagnostic device according to any one of claims 1 to 23, wherein the capture reagent is deposited in a capture zone in a spot having an area equivalent to a circular reaction site having a diameter of 100 to 500 µm.

25. The spot was 140 μg / μm ２ ~1400μg / μm ２ 25. The diagnostic device of claim 24, comprising a capture reagent at a concentration in the range of:

26. 26. The diagnostic device of claim 25, wherein the capture reagent comprises an antibody molecule.

27. The capture reagent comprises an aptamer, and the spots are 0.140 μg / μm ２ ~140 μg / μm ２ 25. The diagnostic device of claim 24, comprising a capture reagent at a concentration in the range of:

28. 28. A diagnostic device according to any one of claims 1 to 27, wherein the diagnostic device comprises a plurality of said capture zones.

29. 30. The diagnostic device of claim 28, wherein the capture reagent bound to each of the capture zones is different from the capture reagent bound to each of the other capture zones.

30. 30. A diagnostic device according to claim 28 or 29, wherein the capture reagent in each capture zone has a different binding specificity for the analyte of interest than the capture reagents in one or more of the other capture zones.

31. 30. A diagnostic device according to claim 28 or 29, wherein the capture reagent in each capture zone has a different binding specificity for the analyte of interest than the capture reagent in each of the other capture zones.

32. 31. A diagnostic device according to any one of claims 28 to 30, wherein the capture reagent in at least a first capture zone has binding specificity for a nucleic acid analyte and the capture reagent in at least a second capture zone has binding specificity for a protein analyte.

33. 33. The diagnostic device of claim 32, wherein the capture reagent in the first capture zone binds to the nucleic acid analyte via nucleic acid hybridization and the capture reagent in the second capture zone is an aptamer and binds to the protein analyte via an aptamer-protein interaction.

34. 34. The diagnostic device of any one of claims 1 to 33, wherein the diagnostic device is a lateral flow device.

35. 35. The diagnostic device of claim 1, further comprising a calibration zone comprising different predetermined concentrations of calibration molecules for at least one target analyte, which, upon contact with a sample buffer, allows comparison and quantification of the concentration of the target analyte in the capture zone.

36. 1. A diagnostic device comprising: a microporous phase material; a capture reagent bound to a capture zone of the microporous material, the capture reagent for binding to an analyte of interest in a test sample; and a calibration zone comprising a plurality of different reference concentrations of calibration molecules for the analyte of interest, wherein in use the concentration of the analyte of interest in a test sample bound to the capture reagent in the capture zone can be determined by comparing a signal produced by binding of the analyte of interest in the test sample in the capture zone with a signal produced by one or more calibration molecule concentrations in the calibration zone.

37. 37. The diagnostic device of claim 35 or 36, wherein the calibration molecules comprise a predetermined concentration of the analyte of interest bound to a microporous phase material in a calibration zone.

38. 37. The diagnostic device of claim 35 or 36, wherein the calibration molecule comprises a predetermined concentration of the capture reagent known to bind to a particular concentration of the analyte, and the calibration molecule is bound to the microporous phase material in the calibration zone.

39. 40. A diagnostic device according to any one of claims 35 to 39, wherein the signals generated by the plurality of calibration molecules in the calibration zone can be used to generate a calibration curve against the signal generated by the analyte in the test sample bound to the capture reagent in the capture zone and compared, allowing the analyte concentration in the test sample to be determined if necessary.

40. 37. A diagnostic device according to claim 36, which is a device according to any one of claims 1 to 35.

41. 1. A method for determining the concentration of an analyte of interest in a test sample, the method comprising: (i) providing a diagnostic device according to any one of claims 35 to 40; (ii) contacting the test sample with a capture reagent in a capture zone; (iii) comparing the signal produced by binding of the analyte of interest in the test sample in the capture zone with the signal produced by one or more reference calibration molecule concentrations in a calibration zone; 1. A method for determining the concentration of an analyte of interest in a test sample, comprising:

42. Step (iii) is generating a calibration curve from the signals of the calibration molecules in the calibration zone; and comparing the signal generated by the analyte bound to the capture reagent in the capture zone to a calibration curve, thereby enabling the concentration of the analyte in the test sample to be determined.

43. 1. A method of manufacturing a diagnostic device, the method comprising: (a) providing a microporous substrate; (b) contacting a capture reagent with the microporous substrate, the capture reagent comprising a linker moiety and a covalently attached moiety at a terminus of the linker moiety, the covalently attached moiety comprising an electromagnetic radiation (EMR)-activatable functional group, each linker moiety comprising or consisting of an oligonucleotide moiety, a hydrocarbon chain, or an oligonucleotide moiety linked to a hydrocarbon chain; (c) exposing the microporous substrate to a source of electromagnetic radiation, wherein the exposure covalently binds the capture reagent to a capture zone of the microporous substrate; A method comprising:

44. 44. The method of claim 43, wherein during the step of exposing to electromagnetic radiation, zones of the microporous substrate other than predetermined capture zones are masked to prevent activation of EMR-activated functional groups in such zones other than the capture zones.

