WO2014027968A1

WO2014027968A1 - Electrochemical detection of virus subtypes or antibodies based on nano-fluidic process in antibody- or antigen-grafted porous membranes

Info

Publication number: WO2014027968A1
Application number: PCT/SG2013/000352
Authority: WO
Inventors: Chee Seng Toh; Thi Thanh Binh Nguyen
Original assignee: Nanyang Technological University
Priority date: 2012-08-16
Filing date: 2013-08-16
Publication date: 2014-02-20

Abstract

Disclosed is a membrane. The membrane comprises a porous body and a plurality of nanochannels. The plurality of nanochannels extends therethrough to allow passage of one or more analytes through the nanochannels. The nanochannels have one or more binding agents provided therein to bind to the one or more analytes. Also disclosed is a method of making the membrane. Also disclosed are a membrane biosensor system and methods for detecting the presence of analytes in a sample.

Description

ELECTROCHEMICAL DETECTION OF VIRUS SUBTYPES OR ANTIBODIES BASED ON NANO-FLUIDIC PROCESS IN ANTIBODY- OR ANTIGEN-GRAFTED POROUS MEMBRANES CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Singapore patent application No. 201206127-1, filed 16 August 2012, the contents of it being hereby incorporated by reference in its entirety for all purposes. FIELD OF THE INVENTION

[002] The present invention relates to an electro-membrane and a method of making an electro-membrane. The present invention also relates to a biosensor and a method for detecting analytes. BACKGROUND OF THE INVENTION

[003] Traditional immune-based methods rely on capture mechanism using equilibrium conditions, which can provide identification of antibodies formed in response to the disease and in some cases, viruses. The most common method utilizes immune-based methods that operate on one-step binding principle. However, cross-reactivity of antibodies is common, which results in non-target being captured by the antibody or antigen probe, thus giving rise to false positive results that limit the detection selectivity of these immune-based methods.

[004] In attempt to overcome the disadvantages of one-step binding method, the two-step binding method (for example the sandwich method) has been developed and was found to reduce non-specific cross-reactivity. For example, if the probability for the probe layer binding to a non-target during the first binding step is 10%, and that the non-target has 10% probability of binding to a second anti-target probe, then the overall chance of the second probe in binding to a non-target captured during the first binding step will just be 1%. At the same time, it is unlikely that a correctly captured target is incorrectly detected during the second binding step process. This is because in most cases, the reagent used in the second binding step process would only recognize the reagent that is used in the first binding step.

[005] Although the magnitude of error is significantly reduced in a two-step binding approach, it is still considered insufficient in overcoming false positive results that may occur due to similarities in the structures of virus subtypes or antibodies. Non-specific binding is also known to occur in high frequency in protein-based compounds that share structure similarities. Until now, there are little advances toward the development of rapid, real-time immune-based diagnostic systems that can provide unambiguous identification of structurally similar antibody or viral serotypes. Accordingly, there is a need to provide an alternative method of detecting analytes, a biosensor for detecting analytes, a membrane to be used in the method of detection and a method for making the membrane.

SUMMARY OF THE INVENTION

[006] In one aspect, there is provided a membrane. The membrane comprises a porous body and a plurality of nanochannels. The plurality of nanochannels extends there through to allow passage of one or more analytes through the nanochannels. The nanochannels have one or more binding agents provided therein to bind to the one or more analytes.

[007] In another aspect, there is provided a method of making a membrane. The method of making the membrane comprises the step of contacting a solution comprising one or more binding agents with a membrane comprising a porous body and a plurality of nanochannels extending there through for allowing passage of one or more analytes through the nanochannels.

[008] In another aspect, there is provided a membrane biosensor system. The membrane biosensor system comprises a reservoir; and one or more membranes positioned within the reservoir. The membrane further comprising one or more detectors. The one or more membranes may be positioned within the reservoir so as to divide the reservoir into at least two separate compartments. Each membrane may comprise a porous body and a plurality of nanochannels extending there through the porous body for allowing passage of one or more analytes through the nanochannels. The nanochannels have one or more binding agents provided therein to bind to the one or more analytes.

[009] In another aspect, there is provided a method of detecting the presence of one or more analytes in a sample. The method comprises the steps of: (a) allowing one or more analytes in the sample to pass through a membrane comprising a porous body and a plurality of nanochannels extending there through for allowing passage of one or more analytes through the nanochannels. The method further comprises step (b) measuring the change in differential pulse voltammetry (DPV) peak current. The method further comprises step (c) comparing the DPV peak current readings obtained from step (b) with the DPV readings obtained from a control sample. A delayed DPV peak current in the sample compared to the control sample may indicate the presence or absence of the one or more analytes in the sample. The nanochannels have one or more binding agents therein to bind to the one or more analytes.

[0010] In another aspect, there is provided a method of detecting the presence of one or more analytes in a sample. The method comprises the steps of: (a) allowing one or more analytes in the sample to pass through a membrane comprising a porous body and a plurality of nanochannels extending there through for allowing passage of one or more analytes through the nanochannels. The method further comprises step (b) allowing one or more labeled binding probes to pass through the membrane. The method further comprises step (c) measuring the change in differential pulse voltammetry (DPV) peak current. The method further comprises step (d) comparing the DPV peak current readings obtained from step (c) with the expected DPV readings obtained from a control sample. A delayed DPV peak current in the sample compared to the control sample indicates the presence or absence of the one or more analytes in the sample. The nanochannels have one or more binding agents therein to bind to the one or more analytes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0012] Fig. 1 is a schematic diagram of an example of virus-grafted nanochannels, followed by addition of labeled antibodies on the feed side of a two-compartment cell. Eluted redox- labeled antibody is detected at the receiver side by electrochemical detection. (B) shows a schematic diagram of an example of antibody-grafted nanochannels, followed by incubation in a sample containing unlabeled viruses, followed by elution experiment of labeled antibody as in (A). (C) shows a schematic diagram of an example of an experimental setup (feed compartment: 100 μί, of Fc-IgG, receiver compartment: 500 μΐ of PBS pH 7.4, WE: GC electrode, RE: Ag/AgCl( 1 M KC1), CE: Pt wire mesh).

