Sandwich assay method

Abstract

The present disclosure relates to a sandwich assay method for detecting the presence of a target molecule in a microfluidic device, the method comprising: a) Providing the microfluidic device comprising a micro- or nanoscale channel, wherein an affinity probe is immobilized to an inner surface of the micro- or nanoscale channel; b) Providing a first solution comprising the target molecule into the microchannel; c) Providing a second solution with detection enhancement probe into the microchannel, the detection enhancement probe comprising a nanoparticle and one or more detection probes with affinity for the target molecule; and d) Detecting the presence of the detection enhancement probe along with the target molecule and the affinity probe by recording the changes in streaming current or potential. The present disclosure furthermore relates to a displacement assay method and to kits for use in the methods.

Description

[0001] SANDWICH ASSAY METHOD

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of detection of target molecules and in particular to a sandwich assay method for the detection of a target molecule. The invention also pertains to a kit for carrying out the assay.

[0004] BACKGROUND

[0005] Electrokinetic methods such as streaming current / potential have been explored for the purpose of bio-recognition because they are sensitive and inexpensive methods that do not require a large sample volume (Gevari et al, 2024, Wasilewska and Adamczyk, 2001; Wu et al., 2012). The methods have been utilized for detection of a wide variety of biological targets ranging from antibodies and various other proteins, (Dev et al., 2016), DNA (Li et al., 2018) and extracellular vesicles (Cavallaro et al., 2019) to non-biological targets (Sadlej et al., 2009).

[0006] The key aspect of these methods is exploiting the changes in the electrostatic and hydrodynamic environment at the solid-liquid interface when target molecules bind to the surface. Previous theoretical and experimental investigations (Adamczyk et al., 2010; Sadlej et al., 2009; Sahu et al., 2020) have studied electrokinetic phenomena, in particular the hydrodynamic and the electrostatic influence of particle adsorption on the inner surface of a microchannel, revealing a complex dependence of the electrokinetic signals on these parameters. Using a set of engineered molecules and the theoretical model proposed by Adamczyk et al. (2010), it has been shown in previous studies (Sahu et al., 2020) that the size and the charge parameters of adsorbing particles may either counteract or assist in electrokinetic sensing leading to a very large difference in the limit of detection. The results offer important insight on how to design a sensitive electrokinetic assay.

[0007] However, for any practical biosensor, the issues of specificity and selectivity are also extremely important. Particularly, in case of immunoassays, it is well-known that the detection of a target molecule often suffers from a low selectivity due to various nonspecific interactions and cross-reactivity (Afsahi et al., 2018; Juncker et al., 2014; Lichtenberg et al., 2019; Pei et al., 2013) of the affinity probes with non-target molecules. This is of particular concern when the target is to be detected from a complex medium e.g. plasma or serum. Among the available strategies to mitigate such problems, sandwich immunoassay is widely used in various well-established sensing platforms (Kim and Lee, 2017; Shui et al., 2018; Sun et al., 2019). In this strategy, a target is detected by two affinity probes each directed against different epitopes of the target, and thereby significantly minimizing the responses arising from non-specific and / or cross-reactive interactions with non-target entities.

[0008] One solution to address the issue of insufficient selectivity is an electrical immunosandwich assay using an electrokinetic-based streaming current method for signal transduction (Sahu et al., 2021). This technique measures changes in streaming current as a target molecule binds to capture probes on the inner surface of a silica microchannel, followed by detection probes binding to the target.

[0009] However, further advances are needed to enhance detection sensitivity and signal strength for electrical immuno-sandwich assays.

[0010] SUMMARY

[0011] One objective is to make available a sandwich assay method for detecting the presence of a target molecule in a microfluidic device comprising a micro- or nanoscale channel, the method comprising: a) Providing the microfluidic device comprising the micro- or nanoscale channel, wherein an affinity probe with affinity for the target molecule is immobilized to an inner surface of the micro- or nanoscale channel; b) Providing a first solution comprising the target molecule into the micro- or nanoscale channel such that binding of the target molecule to the first capture probe is enabled; c) Providing a second solution with a detection enhancement probe into the micro- or nanoscale channel, the detection enhancement probe comprising a nanoparticle and one or more detection probes with affinity for the target molecule, such that binding of the detection probe to the target molecule is enabled; and d) Detecting the presence of the detection enhancement probe along with the target molecule and the affinity probe by recording the changes in streaming current or streaming potential. The sandwich assay method according to the present disclosure is based on the findings that by using a detection enhancement probe, comprising a nanoparticle and one or more detection probes with affinity for the target molecule, in a sandwich assay method for detecting the presence of a target molecule in a microfluidic device enhances the assays sensitivity by amplifying the signal and thereby enable detection in samples at lower concentration of the target molecule.

[0012] The method according to the present disclosure is suitable for analyzing samples with any concentration of the target molecule. However, the advantages of the sandwich method according to the present disclosure becomes particularly pronounced when detecting very low concentrations of target molecule in the sample.

[0013] By “streaming current” herein is meant the electrical current generated when an electrolyte is flown over a charged surface by applying a pressure, causing ions to move along the flow direction. This movement creates a measurable current in an external electrical circuit connecting the ends of the microchannel. The current reflects the surface charge properties, ionic mobility at the solid-liquid interface and pressure difference along the fluidic channel. The streaming current maybe measured with an ammeter.

[0014] By “streaming potential” herein is meant the electrical potential (voltage) generated along a fluidic channel containing charged surface when an electrolyte is flown over it under pressure, causing ions to redistribute. This potential reflects the surface charge properties, ionic mobility at the solid-liquid interface and pressure difference along the fluidic channel. The streaming potential may for example be measured by a voltmeter.

[0015] Hence, streaming current measures ion flow as an electrical current, while streaming potential measures voltage (potential difference) generated due to ion displacement. Both are influenced by surface charge, pressure gradient, surface roughness and electrolyte composition and may equally well be used, as herein, to detect the presence of the detection enhancement probe along with the target molecule and the affinity probe.