45. 45. The method of claim 43 or 44, wherein the electromagnetic radiation is visible light, ultraviolet (UV) light, or electron beam radiation.

46. 46. ​​The method of any one of claims 43 to 45, wherein the diagnostic device is a diagnostic device according to any one of claims 1 to 39.

47. 47. The method of any one of claims 43 to 46, further comprising washing the non-covalently bound capture reagent from the microporous substrate.

48. 48. The method of any one of claims 43 to 47, wherein the diagnostic device is a multiplex diagnostic device having multiple capture zones, and the capture reagent in at least a first capture zone has binding specificity for a different target analyte from the capture reagent in at least a second capture zone.

49. 49. The method of claim 48, wherein the capture reagent in at least a first capture zone has binding specificity for a nucleic acid analyte and the capture reagent in at least a second capture zone has binding specificity for a protein analyte, and optionally the capture reagent in the first capture zone binds to the nucleic acid analyte via nucleic acid hybridization and the capture reagent in the second capture zone is an aptamer and binds to the protein analyte via an aptamer-protein interaction.

50. 50. The method of claim 48 or 49, wherein the capture reagent bound to each of the capture zones is different from the capture reagent bound to each of the other capture zones.

51. (a) contacting a first capture reagent with the microporous substrate, the first capture reagent comprising an oligonucleotide moiety and a covalently attached moiety at a 5' or 3' end of the oligonucleotide moiety, the covalently attached moiety comprising an electromagnetic radiation (EMR)-activated functional group; (b) applying a first mask to the microporous substrate, the first mask having a grating corresponding to a first capture zone of the microporous substrate; (c) exposing the first capture zone to a source of electromagnetic radiation, wherein the mask prevents exposure of areas of the microporous substrate other than the first capture zone to the electromagnetic radiation, wherein the exposure covalently bonds the first capture reagent to the first capture zone of the microporous substrate via the EMR-activated functional group; (d) removing the first mask and, if necessary, washing any unbound first capture reagent from the microporous substrate; (e) contacting a second capture reagent with the microporous substrate, the second capture reagent comprising an EMR-activated functional group; (f) applying a second mask to the microporous substrate, the second mask having a grating corresponding to a second capture zone of the microporous substrate; (g) exposing the second capture zone to a source of electromagnetic radiation, wherein the mask prevents exposure of areas of the microporous substrate other than the second capture zone of the microporous substrate to the electromagnetic radiation, wherein the exposure covalently bonds the second capture reagent to the second capture zone of the microporous substrate via the EMR-activated functional group of the second capture reagent; (h) removing the second mask and, if necessary, washing any unbound second capture reagent from the microporous substrate; (i) repeating steps (e)-(h) for the third and subsequent capture reagents, if desired, using a specific mask for each capture zone; 51. The method of any one of claims 48 to 50, comprising:

52. 52. The method of claim 51, wherein each of the capture reagents comprises an oligonucleotide moiety specific for a corresponding analyte of interest.

53. 52. The method of any one of claims 43 to 51, wherein the diagnostic device is a lateral flow device.

54. 53. The method of any one of claims 43 to 52, wherein the diagnostic device comprises one or more of an absorbent pad downstream of the capture zone, a control zone downstream of the capture zone, and a quenching zone upstream of the capture zone.

55. 55. The method of any one of claims 43 to 54, wherein at least one capture zone comprises a reagent specific for an analyte of interest indicative of sepsis, Ebola virus, or coronavirus.

56. 56. The method of any one of claims 43 to 55, wherein reaction of the reagent with the analyte of interest results in a detectable color change.

57. 57. The method of claims 43 to 56, wherein the diagnostic device comprises a calibration zone comprising calibration molecules for at least one target analyte at different predetermined concentrations, in which contact with a sample buffer allows comparison and quantification of the concentration of the target analyte in the capture zone.

58. 58. The method of claim 57, wherein the calibration molecules comprise a predetermined concentration of the analyte of interest bound to the microporous phase material in the calibration zone.

59. 58. The method of claim 57, wherein the calibration molecule comprises a predetermined concentration of the capture reagent known to bind to a particular concentration of the analyte, and the calibration molecule is bound to the microporous phase material in the calibration zone.

60. 60. A method according to any one of claims 57 to 59, wherein the signals generated by the plurality of calibration molecules in the calibration zone can be used to generate and compared to a calibration curve against the signal generated by the analyte in the test sample bound to the capture reagent in the capture zone, thereby enabling the analyte concentration in the test sample to be determined.

61. 61. A diagnostic device manufactured using the method of any one of claims 43 to 60.

62. 10. A method for detecting the presence of an analyte in a sample, the method comprising contacting a sample comprising the analyte with a diagnostic device described in any one of claims 1 to 40 or claims 61, or a diagnostic device manufactured by the method of any one of claims 43 to 60, and detecting a color change in the capture zone.

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