[0013] Fig. 2 (A) shows the concentration level of bovine serum albumin (BSA) in the receiver solution of the two-compartment cell as the protein transverses across the membrane nanochannels measured using bicinchoninic acid (BCA) protein assay method. In particular, increased in BSA concentration (determined using BCA kit assay) in the receiver solution after traversing an unmodified membrane, from the feed solution was observed. Conditions: lmg mL-1 BSA in 0.1 mL PBS (pH 7.4) buffered feed solution and 0.4 mL PBS buffered (pH7.4) receiver solution. (B) shows the signal response of the electrochemical detector toward ferrocene-labelled BSA as it transverse the same nanochannel membrane from three different concentrations of feed solutions prepared in PBS buffer (pH 7.4). Fitted lines are derived from expected increase in current signal toward increasing redox-labeled BSA amount in receiver solution by diffusion through the membrane, with concurrent electrode passivation due to BSA fouling. Thus, Fig. 2 shows that signal output can be used to predict resolution of constituents in multi-components samples.

[0014] Fig. 3 (A) shows electrode signal response toward redox-labeled anti-Denv2 3H5 antibodies as they passes through Denv2- and Denv3 -virus which were grafted onto the nanochannels membranes. (B) shows repeated experiments with reproducible signal profiles of the electrochemical detector after the membranes are regenerated. Thus, Fig. 3 shows the reproducibility of the signal profile of the present disclosure.

[0015] Fig. 4 shows electrode signal response toward redox-labeled anti-Denv2 3H5 antibodies as they pass through anti-Denv2 -antibody-grafted nanochannel membrane after 1 hour incubation with Denv2 or Denv3 virus. Membranes are prepared using APS covalent attachment with (A) 2 mg L-l, 0.5 mg mL-1, 1 mg niL-1 anti-Denv2 antibody. (B) with membrane incubated with human serum samples from patients with Denv2, 3 and 4 infection. Control is obtained from an uninfected human sample. Thus, Fig. 4 shows the proof-of- concept results on these four clinical serum samples using the immune-membrane biosensor system of the present disclosure.

BRIEF DESCRIPTION OF THE TABLES

[0016] Table 1 shows RT-PCR and PanBio ELISA assay results for uninfected and dengue infected patient serum samples.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0017] Immune-based methods of detecting analytes typically rely on a capture mechanism using equilibrium conditions. Whilst simple and cost effective, such methods are prone to false positive results. Accordingly, there is a need to provide an alternative platform for performing methods of detecting the presence of one or more analytes in a sample. Thus, in one aspect, there is provided a membrane suitable for performing such methods of detection. In one aspect, there is provided a membrane. The membrane may comprise a porous body and a plurality of nanochannels extending there through. The plurality of nanochannels allows passage of one or more analytes through the nanochannels. In one example, the nanochannels as described herein may have one or more binding agents provided therein to bind to the one or more analytes. In one example, the membrane may be substantially free of a detector. That is, the membrane does not encapsulate or surrounds the detector. In one example, the membrane is not in contact with a detector. As used herein, the term "detector" may be interchangeably used with the "electrode" or "sensing element", which is used to refer to means of capturing changes in signals arising within the membrane when target analytes bind to the membrane. The membrane of the present disclosure is advantageously free of the detector, thus preventing fouling of the detector by analytes such as, but not limited to proteins. In one example, the membrane is substantially free of the detector such that fouling caused by proteins does not occur or is at least substantially reduced. This allows reuse of membrane. In one example, should protein fouling occur on the detector, such fouling may be easily removed by means of removing protein fouling, such as polishing.

[0018] In one example, the binding of the binding agent and analyte to the labeled binding probe may determine the speed of the binding agent passing through (or traversing) through the nanochannels to the detecting side of the membrane. In one example, the binding of the binding agent and analyte to the labeled binding probe may cause a delay in the appearance of the analyte through the nanochannels to the detecting side of the membrane. When specific binding to analytes occur, the concentration of labeled probes or binding agents would decrease. Thus, when compared to samples without analytes, the concentration of the labeled probes or binding agents at the detector side of the membrane would appear to be lower than the concentration of the labeled probes or binding agents at the detector side of the membrane.

[0019] The term "analyte" as used herein may be used synonymously with "target molecule" and refers without limitation to a substance or chemical constituent of interest in a sample that is to be detected. The analyte may be a biological substance, such as a biomolecule, a biomarker, complexes, cell fraction or cells. In one example, the analyte may be a nucleic acid, a peptide, a polypeptide or protein, a protein or polypeptide fragment, or functional protein or polypeptide domain, a drug molecule, a small molecule or vitamin. In one example, the analyte may be selected from the group consisting of viruses, virus particles, antibodies, peptides, proteins, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens. As used herein, the term "viruses" refers to infective agents comprising virus particles (or virions) including, but not limited to genetic material including DNA and RNA, a protein coat and, optionally, an envelope of lipids that surrounds the protein coat. The term "virus particles", which may be used interchangeably with "virions", refers to at least one of the components that is present in a virus such as, but is not limited to genetic materials such as DNA and RNA, a protein coat and lipid envelope. In one example, the analytes may be labeled. In one example, the analytes may be labeled with ferrocene-based reagents.

[0020] In one example, the labeled binding probes may be binding agents that are labeled with detectable probes. In one example, the labeled binding probes include, but not limited to labeled- viruses, labeled- virus particles, labeled-antibodies, labeled-peptides, labeled-proteins, labeled-hormones, labeled-cytokines, labeled-lipids, labeled-allergens, labeled-carbohydrates, labeled-enzymes, labeled-antigens and labeled-cellular membrane antigens specific to the one or more analytes in the sample. In one example, the one or more labeled binding probes may interact with the one or more analytes bound to the binding agents in a multistep interaction whilst passing through the membrane. The multistep interaction increases assay specificity by reducing the probability of false positives. As shown in the Experimental Section, in particular at Figure 4, the membrane of the present disclosure was able to detect the various dengue virus isotypes that are known to be structurally very similar.

[0021] In one example, the binding agents may include, but are not limited to viruses, virus particles, antibodies, peptides, proteins, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens. In one example, the binding agents may be viruses, virus particles and/or antibodies. In one example, the viruses or virus particles or antibodies may be dengue viruses, or dengue virus particles or antibodies specific to dengue viruses. In one example, the dengue viruses, or dengue virus particles, or antibodies specific to dengue viruses are dengue serotype 1 (Denvl) viruses, or dengue serotype 1 (Denvl) virus particles, or antibodies specific to dengue serotype 1 (Denvl), or dengue serotype 1 (Denvl) viruses, or dengue serotype 1 (Denvl) virus particles, or antibodies specific to dengue serotype 1 (Denvl), or dengue serotype 3 (Denv3) viruses, or dengue serotype 3 (Denv3) virus particles, or antibodies specific to dengue serotype 3 (Denv3), or dengue serotype 4 (Denv4) viruses, or dengue serotype 4 (Denv4) virus particles, or antibodies specific to dengue serotype 4 (Denv4).