[0016] The nanoparticles may be functionalized with the detection probe, to provide affinity to the target molecule and enable binding to the target molecule. The detection probe and / or the affinity probe may be a biomolecule, such as peptides, proteins, nucleic acids, carbohydrates, lipids.

[0017] Optionally, 5 or more detection probes are attached, such as conjugated, to the surface of the nanoparticle. In embodiments of the present disclosure 10 or more, optionally 20 or more, detection probes are attached, such as conjugated, to the surface of the nanoparticle. The fact that the sandwich assay method according to the present disclosure uses a detection enhancement probe including a nanoparticle functionalized with 5 or more detection probes attached to the surface thereof, provides improved selectivity for the target molecule and a significant amplification of the sensing signal in the streaming current / potential measurement.

[0018] The nanoparticle is advantageously a metal or metal oxide nanoparticle or a polymeric nanoparticle.

[0019] The nanoparticle may have a diameter of from 5 nm in diameter, as measured according to the Dynamic Light Scattering (DLS) method. Optionally, the nanoparticle has a diameter of from 15 nm, as measured according to the DLS method. Optionally, the nanoparticle has a diameter of from 15 nm to 500 nm, preferably the nanoparticle has a diameter within the range of from 20 nm to 500 nm, as measured according to the DLS method.

[0020] The nanoparticle may have a zeta potential of -100 mV or more, preferably -80 mV or more, such as within the range of from -80 mV to 80 mV, optionally within -3omV to 30 mV, as measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0021] In a preferred embodiment, the nanoparticle is a charged nanoparticle. It has been found by the present inventors that using a detection enhancement probe comprising a charged nanoparticle in a sandwich assay method according to the present disclosure greatly enhances the assays sensitivity by amplifying the signal and thereby enable detection in samples at very low concentration of the target molecule. The use of nanoparticles in the detection enhancement probe affects the signal by means of interacting with the flow and additionally, when using charged nanoparticles, by altering the surface charge density.

[0022] The charged nanoparticle may be negatively or positively charged. A zeta potential of the charged nanoparticle maybe positive or negative and have a magnitude ranging from 5 mV to 100 mV, i.e. either from -100 mV to -5 mV or from 5 mV to 100 mV. Optionally, the zeta potential of the charged nanoparticle is positive or negative and has a magnitude ranging from 10 mV to 80 mV, or alternatively from 10 mV to 30 mV, as measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0023] Optionally, after step b) of the method according to the present disclosure of providing a first solution comprising the target molecule into the micro- or nanoscale, the method may include an additional step of removing target molecule not bound to the affinity probes, such as by washing of the inner surface of the micro- or nanoscale channel with the same electrolyte used for the streaming current measurement.

[0024] Optionally after step c) of the method according to the present disclosure of providing a second solution with a detection enhancement probe into the micro- or nanoscale channel, the method may include a step of removing detection enhancement probe not bound to the target molecule, such as by washing of the inner surface of the micro- or nanoscale channel with the same electrolyte used for the streaming current measurement.

[0025] Between step a) and step b) in the method according to the present disclosure, the method may include recording a 1stbaseline current or potential. Alternatively, such information of baseline current or potential may alternatively come pre-determined by standardizing the microchip and affinity coating for specific target. Such standard baseline for each microchip and target may be obtained during standardization process of the microchip and the microfluidic device.

[0026] Optionally, the method may include recording a 2ndbaseline current or potential between step b) and step c).

[0027] The 1stbaseline may serve as a reference for detecting changes, in step d) after the target molecules and the detection probe have been introduced. The method may thus comprise recording of a baseline current or potential (streaming current or streaming potential) between step a) and step b), between step b) and step c) and during step d).

[0028] In one embodiment according to the present disclosure, the method comprises recording a 1stbaseline current or potential, as a reference, and recording a baseline current or potential in step d), i.e. as a part of step d) to detect the present of the detection enhancement probe along with the target molecule and the affinity probe by recording the changes in streaming current or streaming potential.

[0029] The measurement of the baseline may be carried out by flowing an electrolyte and the flow maybe achieved by the application of pressure, such as hydrostatic pressure.

[0030] Optionally, the first and / or second solution is an electrolyte solution with ionic strength within the range of from 1 mM to 2 M, optionally within the range of from 1 mM to 1 M, preferably within the range of from 1 mM to 500 mM or within the range of from 1 mM to 400 mM, more preferably within the range of from 1 mM to 200 mM.

[0031] According to a second aspect, the present disclosure relates to a kit for use in a sandwich assay method for detecting the presence of a target molecule in a microfluidic device comprising a micro- or nanoscale channel, the kit comprising:

[0032] - an affinity probe with affinity for the target molecule, where the affinity probe is capable of being immobilized to an inner surface of a micro- or nanoscale channel

[0033] - a detection enhancement probe comprising a nanoparticle and one or more detection probes with affinity for the target molecule.

[0034] Optionally, the kit comprises a microfluidic device comprising a micro- or nanoscale channel.

[0035] The nanoparticle may have a zeta potential of -100 mV or more, preferably -80 mV or more, such as within the range of from -80 mV to 80 mV, optionally within -3omV to 30 mV, as measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0036] Preferably, the nanoparticle is a charged nanoparticle. The charged nanoparticle may be negatively or positively charged. A zeta potential of the charged nanoparticle maybe positive or negative and have a magnitude ranging from 5 mV to 100 mV, i.e. either from -100 mV to -5 mV or from 5 mV to 100 mV. Optionally, the zeta potential of the charged nanoparticle is positive or negative and has a magnitude ranging from 10 mV to 80 mV or alternatively from 10 mV to 30 mV.

[0037] The zeta potential is measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0038] The nanoparticle may be functionalized with the detection probe, to provide affinity to the target molecule and enable binding to the target molecule.

[0039] The detection probe and / or the affinity probe may be a biomolecule, such as peptides, proteins, nucleic acids, carbohydrates, lipids.

[0040] Optionally, 5 or more detection probes are attached, such as conjugated, to the surface of the nanoparticle. In embodiments of the present disclosure 10 or more, optionally 20 or more, detection probes are attached, such as conjugated, to the surface of the nanoparticle. The fact that the sandwich assay method according to the present disclosure uses detection enhancement probes including charged nanoparticles functionalized with 5 or more detection probes attached to the surface thereof, provides improved selectivity for the target molecule and a significant amplification of the sensing signal in the streaming current / potential measurement.

[0041] The nanoparticle is advantageously a metal or metal oxide nanoparticle or a polymeric nanoparticle.

[0042] The nanoparticle may have a diameter of from 5 nm in diameter, as measured according to the DLS method. Optionally, the nanoparticle has a diameter of from 15 nm, as measured according to the DLS method. Optionally, the nanoparticle has a diameter of from 15 nm to 500 nm, preferably the nanoparticle has a diameter within the range of from 20 nm to 500 nm, as measured according to the DLS method.

[0043] The kit according to the second aspect maybe for use in the method according to the first aspect. According to a third aspect, the present disclosure relates to a displacement assay method for detecting the presence of a target molecule in a microfluidic device comprising a micro- or nanoscale channel, the method comprising: a) Providing the microfluidic device comprising the micro- or nanoscale channel, wherein a complementary probe having a sequence complementary to the target molecule is immobilized to an inner surface of the micro- or nanoscale channel, the complementary probe being conjugated to an affinity probe comprising a nanoparticle; b) Determining streaming current or streaming potential in the micro- or nanoscale channel, to establish a baseline; c) Providing a solution comprising the target molecule into the micro- or nanoscale channel such that displacement hybridization of the target molecule to the complementary probe occurs, via release of the affinity probe; and a) Detecting the presence of the complementary probe-target molecule by recording changes in streaming current or streaming potential.

[0044] Effects and features of the displacement method according to the second aspect as disclosed herein are largely analogous to those described above in connection with the sandwich assay method as disclosed herein.

[0045] The complementary probe and / or the affinity probe may be a biomolecule, such as peptides, proteins, nucleic acids, carbohydrates, lipids.

[0046] The displacement hybridization may occur through competition of affinity and / or concentration, conformational change of one of the binder or through introduction of specific enzymes. The skilled person may, based on the general principles of the architecture of displacement hybridization, select a suitable affinity probe and / or optimizing conditions for displacement and thereby ensure that hybridization occurs efficiently.

[0047] The nanoparticle is advantageously a metal or metal oxide nanoparticle or a polymeric nanoparticle.

[0048] The nanoparticle may have a diameter of from 5 nm in diameter, as measured according to the Dynamic Light Scattering (DLS) method. Optionally, the nanoparticle has a diameter of from 15 nm, as measured according to the DLS method. Optionally, the nanoparticle has a diameter of from 15 nm to 500 nm, preferably the nanoparticle has a diameter within the range of from 20 nm to 500 nm, as measured according to the DLS method.

[0049] The nanoparticle may have a zeta potential of -100 mV or more, preferably -80 mV or more, such as within the range of from -80 mV to 80 mV, optionally within -3omV to 30 mV, as measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0050] In a preferred embodiment, the nanoparticle is a charged nanoparticle. It has been found by the present inventors that using an affinity probe comprising a charged nanoparticle in a displacement assay method according to the present disclosure greatly enhances the assays sensitivity by amplifying the signal and thereby enable detection in samples at very low concentration of the target molecule.

[0051] The charged nanoparticle may be negatively or positively charged.

[0052] A zeta potential of the charged nanoparticle maybe positive or negative and have a magnitude ranging from 5 mV to 100 mV, i.e. either from -100 mV to -5 mV or from 5 mV to 100 mV. Optionally, the zeta potential of the charged nanoparticle is positive or negative and has a magnitude ranging from 10 mV to 80 mV, or alternatively from 10 mV to 30 mV, as measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0053] Step a) may either be preceded by a step of first immobilizing the complementary probe to the inner surface of the micro- or nanoscale channel and a subsequent step of providing a solution with the affinity probe comprising the nanoparticle into the micro- or nanoscale channel such that the affinity probe is conjugated to the complementary probe, or alternatively be preceded by a step of conjugating the affinity probe comprising the nanoparticle to the complementary probe and in a subsequent step immobilizing the complementary probe conjugated to the affinity probe to the inner surface of the micro- or nanoscale channel.

[0054] Optionally, after step a) of the method according to the third aspect, the method may include an additional step of removing affinity probes not bound to the complementary probes, such as by washing of the inner surface of the micro- or nanoscale channel with the same electrolyte used for the streaming current measurement.

[0055] Step b) in the method according to the present disclosure includes determining a 1stbaseline current or potential.

[0056] The baseline current or potential either be measured or be provided pre-determined by standardizing the microchip and affinity coating for specific target. Such standard baseline for each microchip and target may be obtained during standardization process of the microchip and the microfluidic device.

[0057] Measurement of the baseline maybe carried out by flowing an electrolyte and the flow maybe achieved by the application of pressure, such as hydrostatic pressure.

[0058] Optionally, the solution is an electrolyte solution with ionic strength within the range of from 1 mM to 2 M, optionally within the range of from 1 mM to 1 M, preferably within the range of from 1 mM to 500 mM or within the range of from 1 mM to 400 mM, more preferably within the range of from 1 mM to 200 mM.

[0059] According to a fourth aspect, the present disclosure relates to t kit for use in a displacement assay method for detecting the presence of a target molecule in a microfluidic device comprising a micro- or nanoscale channel, the kit comprising:

[0060] - a complementary probe comprising a sequence complementary to the target molecule, which complementary probe is capable of being immobilized to an inner surface of a micro- or nanoscale channel, wherein:

[0061] - the complementary probe is conjugated with an affinity probe comprising a nanoparticle; or

[0062] - the kit comprises an affinity probe comprising a nanoparticle, the affinity probe having affinity for the complementary probe.