[0022] In one example, the one or more binding agents may be grafted to the nanochannels. As used herein, the term "grafted" or "graft", or any other grammatical permutations, refer to the addition of the binding agent onto the nanochannels. In one example, the one or more binding agents may be covalently grafted to the nanochannels. In one example, the binding agents may be grafted to the nanochannels by salinizing steps. In one example, the binding agents may be grafted to the nanochannels by using organic compounds containing carboxylic groups, silane-carbodiimide grafting reaction, polymers such as common polymers that are formed by polymerization of one or more monomer including, but not limited to 2-hydroxyethyl methacrylate, glycidyl methacrylate, (polyethylene glycol) methacrylate, (polyethylene glycol) methylether methacrylate, ethylene glycol dimethacrylate and poly(ethylene glycol) dimethacrylate. In one example, the binding agents may be grafted to the nanochannels by using silane-carbodiimide grafting reaction.

[0023] In one example, the membranes of the present disclosure may be modified with modifications that include (a) high molecular weight poly(ethyleneimine) and poly(ethyleneglycol) (PEI-PEG), (b) formation of a self-assembled monolayer using a silane coupling reaction with PEG (silane-PEG), (c) (ω-methoxy terminated PEG) trimethoxysilane and the like.

[0024] As used herein, the term "porous", pores, or any other grammatical variation refers to having a plurality of holes, voids or empty spaces within a body such that the body is permeable to fluid, liquid or air. In one example, the porous body comprises a front surface and a rear surface. In one example, the dimension between the front and rear surfaces of the porous body may be in the range of 1 to 200 microns, 1 to 150 microns, 1 to 100 microns, 1 to 50 microns, 10 to 200 microns, 50 to 200 microns, 100 to 200 microns, 150 to 200 microns, 10 to 150 microns, or 50 to 100 microns. In one example, the front surface and rear surface may be substantially flat. In one example, the front surface and rear surface may be generally parallel to each other. In one example, the front surface and rear surface may not be substantially flat such that the front surface and rear surface may not be generally parallel to each other.

[0025] In one example, the nanochannels may extend through the porous body between the front and rear surface. In one example, there may be a plurality of nanochannels provided in an ordered array extending through the porous body. In one example, the nanochannels may have a diameter of or equivalent diameter thereof in the range of 10 nm to 200 nm, 10 nm to 150 nm, 10 nm to 100 nm, 10 nm to 50 nm, 50 nm to 200 nm, 100 nm to 200 nm, 150 nm to 200 nm, 50 nm to 150 nm, or 50 to 100 nm. [0026] In one example, the porous body includes, but is not limited to a ceramic material, a semiconductive material and a polymeric material. In one example, the ceramic material may include, but is not limited to alumina membrane, a titanium dioxide membrane and a zirconia membrane. In one example, the porous body comprises an alumina membrane.

[0027] In one example, the porous body may have a substantially regular pore structure. In another example, the porous body may have a substantially irregular pore structure. In one example, the pore density of the porous body may be any one of the range of 10³ to 10" pores per cm², 10⁴ to 10¹¹ pores per cm², 10⁵ to 10¹¹ pores per cm², 10⁶ to 1.0¹¹ pores per cm ,10⁷ to 10" pores per cm², 10⁸ to 10ⁿ pores per cm²; 10⁹ to 10¹¹ pores per cm², lO¹⁰ to 10¹¹ pores per cm², 10³ to 10¹⁰ pores per cm², 10³ to 10⁹ pores per cm², 10³ to 10⁸ pores per cm 2 , 103 to 107 pores per cm 2 , 103' to 106 pores per cm 2 , 103 to 105 pores per cm 2 , or 103 to 10⁴ pores per cm².

[0028] In one example, the sample may be flowed through the porous body via any means that would allow the sample to traverse or pass through the porous body from one end to another in one direction. In one example, such means may include, but is not limited to simple diffusion principle such as concentration gradient and pump.

[0029] In one example, the membranes of the present disclosure may be reused in a subsequent assay. In one example, the labeled binding agents as used herein may be reused in a subsequent assay. In one example, the membranes of the present disclosure may be reused by removing the binding agent from the membrane by using an agent capable of removing the binding agents. In one example, the agent capable of removing the binding agents includes, but is not limited to cation chelator, high ionic strength agents, low pH agents, high pH agents, oxidizing agents, reducing agents and agents that may denature the binding probes or binding agents (such as sodium hydroxide). In one example, the oxidizing agent has an oxidation potential of at least about 1.3 volts and may include, but is not limited to periodate, peroxodisulfate, hypochlorite, chromate, permanganate, and perchlorate. In one example, the reducing agent includes, but is not limited to thiosulfate, dithionite, dithiothreitol, dithioerythritol and mercaptoethanol. In one example, the agent capable of removing binding agent includes, but is not limited to EDTA, KC1, NaCl, MgCl₂, HC1, Glycine, H₃P0₄, HBS- EP Buffer (lOmM Hepes, 150mM NaCl, 3mM EDTA, 0.005% Tween-20), SDS, DMSO, NaOH, guanidine, urea, sodium borate, salt of a quaternary ammonium compound such as stearyltrimethylammonium chloride, cetyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, benzalkonium chloride, benzyldimethyltetradecylammonium chloride, benzethonium chloride, and myristyltrimetylammonium bromide, and coconut amine acetate (for example: RNH₂ CH₃COOH where R = Cs to C₁₈). In one example, the agent capable of removing the binding agents may be sodium hydroxide.

[0030] In one aspect, there is provided a method of detecting the presence of one or more analytes in a sample. As used herein, the term "detecting" refers to locating or the act of identifying the presence or absence of an analyte of interest. In one example, the method of detecting the presence of one or more analytes comprises the use of the membrane as described herein. In one example, the method may comprise the steps of: (a) allowing one or more analytes in the sample to pass through a membrane comprising a porous body and a plurality of nanochannels. The nanochannels extend there through for allowing passage of one or more analytes through the nanochannels. In one example, the nanochannels have one or more binding agents therein to bind to the one or more analytes. The method further comprises step (b) measuring the change in differential pulse voltammetry (DPV) peak current. The method further comprises step (c) comparing the DPV peak current readings obtained from step (b) with the DPV readings obtained from a control sample.