[0063] Generally, all terms used in the claims are to be interpreted according to their ordinary meaning in the technical field, unless explicitly defined otherwise herein. All references to "a / an / the element, apparatus, component, means, step, etc." are to be interpreted openly as referring to at least one instance of the element, apparatus, component, means, step, etc., unless explicitly stated otherwise. The steps of any method disclosed herein do not have to be performed in the exact order disclosed, unless explicitly stated. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Aspects and embodiments are now described, by way of example, with reference to the accompanying drawings, in which:

[0065] Fig. 1 schematically illustrates a microfluidic device and a microchannel.

[0066] Fig 2 schematically illustrates a sandwich assay according to the prior art.

[0067] Figs. 3A-C schematically illustrate a sandwich assay according to the present disclosure.

[0068] Figs. 4A-B illustrate a schematic experimental set up for use in the method according to the present disclosure.

[0069] Fig. 5 illustrates the streaming current signal progression from comparative experiments between a sandwich assay according to the prior art and a sandwich assay according to the present disclosure.

[0070] Fig. 6 is a graph showing the result from the comparative experiments between a direct assay according to the prior art and a sandwich assay according to the present disclosure.

[0071] Figs. 7A-C schematically illustrate a displacement assay according to the present disclosure.

[0072] DETAILED DESCRIPTION

[0073] The aspects of the present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which certain embodiments of the invention are shown.

[0074] These aspects may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided by way of example so that this disclosure will be thorough and complete, and to fully convey the scope of all aspects of invention to those skilled in the art.

[0075] The present disclosure relates to a sandwich assay method for detection of a target molecule in a microfluidic device comprising a micro- or nanoscale channel. The target molecule may be any biomolecule for which it is possible to obtain affinity probes. For example, the target molecule maybe a protein or an enzyme, hormones, cytokines, small molecules, antigens including infectious disease markers or tumor markers, antibodies, peptides, nucleic acids, liposomes, exosomes, lipid particles, exomers.

[0076] The nanoparticle may have a zeta potential of -100 mV or more, preferably -8o mV or more, such as within the range of from -8o mV to 8o mV, optionally within -3omV to 30 mV, as measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0077] It has been found advantageous if the nanoparticle is a charged nanoparticle. A zeta potential of the charged nanoparticle may be positive or negative and have a magnitude ranging from 5 mV to 100 mV, i.e. either from -100 mV to -5 mV or from 5 mV to 100 mV. Optionally, the zeta potential of the charged nanoparticle is positive or negative and has a magnitude ranging from 10 mV to 80 mV or alternatively from 10 mV to 30 mV.

[0078] The zeta potential is measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS) measured by Zetasizer from Malvern Panalytical, in a PBS solution and at a pH 7,4.

[0079] The nanoparticle may be a metal or metal oxide nanoparticle or a polymeric nanoparticle. The nanoparticle may for example be a silver nanoparticle (AgNPs), gold nanoparticle (AuNPs), platinum nanoparticles (PtNPs), Copper nanoparticles (CuNPs), Iron Oxide (Fe2O3and Fe2O4) nanoparticle, Titanium Oxide (Ti02) nanoparticle, Zinc Oxide (ZnO) nanoparticle, Aluminum Oxide (A12O3) nanoparticle, a Silica nanoparticle such as a Silicon Dioxide (Si02) nanoparticle, Poly(lactic-co- glycolic acid) (PLGA) nanoparticle, polystyrene nanoparticle and lipid nanoparticle.

[0080] The sandwich assay method according to the present invention involves several steps, which are preferably performed in the set-out order. While certain steps must follow the outlined order, additional steps may be incorporated into the method. These additional steps can be performed prior to carrying out the method of the invention, between the steps, subsequent to the formation of a complex between the affinity probe and the target molecule or subsequent or prior to the formation of the complex with the affinity probe-target molecule-detection enhancement probe. The first step may for example be carried out by the pre-steps of: - Providing the microfluidic device comprising the micro- or nanoscale channel;

[0081] - Providing an affinity probe with affinity for the target molecule, which affinity probe is capable of being immobilized to an inner surface of the micro- or nanoscale channel; and

[0082] - Immobilizing the affinity probe to the inner surface of the micro- or nanoscale channel;

[0083] The immobilization of the affinity probe to the inner surface of the micro- or nanoscale channel may be carried out prior to carrying out the sandwich assay method and be supplied with the affinity probe immobilized to the inner surface of the micro- or nanoscale channel or during the course of the method.

[0084] Fig. 1 is a schematic illustration of a microchannel 2 of a microfluidic device 1 and the principles of a microfluidic device.

[0085] The microfluidic device is a system manipulating small amounts of fluid, such as in the order of io_3 to io-18liters, using microchannels with sizes of io to hundreds micrometer or nanochannels with sizes of io to hundreds nanometer. The microfluidic devices generally use a pump and chip with micro- or nanoscale channels. The pump is used to precisely move liquid inside the channels of the chip. Electrodes are placed at each end of the micro- or nanoscale channel to detect and measure changes in the streaming current or streaming potential and these electrodes are connected to a measuring device, such as an ammeter or a voltmeter, which may detect small changes caused by the flow of ions along the channel wall.

[0086] As shown in Fig. 1 there is a layer of electrical charges present on the inner surface of a microchannel and at the interface between the solid and the liquid. Materials like silica or glass often develop a negative charge (depending on the pH) when in contact with an aqueous solution due to ionization of surface groups. This surface charge affects how molecules in the sandwich assay, including the affinity probes, bind to or interact with the inner surface of the microchannel and it also affects the streaming current or streaming potential.

[0087] The surface charge of the micro- or nano channel 2 attracts counter-ions from the solution, forming the electric double layer (Stern and diffuses layer) along the channel inner surface 3. Fig. 2 schematically illustrates a prior art sandwich assay, wherein affinity probes 4 have been immobilized to the inner surface 3 of a microchannel in a microfluidic device, see Fig. 1 and Figs. 4A-B. As shown in Fig. 2, in the prior art sandwich assay, the affinity probe 4 binds with the first region of a target molecule 5 and a detection probe 6’ binds to a second region of the target molecule 5, which reduces false positive signals arising from non-specific bindings 8 which are not related to the target molecule.

[0088] Figs. 3A-C schematically illustrate a sandwich assay according to the present disclosure. The method according to the present disclosure is carried out on the inner surface 3 of a microchannel 2, see Fig. 1, where the affinity probe 4 is capable of being immobilized. The microchannel 2, and consequently its inner surface 3, may be composed of silica or glass. To facilitate immobilization of the affinity probe 4 to the inner surface 3, a surface activation maybe carried out. For a silica surface, examples of surface activations are short oxygen plasma treatment or cleaning with RCA standard recipe (DOI 10.1149 / 1.2086825). This maybe followed by a chemical surface functionalization step such as coating the surface with a layer of silane-PEG- Biotin or Poly-L-Lysin-PEG-Biotin (see, Gevari et al, 2024), followed by an affinity conjugation with either streptavidin or avidin or neutravidin.

[0089] During the immobilization of the affinity probe 4, the affinity probe 4 may for example be introduced into the microchannel by flowing or incubating the microchannels with the affinity probes. The micro- or nanochannels may also be open channels comprising a removable top, enabling easy access and precise positioning of the affinity probe 4.

[0090] Fig. 3A illustrates a step a) wherein a microfluidic device 1 comprising the micro- or nanochannel 2 is provided and an affinity probe 4 with affinity for a target molecule 5 is immobilized to an inner surface 3 of the microchannel 2.

[0091] In a step, not shown, after the step illustrated in Fig. 3A, the microfluidic device may record streaming current / potential to provide a baseline signal. This baseline may serve as a reference for detecting changes.

[0092] The measurement of the baseline may be carried out by flowing an electrolyte and the flow maybe achieved by the application of pressure, such as hydrostatic pressure. Fig. 3B illustrates a step b) of introducing the target molecule 5 into the microchannel 2, shown in Fig. 1. The target molecule 5 may be introduced with a solution comprising the target molecules 5. The solution may be an aqueous solution. Optionally, the solution is an electrolyte solution. The electrolyte may have an ion concentration within the range of from 1 mM to 2 M, optionally within the range of from 1 mM to 1 M, preferably within the range of from 1 mM to 500 mM or within the within the range of from 1 mM to 400 mM, more preferably within the range of from 1 mM to 200 mM. An example of a suitable electrolyte solution is phosphate buffer saline (PBS).

[0093] When the solution comprising the target molecule 5 is provided into the microchannel, a first region of the target molecule 5 binds to the affinity probe 4 immobilized to the inner surface 3 of the microchannel. The binding event changes the electrical and hydrodynamic environment along the channels inner surface 3, which is reflected as a change in the streaming current or potential.

[0094] The method may subsequently include a washing step, not shown, of removing target molecule not bound to the affinity probe by washing the inner surface of the micro- or nanochannel.

[0095] In a, optional step, not shown, and after the step illustrated in Fig. 3B, the microfluidic device may record streaming current / potential to provide a baseline current or potential. This baseline may serve as a reference for detecting changes due to target binding to the first antibody, 4 in Fig 3B.

[0096] Fig. 3C illustrates a subsequent step of introducing a detection enhancement probe 7, the detection enhancement probe 7 comprising a nanoparticle 9 with a detection probe 6 conjugated to the surface of the nanoparticle 9. The detection probe 6 has affinity for the target molecule 5, such that binding of the detection probe 6 to a second region of the target molecule 5 is enabled. The fact that sandwich assays require binding of the affinity probe 4 and the detection probe 6 to the target molecule 5 reduces false positive signals arising from non-specific bindings 8 which are not related to the target molecule. The detection enhancement probe 7 may be introduced with a solution comprising the detection enhancement probe 7. The solution may be an aqueous solution. Optionally, the solution is an electrolyte solution. The electrolyte may have an ion concentration within the range of from 1 mM to 2 M. An example of a suitable electrolyte solution is phosphate buffer saline (PBS).

[0097] The binding of the detection enhancement probe 7 to the already captured target molecule 5 causes another change in streaming potential.

[0098] The method may subsequently include a washing step, not shown, of removing second detection enhancement probe not bound to the target molecule by washing the inner surface of the micro-or nanochannel.

[0099] The difference in the streaming potential / current between the initial baseline and after the detection probe binds is measured by the electrodes. This change in potential / current is closely associated with the concentration of the target molecule in the sample.

[0100] Additionally, the streaming current / potential detection maybe carried out by a Field- Effect Transistor (FET), such as by using electrokinetic gating effect (DOI: io.ioi6 / j.snb.2oi8.02.017).

[0101] Fig. 7A illustrates a step a), in which a microfluidic device comprising a micro- or nanochannel (as illustrated in Fig. 1) is provided with a complementary probe 14 that exhibit affinity for a target molecule 15 (shown in Fig. 7C). The complementary probe 14 is immobilized to an inner surface 3 of the microchannel.

[0102] Fig. 7B illustrates a step in which an affinity probe 16, comprising a nanoparticle 9, is conjugated to the complementary probe 14. This step may either occur as a separate step, or the affinity probe 16, comprising the nanoparticle 9, may be conjugated to the complementary probe 14 prior to immobilizing the complex to the inner surface 3 of the microchannel.

[0103] Following the step illustrated in Figa. 7A-B, the microfluidic device records streaming current / potential in a step b) to establish a baseline signal (step not shown). This baseline may serve as a reference for detecting changes.

[0104] The measurements of the baseline may be carried out by flowing an electrolyte and the flow may be achieved by the application of pressure, such as hydrostatic pressure.

[0105] Fig, 7C illustrates step c) in which the target molecule 15 is introduced into the microchannel 2 (shown in Fig. 1). The target molecule 15 maybe introduced with a solution comprising the target molecules 15. The solution maybe an aqueous solution. Optionally, the solution is an electrolyte solution. The electrolyte may have an ion concentration within the range of from 1 mM to 2 M, optionally within the range of from 1 mM to 1 M, preferably within the range of from 1 mM to 500 mM or within the range of from 1 mM to 400 mM, more preferably within the range of from 1 mM to 200 mM. An example of a suitable electrolyte solution is phosphate buffer saline (PBS).