[0031] In one example, the methods as described herein may further comprise a step of labeling one or more analytes. The step of labeling one or more analytes may proceed prior or after to any steps of the methods as described herein. Thus, in one example, the step of labeling the one or more analytes occurs prior to step (a) of the methods as described herein. In one example, the methods as described herein may further comprise prior to step (a), the step of labeling the one or more analytes. In one example, the one or more analytes interact with the one or more binding agents in a multistep interaction whilst passing through the membrane. As illustrated in the Experimental Section and Fig. 4, the multistep interaction ensures increased specificity as it reduces the probability of the magnification of detection of false positive. This is because when each interaction is a binding step and the probability of binding to non-target (i.e. false binding) is 10% of positive target, then for five binding interactions the probe encounters as it passes through (or traverse across) the membrane, then the overall probability of binding of the non-target probe would be 0.001% or 10⁵ times that of the target probe. This is unlike a sandwich-binding or two-step binding immune method that relies upon two antibody-antigen binding steps and a static capture approach, which requires many washing steps in order to avoid any non-specific capture. In contrast, in the method of the present disclosure, any non-specific adsorption is competitively removed or washed out by a specific capture that would have higher binding affinity. Furthermore, the method of the present disclosure may reduce the non-specific binding of other species such as non-specific binding of probes to analytes that have very similar binding sites or structures as the target analytes.

[0032] In one example, the one or more analytes passing through the membrane may interact with the one or more binding agents in a multistep interaction. In one example, the one or more analytes passing through the membrane may bind to the binding agents in the nanochannels reversibly or dynamically, in a non-static capture approach. In one example, the term "multistep" refers to the binding of a target analyte to a first binding agent (i.e. a first step), which further binds to a first binding probe (i.e. a second step). As the nanochannels are arranged such that analytes flow uni-directionally (for example through diffusion or concentration gradient), the analytes would detach from the first binding agent and the first binding probe and binds to a further second binding agent or second binding probe along the length of the nanochannels, thus allowing the analytes to bind to multiple binding agent or probe multiple times as they passes through the nanochannels (i.e. multistep binding). Thus, the analytes would travel, pass-through or traverses along the nanochannels and would fluidly bind to multiple binding agents and/or binding probes reversibly and non-statically. When the target analyte binds to binding agent and/or binding probes, a change in signal such as a differential pulse voltammetry (DPV) may occur, thus indicating the presence or absence of the analytes as they passes through the nanochannel. As used herein, the term "differential pulse voltammetry (DPV)" refers to an electrochemical technique where the cell current is measured as a function of time and as a function of the potential between the indicator and reference electrodes. The potential may be varied using pulses of increasing amplitude and the current may be sampled before and after each voltage pulse. In one example, the delayed DPV peak current in the sample compared to the control sample may indicate the presence or absence of the one or more analytes in the sample. In one example, the DPV peak current in the sample may be compared to the control sample thus indicating the presence of the one or more analytes in the sample. In one example, the method may further comprise the step of labeling the one or more analytes. In one example, the labeling of one or more analytes may be performed before passing the sample through the membrane.

[0033] In another aspect, there is provided a method of detecting the presence of one or more analytes in a sample. The method comprises the steps of: (a) allowing one or more analytes in said sample to pass through a membrane comprising a porous body and a plurality of nanochannels extending there through for allowing passage of one or more analytes through the nanochannels, wherein the nanochannels have one or more binding agents therein to bind to said one or more analytes; (b) allowing one or more labeled binding probes to pass through the membrane; (c) measuring the change in differential pulse voltammetry (DPV) peak current; (d) comparing the DPV peak current readings obtained from step (c) with the expected DPV readings obtained from a control sample. In one example, the delayed DPV peak current in the sample compared to the control sample may indicate the presence or absence of the one or more analytes in the sample. In one example, the delayed DPV peak current in the sample compared to the control sample in step (d) indicates the presence of the one or more analytes in the sample.

[0034] In one example, the plurality of nanochannels is provided in an ordered array extending through the porous body.

[0035] In one example, the method of detecting analytes as described herein may be used as follows: labeled antibodies or antigen probes flow through virus- or antibody-coated nanoporous materials (such as nanoporous alumina membrane) using, for example, simple diffusion principle driven by concentration difference of labeled antibody probe in the 2- compartment cell separated by the virus- or antibody-coated nanoporous membrane material. As the virus (or antigenic viral protein) or antibody bound within the membrane material can interact reversibly and dynamically in multiple steps as the antibody or antigen probe moves through the nanoporous material, its rate of movement is strongly influenced by the binding affinities between the diffusing probe and the bound antigen or antibody (see Fig. 1 A,B).

[0036] In one example, the method as described herein may be used to determine, identify or detect a dengue serotype in a sample. The present disclosure demonstrates general virus identification utility despite highly conserved amino acid residue sequences within the common serotypes of dengue virus. In one example, the method as described herein successfully achieves the identification of dengue serotype 2 (Denv2) from dengue serotypes 3 and 4 viruses (Denv3 and Denv4) in serum samples obtained from infected patient. In one example, the methods or system as described herein comprises a low cost detector, such as a potentiostat or galvanostat, no flow-injection instrumentation and a nanochannel membrane separating a two-compartment cell (Fig. 1C). As shown in Fig. 4, the method as disclosed herein can differentiate one dengue virus serotype from the other serotypes within short analysis time of less than 2 hr. It is envisaged that the method of the present disclosure may have shorter assay period if the sample size used is smaller and the sensing elements are reconfigured appropriately as known by the skilled person in the art. Furthermore, the methods or systems of the present disclosure are comparable to some current state-of-the-art real-time polymerase chain reaction (PCR) methods.

[0037] Without wishing to be bound by theory, one of the advantages of the present disclosure is the ability to differentiate between the similar antibodies/antigens using a diffusing probe, which interacts with the antibodies/antigens with multiple binding steps interaction. This is unlike existing immune-based methods, which rely on a single step or two-step binding such as the sandwich method. The multistep method ensures reduction in the number of false positive targets detection by the detector. The multistep method advantageously reduces the traverses speed of the binding agents or binding probes through the membrane and keep the binding agents, probes and analytes stay longer in the nanochannels (similarly to what happen in the chromatography column). The reduction of the traverse speed of the binding probes, analytes and agents leads to the delay in the observed differential pulse voltammetry (DPV) peak height. The change of DPV peak height indicates the presence of analytes. Additionally, the multistep process may be applied when different binding probes are used.

[0038] Furthermore, because of thin membrane thickness (tens of micrometer), the time taken for the probe to diffuse through is in range of few seconds to one or two hours. So appropriate sensing elements (electrochemical or optical) can be used to measure the rate at which the probe passes through the membrane.

[0039] Furthermore, also disclosed is a membrane biosensor system. The membrane biosensor system may comprise a reservoir, one or more membranes as described herein and one or more detectors. In one example, the membranes may be positioned within the reservoir so as to divide the reservoir into at least two separate compartments. In one example, a concentration gradient of the one or more analytes may be present between the separate compartments. As used herein, "a concentration gradient" refers to the graduated difference in concentration of an analyte, binding agents or binding probes per compartments.

[0040] As used herein, the term "reservoir" refers to a receptacle or chamber for storing the sample. In one example, the reservoir comprises three, four, five, six, seven, or eight separate compartments. In one example, the chamber or receptacle may be formed by the positioning of the membranes in series thus acting as the walls of the chamber or receptacle.

[0041] In one example, there may be at least two, three, four, five or six detectors. As used herein, the detectors or sensing element may include, but is not limited to electrochemical analyzer, acoustics mass analyzers and optical detectors. In one example, the detector is adjacent to the membrane. In one example, the detector is not in contact with the membrane. In one example, the detector is adjacent to the membrane but is not in contact with the membrane. The electrode, sensing element or detector may be at least 1 nm, at least 5 nm, at least 10 nm, at least 50 nm, at least 100 nm, at least 1000 nm, at least 5 μπι, at least 10 urn, at least 50 μηι, at least 100 μιη, at least 500 μηι, at least 1000 μπι, at least 5 mm, at least 10 mm, of at least 50 mm apart from the membrane. In one example, the electrode, sensing element or detector is about 1 to about 1000 nm apart from the membrane.

[0042] In view of the methods of detecting analytes using the membrane as provided herein, the present disclosure also aims to provide the method of making the membrane of the present disclosure. Thus, disclosed is the method of making the membrane of the present disclosure. The method of making the membrane of the present disclosure comprises the step of: contacting a solution comprising one or more binding agents with a membrane comprising a porous body and a plurality of nanochannels extending there through for allowing passage of one or more analytes through the nanochannels.

[0043] In one example, the method as described herein may further comprises prior to the step of contacting said solution comprising one or more binding agents with the membrane, the step of salinizing the membrane to allow the one or more binding agents to adhere to the nanochannels. In one example, the step of salinizing the membrane covalently grafts the one or more binding agents to the nanochannels. In one example, the salinizing step may be performed by immersing the membrane in silane functionalized agents such as, but are not limited to propyltrimethoxysilane, butyltrimethoxysilane, propyltriethoxysilane, butyltriethoxysilane, propyltrichlorosilane, propyltribromosilane, butyltrichlorosilane, butyltribromosilane, 1,1-hexadecyltrichlorosilane, 1,5-pentadecenyltrichlorosilane, 1,1- bromoundecyltrichlorosilane, monochlorosilane, dichlorosilane and 3- aminopropyltriethoxysilane. In one example, the salinizing step may be performed using suitable solvents such as, but are not limited to any dry organic solvent, including aromatic hydrocarbons such as toluene, hexadecane, benzene, naphthalene, xylene. In one example dry ketones may include, but not are limited to acetone and the like. In one example, the solvent used may be hexadecane.

[0044] In one example, prior to the step of contacting the membrane with a solution comprising one or more binding agents, the steps of washing and drying the membrane to remove the 3-aminopropyltriethoxysilane in acetone. In one example, the method may further comprises the step of contacting the membrane with a solution comprising one or more binding agents, the steps of washing and drying the membrane to remove the solution comprising one or more binding agents. In one example, the washing step may be performed by immersing the membrane in a solution. In one example, the solution used in the washing step may be a salt solution, including, but not limited to NaCl, KC1, MgCl₂, Mg S0₄, Na₂C0₃ and Na₂S0₄. In one example, the washing step may be performed by immersing the membrane in NaCl.

[0045] In one example, the method further comprises a drying step that may be performed using a normal oven (i.e. oven dry method), a vacuum oven, air drying, a centrifuge drying, a drum drying, freeze drying, microwave- and hybrid microwave drying. In one example, when the method comprises oven drying, the temperature of the oven may not be higher than 50 degree Celsius. In one example, the method comprises a drying step that may be performed by drying the membrane in nitrogen

[0046] As used herein, the term "about", in the context of diameter or dimensions of the porous body, nanochannels or distance of membrane to detector, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

[0047] As used herein, the term "one or more" refers to one, two, there, four, five, six, seven, eight, nine, ten or more possible binding agents, analytes, labeled binding probes or any other feature that is recited as "one or more".

[0048] As used herein, the term "sample" as used herein refers to any sample, which includes an analyte as defined herein. Such samples may, for example, include samples derived from or comprising stool, whole blood, serum, plasma, tears, saliva, nasal fluid, sputum, ear fluid, genital fluid, breast fluid, milk, colostrum, placental fluid, amniotic fluid, perspirate, synovial fluid, ascites fluid, cerebrospinal fluid, bile, gastric fluid, aqueous humor, vitreous humor, gastrointestinal fluid, exudate, transudate, pleural fluid, pericardial fluid, semen, upper airway fluid, peritoneal fluid, fluid harvested from a site of an immune response, fluid harvested from a pooled collection site, bronchial lavage, urine, biopsy material, e.g. from all suitable organs, e.g. the lung, the muscle, brain, liver, skin, pancreas, stomach, etc., a nucleated cell sample, a fluid associated with a mucosal surface, hair, or skin. In addition, samples from environmental sources, e.g. water samples, meat or poultry samples, samples from sources of potential contamination etc. may be used. [0049] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to. be within the scope of this invention.

[0050] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0051] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION Grafting of dengue virus particles or antibody onto membrane nanoehaimels.