[0106] When the solution comprising the target molecule 15 is provided into the microchannel, displacement hybridization occurs wherein the target molecule 15 binds to the complementary probe 14 with higher affinity than the affinity probe 16. As a result, the affinity probe 16, carrying the nanoparticle 9, is released from the complementary probe 14. The displacement may alternatively occur after, for example, the introduction of an enzyme when both the target molecule and the affinity probe comprising the nanoparticle are conjugated to the complementary probe causing release of the affinity probe.

[0107] The displacement changes both the electrical and hydrodynamic environment along the inner surface 3 of the channel. These changes are reflected as a change in the streaming current or potential.

[0108] EXPERIMENTAL SECTION

[0109] Example

[0110] Synthesis and purification of PNA-peptide probes

[0111] PNA and peptide synthesis was microwave assisted and conducted using a fully automated Biotage Initiator+ Alstra microwave peptide synthesizer. The resin used was Rink Amide Chemmatrix (0.42 mmol / g purchased from Biotage, Sweden) at a 0.1 mol scale in a 10 mL reactor vial. The coupling strategy followed basic 9- fluorenylmethoxycarbonyl (Fmoc) chemistry in an iterative 3 step process including: residue-coupling, capping and Fmoc-deprotection.

[0112] The PNA monomers including Fmoc-A(Bhoc)-aeg-OH, Fmoc-C(Bhoc)-aeg-OH, Fmoc-G(Bhoc)-aeg-OH and Fmoc-T-aeg-OH were purchased from PNA Bio (CA, USA). Fmoc-Glu(OtBu)-OH, Fmoc-Tyr(OtBu)-OH and Fmoc-Cys(Trt)-OH were purchased from Novabiochem (Germany) and the {2-[2- (Fmocamino)ethoxy]ethoxy}acetic acid (AEEA) was purchased from Asta Tech Inc. (USA).

[0113] All PNA monomer couplings were conducted with diisopropylcarbodiimide (DIC) and Oxyma Pure with 4 times molar excess at a concentration of 0.07 M in dimethylformamide (DMF) or in N-methyl-2-pyrrolidone (NMP) for the C monomer. Amino acids and AEEA were coupled in the same way but at 6 times molar excess and in a concentration of 0.17 M in DMF. The coupling reagents (DIC and Oxyma Pure) were added in same molar excess as amino acid residues, AEEA or PNA monomers. All couplings were microwave assisted and conducted for 10 minutes at 75°C.

[0114] Each coupling step was subsequently followed by a ninhydrin test to see if an additional coupling was required. Following a positive ninhydrin test the coupling step was repeated while with a negative ninhydrin test any unreacted peptide was capped using NMP:2,6-lutidine:acetic anhydride (89:6:5) for 2 minutes at room temperature. Coupled residues were Fmoc-deprotected with DMF:piperidine (4:1) for 3 minutes followed by an additional treatment for 10 minutes.

[0115] PNA probes containing Cys were cleaved off the resin for 3 hours at room temperature in Trifluoroacetic acid (TFA):Triisopropylsilane (TIS): Ethanedi thiol (EDT):MilliQ (92.5:2.5:2.5:2.5), while probes not containing Cys were cleaved in the same condition but with a mix of TFA:TIS:MilliQ (95:2.5:2.5). Cleaved probes were precipitated in 10 times volume excess of cold diethyl ether, pelleted and washed twice by centrifugation at 4oooxg for 5 minutes at 4°C, then resuspended in fresh, cold diethyl ether to remove excess TFA. Following the last centrifugation excess diethyl ether was poured off and the pellet allowed to try by evaporation within a fume hood. Dried pellets were resuspended in 10 mL MilliQ: Acetonitrile (ACN) (1:1) with 0.1% TFA, frozen at -8o°C and freeze-dried overnight.

[0116] Crude, freeze-dried PNA probes were purified using reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C18 semipreparative columns (300SB- C18, 9.4 x 250 mm2, 5 pm pore size; Agilent, Santa Clara, CA, USA). The crude probes were resuspended in 100% A-buffer (MilliQ with 0.1% TFA), injected on the column and eluted with a gradient of 10-60% B-buffer (ACN with 0.1% TFA) over 50 min at a flow-rate of 3 mL / min and a constant temperature of 6o°C. UV-peaks at 260 nm were collected in fractions of 1.5 mL each and analyzed with matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) (4800 MALDI-TOF / TOF, Sciex, Framingham, MA, USA) using an a-cyano-4-hydroxycinnamic acid matrix (CHCA). Fractions containing only the desired PNA probe product were pooled together, frozen at -8o°C and freeze-dried overnight.

[0117] Conjugation of PNA probes to silica nanoparticles

[0118] Conjugation of purified PNA probes to silica nanoparticles (NPs) was done using carbodiimide chemistry. DiagNano™ Carboxyl Silica Nanoparticles, 100 nm, were purchased from CD Bioparticles, USA. 1 mL of NP suspension (25 mg / mL) was transferred to a sterile 1.5 mL Eppendorf tube. 4 mg of i-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride) (EDC) and 8 mg of (N- hydroxysuccinimide) (NHS) in 250 pL 2-(4-morpholino) ethanesulphonic acid (MES) buffer at pH 6.3 was added to the NP suspension and incubated on a rotamixer at room temperature for 45 minutes. The activated NPs were then washed by pelleting with centrifugation at 20 000 xg for 15 minutes, supernatant discarded, NPs resuspended by vortexing in PBS buffer (0.01 M PBS, pH 7.4), then once more centrifuged and supernatant discarded. Subsequently, 12.55 pg of PNA probe was dissolved in 1 mL PBS buffer and added to the pelleted NPs. The NPs were resuspended in the PNA mix by vortexing until visibly resuspended then incubated with continuous mixing for 3 hours at room temperature. Following the conjugation process, NPs were washed once more with PBS buffer using the same method as described earlier. Pelleted NPs were then resuspended in 200 pL of 25 mM glycine in PBS buffer and incubated with continuous mixing for 30 minutes at room temperature. After 30 minutes, glycine was removed from the mix by an additional wash step with PBS buffer. NPs were finally resuspended in 200 pL PBS buffer and kept at room temperature until used.