[0052] Grafting of viruses and antibody in alumina membrane nanochannels were carried out using usual silane-carbodiimide grafting reaction as follows. The platinum-coated alumina membranes were immersed in 5% 3-aminopropyltrimethoxysilane in acetone for an hour. After an hour, the membranes were washed thoroughly with acetone and dried with nitrogen. The membranes were further dried in oven for 30 minutes at 45 °C. The membranes were then immersed in glutaraldehyde for 6 hours, followed by thorough washing with ultrapure and drying with nitrogen. [0053] 30 μΐ of 100 pfu mL-1 dengue virus solution was drop-casted onto the membrane and kept at high humidity overnight. The membranes were subsequently rinsed with 1 M NaCl to remove any non-specifically absorbed dengue virus particles and dried in nitrogen. 10-5 M of propylamine was added to the membranes (30 μΐ for a membrane) and kept at high humidity for 6 hr to remove excess glutaraldehyde and improve hybridization efficiency. After which, the membranes were thoroughly washed using 1 M NaCl, followed by ultrapure water and dried with nitrogen. The same procedure was applied for the immobilization of anti-Denv2 antibody to the membrane as follows.

[0054] 100 μΜ of anti-Denv2 antibodies was dropped onto the surface (30 μΐ for a membrane) and kept at high humidity overnight. The membranes were subsequently rinsed with 1 M NaCl to remove any non-specific absorbed Dengue virus and dried in nitrogen. 10⁵ M of propylamine was added to the membranes (30 μΐ. for a membrane) and kept at high humidity for 6 hours. This was done to improve the hybridization efficiency since propylamine was reported to react with the excess glutaraldehyde. After which, the membranes were thoroughly washed using 1 M NaCl, followed by ultrapure water and dried with nitrogen.

Preparation of IgG/BSA labeled with Fc-COOH.

[0055] Anti-Denv2 IgG (1 mg mL-1, 900 μg) was concentrated to ca. 3 mg mL-1, 300 μΐ. using Eppendorf concentrator 5301 at 4°C (solution A). Fc-COOH (23 mg) was dissolved in 1 ml DMSO (solution B). EDC (0.14 mmol) and NHS (0.14.mmol) were dissolved in 1 ml DMSO (solution C). Solution B was dropped into solution C with stirring for 2 h at 25 °C. The mixture was added to solution A and stirred at 4 °C overnight. Free Fc-COOH molecules were removed using Econo-Pac® 10DG column. Two bands were observed in the column. The first band contained IgG labeled with Fc-COOH (IgG-Fc) and the second band contained the free Fc-COOH molecules. The first band was eluted out using PBS as eluent and collected in fractions of 1 mL. BCA protein assay method was used to identify the IgG-Fc fractions which were combined and concentrated to ca. 2 mL using Eppendorf concentrator 5301 at 4 °C. The final concentration of IgG-Fc was determined by BCA protein assay method using Agilent Technologies, Carry 100 UV- Visible Spectrophotometer. Cyclic voltammetry of IgG-Fc was performed using CH Instruments, Electrochemical Analyzer model 750D.For the BSA labeling, BSA solution (3 mg mL-1, 300 μΐ) was used to react with solution B following the same procedure above. Analysis procedure of the immune-membrane biosensor system.

[0056] All analyses were carried out at room temperature and in the custom-made membrane cell where the modified membrane was clamped between the feed and receiver compartments (Fig. 1). At start of the experiment, the feed compartment contained 100 μΙ_- of Fc-IgG in PBS (pH 7.4) and the receiver compartment contained 500 μΐ,, PBS (pH 7.4) solution. Home-made glass-carbon (GC) working electrode (WE), Pt mesh auxiliary electrode (CE) and Ag/AgCl (1 M KC1) reference electrode from CHI Instrument (RE) were placed in the receiver solution. The solution in the receiver compartment was stirred constantly throughout the analysis. The amount of labelled antibody (IgG-Fc) that transverse across to the receiver compartment was measured using differential pulse voltammetry (10 mV increment potential, 50 mV amplitude, (CHI Instrument 750D), from 0.3-1.3 V versus Ag/AgCl(l M KC1). Repeated DPV scans were applied at an interval of 1 min to measure the increase in IgG-Fc concentration with time in the receiver compartment. Real sample analysis.

[0057] Four clinical serum samples derived from Denv2, Denv3 and Denv4 infected patients, and an uninfected patient were used in the real sample analyses. These serum samples were collected from patients between 3-5 days after fever onset and the virus serotype is validated using RT-PCR assays and the amount of IgG associated with secondary infection is confirmed using PanBio ELISA assay. 15 μΐ-, of each serum sample was first diluted 2 times with PBS (pH 7.4) buffer and subsequently drop-casted onto the antibody-grafted membrane, followed by 1 h incubation and the analysis procedure. The National Healthcare Group (Singapore) Domain Specific Review Boards approved the Prospective Adult Dengue Study (Reference No.: 2009/00432) from which clinical samples were derived. Informed consent was obtained for all participants.

RESULTS

Characterization of the immune-membrane biosensor setup.

[0058] Fig. 2A shows the concentration level of bovine serum albumin (BSA) in the receiver solution of the 2-compartment cell as the protein transverses across the membrane nanochannels, measured using BCA protein assay method. To achieve a peak signal output that varies in peak times according to the diffusion rate, the electrochemical detector is selected which can respond sensitively to a redox-labeled protein while at same time undergoes surface fouling. The opposing effect of the increasing level of the redox-labeled protein in the receiver solution and the depression of signal due to electrode fouling by the same protein will create a peak signal. Fig. 2B shows the signal response of the electrochemical detector toward ferrocene-labeled BSA as it passes through the same nanochannel membrane from 3 different concentrations of feed solutions. The signal peal- shifts toward shorter times as the protein concentration in the feed solution increases, as expected due to faster protein diffusion and electrode fouling. Thus, the signal output can be measured as a form of elution time that depends on the rate of diffusion of the protein transversing the nanochannel membrane. Such a parameter is particularly useful to describe separation efficiency, to derive physical parameter such as partition equilibrium constants between eluting species and the stationary surface, and to predict resolution of constituents in multi-components samples.

Specificity.