[0119] Fluidic and electrical measurement

[0120] The center piece of the sensing platform is a silicon-based microfluidic device 1, here illustrated as a microchip with four interconnected microchannels 2. In the illustrated microchip 1, a top of the microchip 1 is removable, allowing the solutions comprising the affinity probes, target molecule and detection enhancement probe to be drop cast and subsequently incubated.

[0121] The four interconnected microchannels 2 share a common inlet 101 in the center, and have separate outlets 102’, 102”. The biosensor is installed on a chip manifold 103 to ensure a seamless microfluidic interfacing. A platinum hollow tube electrode 105a is placed at the inlet 101 of the microchip 100 and another one 105b, 105c is placed at each outlet 102’, 102” to help measure the streaming current from each of the four channels 2.

[0122] The schematic of the experimental setup consisting of the chip manifold 103, Platinum electrodes iO5a-c, digital ammeter 106, pressure regulator 107, flow sensor 108, analyte reservoir 109, and data acquisition system no.

[0123] The main components used for the measurements are listed below:

[0124] - Keithley picoammeter (model no. 2636A)

[0125] - Elveflow OBi flow controller

[0126] - flow sensor (Elveflow, MSF3)

[0127] The silicon-based microchip was prepared according to the description in Gevari et al. 2024, which is incorporated herein as reference.

[0128] Immobilization of affinity probes

[0129] Prior to immobilizing the affinity probes, the microchip surface was cleaned using a solution of H2O, H2O2, and NH4OH (ratio 5:1:1) at 8o°C for 20 minutes. Following the cleaning, the microchip was immersed overnight in a 1 mg / mL solution of commercially purchased silane-PEG-thiol (SPT) dissolved in 95% ethanol. During this step, the silane molecules underwent a hydrolysis reaction, forming a coating on the microchip surface. After thorough washing, the microchip was incubated for 1 hour with a 100 nM solution of thiol-conjugated PNA probes. The thiol groups on the SPT and PNA probes participated in a disulfide bonding reaction. Finally, the chip was washed thoroughly with the buffer to remove any unbound probes and stored at 4°C for DNA detection. Sandwich assay procedure

[0130] To investigate the effect of using charged nanoparticles in the sandwich assay method according to the present disclose, two experimental set ups were conducted. In Example i, the detection probe in the sandwich assay consists of PNA probes conjugated to charged silica particles, whereas in the Comparative Example, the signal is from a direct assay, i.e. without any detection enhancement probe.

[0131] In both Example 1 and the Comparative Example, the inner surface of the microchannels were coated with affinity probes (PNA prepared as previously described) to achieve immobilization. Following this preparation, the streaming current (Li) was measured in each of the examples by flowing PBS through the channels. The signal (Li) is represented as “PNA” in Fig. 5

[0132] Similarly, in each of the examples, a PBS solution containing complementary ss-DNA as target molecules was introduced into the microchannels to allow hybridization with the PNA affinity probes. The streaming current was then measured again (Is2), after washing off the unbound ssDNA. In Fig. 5, the signal (Is2) obtained at this step is labelled as DNA. The signal represents the comparative example.

[0133] In Example 1, the detection enhancement probe according to the present disclosure, i.e. PNA+NP in a PBS solution, was provided to the microchannel and incubated. After removing any unbound detection enhancement probe, the streaming current (Is3) was measured in the same manner. The signal (Is3) is shown in the graph in Fig. 5, and labelled as NP.

[0134] It should be noted that, in a separate experiment, and to evaluate non-specific interaction between the nanoparticles and the background, the microchannel surface was coated by the PNA probes in an identical manner. Next, the detection enhancement probes (PNA+NP) was provided to assess the level of the non-specific interaction of the nanoparticles with the background. The streaming current was recorded in a similar manner after removing any unbound detection enhancement probe. The signal is represented as “ctrl” in Fig. 5. Results

[0135] Fig. 6 is a graph illustrating the results from the comparative measurements between Examples i, i.e. a sandwich assay method according to the present disclosure, i.e. as shown in Fig. 3C and a Comparative Example represented by a direct assay. The graph illustrates the sensing signal (streaming current) in detection of single stranded DNA (ss-DNA) with the solid curve showing the net signal, i.e. (AIDNA=IS3- ISI) in detection of various concentrations of ss-DNA for Example 1 i.e., using amplification with the nanoparticle-based amplification probe. The dashed curve shows the signal for the comparative example, i.e. the net signal (AIDNA=IS2-ISI) obtained from the direct assay without any amplification probe. As may be seen from this graph, the direct assay was not capable of sensing any DNA at concentrations lower than about 0.2 nM (2230 pg / mL), while the use of the detection enhancement probe according to the present disclosure enabled sensing much lower concentration of the target ss-DNA. A further optimization of the method (i.e. the use of detection amplification probe) allowed to detect up to a concentration of 8.5 pM (95 pg / mL).

[0136] REFERENCES

[0137] • Adamczyk, Z., Sadlej, K., Wajnryb, E., Nattich, M., Ekiel-Jezewska, M.L., Blawzdziewicz, J., 2010. Adv. Colloid Interface Sci. 153, 1-29.

[0138] • Afsahi, S., Lerner, M.B., Goldstein, J.M., Lee, J., Tang, X., Bagarozzi, D.A., Pan, D., Locascio, L., Walker, A., Barron, F., Goldsmith, B.R., 2018. Biosens. Bioelectron. 100, 85-88.