[0059] 3H5 antibody binds to the envelope protein domain III which is the neutralizing domain of dengue virus. Therefore, the redox-labeled 3H5 antibody was used as probe molecules to interact specifically or non-specifically with nanochannel-grafted target or non- target virus particles, respectively (Fig. 1). Fig. 3 A shows that using the systems or methods of the present disclosure, the diffusion of serotype 2 antibody across the Denv2-grafted nanochannels gives a delayed peak with an elution time of 30 min. This is distinctly different from the control membrane without any grafted virus (8 min). To test the specificity of this method, the same experiment was carried out using the non-specific dengue 3 virus (Denv3) with high structural similarity as Denv2. The 3H5 antibody which neutralizes Denv2 is however not neutralizing for the other three serotypes because of differences in the domain III envelope proteins. It was observed that the Denv3-grafted membrane clearly gave a different elution time compared to the Denv2-grafted membrane. Specific interaction with the nanochannel-grafted Denv2 virus slowed down the transversing (or passing through) rate of the labeled anti-Denv2 antibody molecules, compared to the nanochannel-grafted Denv3 virus. The virus-grafted nanochannel membrane can be regenerated by removing the virus- bound antibody from the grafted virus particles by passing 5 mL of 0.01 M HCl through the membrane at a slow rate of 0.3 mL min-1. Fig. 3B shows the reproducible signal profile derived at the electrochemical detector before and after the regeneration procedure. The slight increase in elution time is attributed to the incomplete removal of the dengue 2 antibodies from within the membrane.

Non-labeled virus target.

[0060] In real sample analyses, direct detection of non-labeled target analytes is highly desired because it achieves minimal sample preparation and pre-treatment. Using the same grafting method, 3H5 monoclonal serotype 2 antibody was attached onto the alumina nanochannel walls, followed by the incubation of the membrane in Denv2 virus solution and subsequent Denv2 antibody probe elution experiment. Unlike the virus grafting approach which requires overnight incubation of the membrane in the virus solution, this antibody- virus-antibody approach achieves virus capture within a short time of 60 min, followed by the immune-membrane biosensing procedure of ~30 min, with a total analysis time of less than 2 h. Because the virus is specifically captured using antibody-virus interaction, the grafted antibody approach is potentially applicable to direct detection of unlabeled virus in real sample analysis unlike the grafted virus approach (Fig. 4A).

[0061] To test the utility of the method in clinical analysis, four serum samples were collected from an uninfected patient and Denv2, Denv3 and Denv4 virus infected patients between 3-5 days after onset of fever and were validated using reverse transcriptase- polymerase chain reaction, RT-PCR (Table 1). PanBio ELISA assays indicated the presence of immunoglobulin M (IgM) associated with current dengue infection in the Denv2 and Denv4 samples and substantial amount of immunoglobulin G (IgG) associated with a previous dengue infection in the Denv4 sample (Table 1). Fig. 4B shows the proof-of- concept results on these four clinical serum samples using the membrane and/or methods and/or system of the present disclosure. The anti-Denv2 antibody probe eluted at the longest time for the Denv2 sample which was clearly distinguished from the other samples, thus demonstrates the excellent selectivity of the simple procedure relevant to real sample analysis. [0062] Table 1. RT-PCR and PanBio ELISA assay results for uninfected and dengue infected patient serum samples.

Claims

1. A membrane comprising:

a porous body and a plurality of nanochannels extending therethrough for allowing passage of one or more analytes through the nanochannels, wherein said nanochannels have one or more binding agents provided therein to bind to said one or more analytes.

2. The membrane according to claim 1, wherein the membrane is not in contact with a detector.

3. The membrane according to claim 1 or 2, wherein said porous body comprises a front surface and a rear surface and said nanochannels extend through said porous body between said front and rear surfaces.

4. The membrane according to any one of claims 1 to 2, wherein said plurality of nanochannels are provided in an ordered array extending therethrough said porous body.

5. The membrane according to any one of claims 1 to 4, wherein the porous body is selected from the group consisting of a ceramic material, a semiconductive material and a polymeric material.

6. The membrane according to claim 5, wherein the ceramic material is selected from the group consisting of an alumina membrane, a titanium dioxide membrane and a zirconia membrane.

7. The membrane according to claim 6, wherein the porous body comprises an alumina membrane.

8. The membrane according to any one of the preceding claims, wherein the nanochannels have a diameter or equivalent diameter thereof in the range of 10 nm to 200 nm.

9. The membrane according to any one of claims 2 to 8, wherein the dimension between the front and rear surfaces of said porous body is in the range of 1 to 200 microns.

10. The membrane according to any one of claims 2 to 9, wherein the front surface and rear surface are substantially flat and are generally parallel to each other.

11. The membrane according to any one of the preceding claims, wherein the porous body has a substantially regular pore structure.

12. The membrane according to any one of the preceding claims, wherein the pore density of the porous body is in the range of 10³ to 10" pores per cm².

13. The membrane according to any one of the preceding claims, wherein said binding agents are selected from the group consisting of viruses, virus particles, antibodies, proteins, peptides, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens.

14. The membrane according to claim 13, wherein said binding agents are viruses, or virus particles or antibodies.

15. The membrane according to claim 14, wherein said viruses, or virus particles, or antibodies are dengue viruses, or dengue virus particles or antibodies specific to dengue viruses.

16. The membrane according to claim 15, wherein said dengue viruses, or dengue virus particles, or antibodies specific to dengue viruses are dengue serotype 2 (Denv2) viruses, or dengue serotype 2 (Denv2) virus particles, or antibodies specific to dengue serotype 2 (Denv2).

17. The membrane according to any one of the preceding claims, wherein said analytes are selected from the group consisting of viruses, virus particles, antibodies, proteins, peptides, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens.

18. The membrane according to any one of the preceding claims, wherein said one or more analytes are labeled.

19. The membrane according to claim 18, wherein the one or more analytes are labeled with ferrocene-based reagents.

20. A method of making a membrane according to claim 1, comprising the step of:

contacting a solution comprising one or more binding agents with a membrane comprising a porous body and a plurality of nanochannels extending therethrough for allowing passage of one or more analytes through the nanochannels.

21. The method according to claim 20, further comprising, prior to the step of contacting said solution comprising one or more binding agents with said membrane, the step of silanizing the membrane to allow said one or more binding agents to adhere to said nanochannels.

22. The method according to claim 21, wherein the step of silanizing the membrane covalently grafts said one or more binding agents to said nanochannels.

23. The method according to any one of claims 21 to 22, wherein the silanizing step is performed by immersing the membrane in 3-aminopropyltriethoxysilane in acetone.

24. The method according to claim 23, further comprising, prior to the step of contacting the membrane with a solution comprising one or more binding agents, the steps of washing and drying the membrane to remove the 3-aminopropyltriethoxysilane in acetone.

25. The method according to any one of claims 20 to 24, further comprising, after the step of contacting the membrane with a solution comprising one or more binding agents, the steps of washing and drying the membrane to remove the solution comprising one or more binding agents.