[0139] • Cavallaro, S., Horak, J., Haag, P., Gupta, D., Stiller, C., Sahu, S.S., Gorgens, A., Gatty, H. K., Viktorsson, K., El-Andaloussi, S., Lewensohn, R., Eriksson Karlstrom, A., Linnros, J., Dev, A., 2019. ACS Sensors acssensors.

[0140] • Dev, A., Horak, J., Kaiser, A., Yuan, X., Perols, A., Bjork, P., Karlstrom, A.E., Kleimann, P., Linnros, J., 2016. Biosens. Bioelectron. 82, 55-63.

[0141] • Gevari M.T., Sahu, S.S., Stridfeldt F., Haag, P., Petris L.D., Viktorsson K., Lewensohn R., Gori A., Cretich M., Dev A., 2024. ACS Sens. 9, 2935-2945.

[0142] • Juncker, D., Bergeron, S., Laforte, V., Li, H., 2014. Curr. Opin. Chem. Biol. 18, 29-37-

[0143] • Li, Y., Lai, S.N., Zheng, B., 2018. Sensors Actuators B Chem 259, 871-877.

[0144] • Lichtenberg, J.Y., Ling, Y., Kim, S., 2019. Sensors (Switzerland).

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Claims

25CLAIMS1. A sandwich assay method for detecting the presence of a target molecule (5) in a microfluidic device (1) comprising a micro- or nanoscale channel (2), the method comprising: a) Providing the microfluidic device comprising the micro- or nanoscale channel, wherein an affinity probe (4) with affinity for the target molecule is immobilized to an inner surface (3) of the micro- or nanoscale channel; b) Providing a solution comprising the target molecule into the micro- or nanoscale channel such that binding of the target molecule to the affinity probe is enabled; c) Providing a second solution with a detection enhancement probe (7) into the micro- or nanoscale channel, the detection enhancement probe comprising a nanoparticle (9) and one or more detection probes (6) with affinity for the target molecule, such that binding of one of the one or more detection probes to the target molecule is enabled; and d) Detecting the presence of the detection enhancement probe along with the target molecule and the affinity probe by recording the changes in streaming current or streaming potential.

2. The sandwich assay method according to claim 1, wherein the one or more detection probes is / are conjugated to the surface of the nanoparticle.

3. The sandwich assay method according to claim 1, wherein 5 or more detection probes are conjugated to the surface of the nanoparticle.

4. The sandwich assay method according to any one of the preceding claims, wherein the nanoparticle is a charged nanoparticle.

5. The sandwich assay method according to claim 4, wherein a zeta potential of the charged nanoparticle is positive or negative and has a magnitude ranging from 5 mV to 100 mV, optionally from 10 mV to 80 mV, as measured according to electrophoretic mobility by using Electrophoretic Light Scattering (ELS), in a PBS solution and at a pH 7,4, as described herein.

6. The sandwich assay method according to any one of the preceding claims, wherein the nanoparticle has a diameter of 5 nm or more, as measuredaccording to the Dynamic Light Scattering (DLS) method, optionally within the range of from 5 nm to 500 nm.

7. The sandwich assay method according to any one of the preceding claims, wherein the nanoparticle is a metal or metal oxide nanoparticle or a polymeric nanoparticle.

8. The sandwich assay method according to any one of the preceding claims, wherein the first and / or second solution is an electrolyte solution with ion concentration within the range of from 1 mM to 2 M.

9. A displacement assay method for detecting the presence of a target molecule (15) in a microfluidic device (1) comprising a micro- or nanoscale channel (2), the method comprising: a) Providing the microfluidic device comprising the micro- or nanoscale channel, wherein a complementary probe (14) having a sequence complementary to the target molecule is immobilized to an inner surface (3) of the micro- or nanoscale channel, the complementary probe being conjugated with an affinity probe (16) comprising a nanoparticle (9); b) Determining streaming current or streaming potential in the micro- or nanoscale channel (2), thereby recording a baseline; c) Providing a solution comprising the target molecule into the micro- or nanoscale channel such that displacement hybridization of the target molecule to the complementary probe is enabled, via release of the affinity probe; and d) Detecting the presence of the complementary probe-target molecule (14,15) by recording the changes in streaming current or streaming potential.

10. The displacement assay method according to claim 9, wherein the nanoparticle is a nanoparticle according to any one of claims 4 to 7. ii. A kit for use in a sandwich assay method for detecting the presence of a target molecule in a microfluidic device comprising a micro- or nanoscale channel, the kit comprising:- an affinity probe with affinity for the target molecule, which affinity probe is capable of being immobilized to an inner surface of a micro- or nanoscale channel- a detection enhancement probe comprising a nanoparticle and one or more detection probes with affinity for the target molecule.

12. A kit for use in a displacement assay method for detecting the presence of a target molecule (15) in a microfluidic device comprising a micro- or nanoscale channel (2), the kit comprising:- a complementary probe (14) comprising a sequence complementary to the target molecule, which complementary probe is capable of being immobilized to an inner surface (3) of a micro- or nanoscale channel, wherein:- the complementary probe is conjugated with an affinity probe comprising a nanoparticle; or- the kit comprises an affinity probe comprising a nanoparticle, the affinity probe having affinity for the complementary probe.

13. The kit according to claim 11, wherein the one or more detection probes is / are conjugated to the surface of the nanoparticle.

14. The kit according to claim 11, wherein 5 or more detection probes are conjugated to the surface of the nanoparticle.

15. The kit according to any one of claims 11 to 14, wherein the nanoparticle is a charged nanoparticle.

16. The kit according to claim 15, wherein a zeta potential of the charged nanoparticle is positive or negative and has a magnitude ranging from 5 mV to 100 mV, optionally from 10 mV to 80 mV, as measured according to electrophoretic mobility by using ELS, in a PBS solution and at a pH 7,4, as described herein.

17. The kit according to any one of claims 11 to 16, wherein the nanoparticle has a diameter of 5 nm or more, as measured according to the DLS method, optionally within the range of from 5 nm to 500 nm.

18. The kit according to any one of claims 11 to 17, wherein the nanoparticle is a metal or metal oxide nanoparticle or a polymeric nanoparticle.

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