26. The method according to any one of claims 24 to 25, wherein the washing step is performed by immersing the membrane in NaCl.

27. The method according to any one of claims 24 to 26, wherein the drying step is performed by drying the membrane in nitrogen.

28. The method according to any one of claims 20 to 27, wherein the one or more binding agents are selected from the group consisting of viruses, virus particles, antibodies, proteins, peptides, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens.

29. A membrane biosensor system, comprising: a reservoir;

one or more membranes positioned within the reservoir so as to divide the reservoir into at least two separate compartments, each membrane comprising a porous body and a plurality of nanochannels extending therethrough said porous body for allowing passage of one or more analytes through the nanochannels, wherein the nanochannels have one or more binding agents provided therein to bind to said one or more analytes; and

one or more detectors.

30. The system according to claim 29, wherein said porous body comprises a front surface and a rear surface and said nanochannels extend through said porous body between said front and rear surfaces.

31. The system according to any one of claims 29 to 30, wherein said plurality of nanochannels are provided in an ordered array extending therethrough said porous body.

32. The system according to any one of claims 29 to 31, wherein the reservoir comprises at least three separate compartments.

33. The system according to any one of claims 29 to 32, comprising at least two detectors.

34. The system according to any one of claims 29 to 33, wherein a concentration gradient of said one or more analytes is present between the separate compartments.

35. The system according to any one of claims 29 to 34, wherein said one or more detectors are selected from the group consisting of electrochemical analyzers, acoustics mass analyzers and optical detectors.

36. The system according to any one of claims 29 to 35, wherein the detector is not in contact with the membrane.

37. The system according to any one of claims 29 to 36, wherein the detector is adjacent to the membrane.

38. A method of detecting the presence of one or more analytes in a sample, comprising the steps of:

allowing one or more analytes in said sample to pass through a membrane comprising a porous body and a plurality of nanochannels extending therethrough for allowing passage of one or more analytes through the nanochannels, wherein the nanochannels have one or more binding agents therein to bind to said one or more analytes;

measuring the change in differential pulse voltammetry (DPV) peak current;

comparing the DPV peak current readings obtained from step (b) with the DPV readings obtained from a control sample, wherein a delayed DPV peak current in the sample compared to the control sample indicates the presence or absence of said one or more analytes in the sample.

39. The method according to claim 38 wherein the membrane is not in contact with a detector.

40. The method according to claim 38 or 39 wherein a delayed DPV peak current in the sample compared to the control sample in step (c) indicates the presence of said one or more analytes in the sample.

41. The method according to any one of claims 38 to 40, further comprising prior to step (a), the step of labeling said one or more analytes.

42. The method according to claim 41, wherein said one or more analytes are labeled with 5 ferrocene-based reagents.

43. The method according to any one of claims 38 to 41, wherein said one or more analytes interact with said one or more binding agents in a multistep interaction whilst passing through the membrane.

10

44. A method of detecting the presence of one or more analytes in a sample, comprising the steps of:

allowing one or more analytes in said sample to pass through a membrane comprising a porous body and a plurality of nanochannels extending therethrough for allowing 15 passage of one or more analytes through the nanochannels, wherein the nanochannels have one or more binding agents therein to bind to said one or more analytes;

allowing one or more labeled binding probes to pass through the membrane; measuring the change in differential pulse voltammetry (DPV) peak current; ^■ comparing the DPV peak current readings obtained from step (c) with the expected

20 DPV readings obtained from a control sample, wherein a delayed DPV peak current in the sample compared to the control sample indicates the presence or absence of said one or more analytes in the sample.

45. The method according to claim 44 wherein the membrane is not in contact with a 25 detector.

46. The method according to claim 44 or 45, wherein a delayed DPV peak current in the sample compared to the control sample in step (d) indicates the presence of said one or more analytes in the sample.

30

47. The method according to any one of claims 44 to 46, wherein the one or more labeled binding probes in step (b) are one or more binding agents such as viruses, virus particles, antibodies, peptides, proteins, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens specific to said one or more analytes in the sample.

48. The method according to any one of claims 44 to 47, wherein said one or more analytes passing through the membrane in step (a) interact with said one or more binding agents in a multistep interaction.

49. The method according to any one of claims 44 to 48, wherein said one or more analytes passing through the membrane in step (a) are bound by the binding agents in said nanochannels.

50. The method according to claim 49, wherein said one or more labeled binding probes in step (b) interact with said one or more analytes bound to the binding agents in a multistep interaction whilst passing through said membrane.

51. The method according to any one of claims 38 to 50, wherein said one or more binding agents in step (a) are covalently grafted to said nanochannels.

52. The method according to any one of claims 38 to 51, wherein said porous body comprises a front surface and a rear surface and said nanochannels extend through said porous body between said front and rear surfaces.

53. The method according to any one of claims 38 to 52, wherein said plurality of nanochannels are provided in an ordered array extending therethrough said porous body.

54. The method according to any one of claims 38 to 53, wherein the porous body is selected from the group consisting of a ceramic material, a semiconductive material and a polymeric material.

55. The method according to claim 54, wherein the ceramic material is selected from the group consisting of an alumina membrane, a titanium dioxide membrane and a zirconia membrane.

56. The method according to claim 55, wherein the porous body comprises an alumina membrane.

57. The method according to any one of claims 38 to 56, wherein the nanochannels have a diameter or equivalent diameter thereof in the range of 10 nm to 200 nm.

58. The method according to any one of claims 52 to 57, wherein the dimension between the front and rear surfaces of said porous body is in the range of 1 to 200 microns.

59. The method according to any one of claims 52 to 58, wherein the front surface and rear surface are substantially flat and are generally parallel to each other.

60. The method according to any one of claims 38 to 59, wherein the porous body has a substantially regular pore structure.

61. The method according to any one of claims 38 to 60, wherein the pore density of the porous body is in the range of 10³ to 10¹¹ pores per cm².

62. The method according to any one of claims 38 to 61, wherein said binding agents are selected from the group consisting of viruses, virus particles, antibodies, peptides, proteins, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens.

63. The method according to claim 62, wherein the binding agents are viruses or antibodies.

64. The method according to any one of claims 38 to 63, wherein said analytes are selected from the group consisting of viruses, virus particles, antibodies, peptides, proteins, hormones, cytokines, lipids, allergens, carbohydrates, enzymes, antigens and cellular membrane antigens.