A device for detecting an analyte

Abstract

A device for detecting an analyte is provided. The device comprises: an optical system having a point spread function, a substrate, a passivation layer provided on the substrate, and a plurality of nanoholes distributed within the passivation layer.

Description

[0001] A DEVICE FOR DETECTING AN ANALYTE

[0002] TECHNICAL FIELD

[0003] The present invention relates to a device for use in detection of analytes, and a method of manufacturing said device. The present invention also relates to a method of biomolecular detection, and an apparatus which can perform such biomolecular detection.

[0004] BACKGROUND TO THE INVENTION

[0005] Mass photometry (MP) is a fast-growing optical technique for label-free mass measurement of single molecules in solution / suspension based on interference of light reflected from the interface and scattered by molecules binding to or interacting with such an interface, disclosed in Young et al., “Quantitative mass imaging of single biological macromolecules”, Science 2018 360: 423-427. The signal of a landed molecule appears on the microscope camera’s field of view (FoV) as a point spread function (PSF) after ratiometric subtraction of the background. The contrast of the PSF can then be related to molecular mass.

[0006] Mass photometry has already found applications in numerous studies and applications, such as studies of intermolecular interactions, dynamics, and stability, all from a single droplet. However, these applications may have intrinsic limitations, and these may derive from uncontrolled binding of the analyte to the glass slide. For example, mass-photometry may only be able study relatively strong interactions under certain conditions. To study low-affinity interactions, higher analyte concentrations may be required, which, in turn, can lead to overcrowding of FoV by PSFs, and mass determination is not possible.

[0007] Previously, partial passivation of the interface has been implemented to try and reduce the frequency of landing events. It will generally be understood that passivation of an interface involves making it inert, substantially preventing non-specific interactions with the analyte. Passivated surfaces for single molecule experiments, preventing non-specific adsorption, have been engineered in multiple ways; one of which being the deposition of Bovine Serum Albumin (BSA) protein on the surface. However, such passivation is far from absolute and produces unwanted background in MP from non-covalently attached and desorbing molecules. Most previous approaches to surface passivation were developed for fluorescence microscopy. Analyte binding the surface for a few milliseconds and subsequently unbinding does not present any problem for fluorescence; however, such events are detected by mass photometry, which still leads to FoV overcrowding at high concentration. If a lower concentration of analyte is used, within the preferred range of the detection apparatus, the concentration of the sample may be found to be insufficient for sufficient reactions to take place to permit detection. There is therefore a requirement to develop a technique and / or a device to permit detection apparatus to operate over a greater range of concentrations, particularly concentrations above 10 nM, 20 nM or 50 nM. For example, if the analyte (e.g. protein) binds / interacts weakly with the surface then a concentration above 10 nM impairs the measurements because proteins unbound from the surface increase event density in the FoV. If the anlaytes (proteins) bind the surface well, then measurements above following concentrations start to be problematic: 20 nM for low mass proteins ~50 kDa, 50 nM for higher mass proteins - 100 kDa. The dependence on protein mass is caused by the need to have a longer averaging window for smaller proteins to distinguish them from the background.

[0008] In order to accurately identify each binding event an area of approximately 1 pm2should be free of other binding events during the averaging time of 10-200 ms. A concentration of the order of 10 nM results in about 1 binding event per 1 pm2per second which results in manageable data and image quality with single molecule mass accuracy. Thus, the analyte concentration is limited to the order of 10nM. In contrast, sample concentrations are often higher than 10nM and this limits the use of mass photometry.

[0009] The diffraction of light ultimately limits the optical performance of microscopes and restricts their ability to interrogate a single molecule in a very crowded environment. The present invention arises from the desire to solve this issue and determine characteristics of an analyte without the need to dilute samples.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention relates to methods for measuring properties of an analyte, and a device for detecting an analyte and measuring the properties and / or characteristics of said analyte. A method of manufacturing the device, as well as a system for measuring properties of the analyte, is also disclosed herein.

[0012] In a first aspect, the present invention provides a method for measuring a property of an analyte, the method comprising the steps of:

[0013] (i) providing a device comprising a substrate and a passivation layer provided on a surface of the substrate, wherein the passivation layer comprises nanoholes with a cross- sectional dimension of less than 200 nm; (ii) adding a solution comprising an analyte to the substrate such that the analyte is received by the nanoholes;

[0014] (iii) illuminating the surface of the device and detecting a signal from the analyte using a detector; and

[0015] (iv) measuring at least one property of the analyte using the detected signal.

[0016] Step (iii) of the method disclosed in the first aspect herein may further comprise measuring an electrochemical signal of the analyte. The electrochemical signal may be electrochemical impedance spectroscopy, differential pulse voltammetry, square wave voltammetry, cyclic voltammetry, chronoamperometry, open circuit potential or chronopotentiometry.

[0017] In any embodiment, detecting a signal from the analyte using a detector may rely on detecting the interference between scattered and reference light, or pure light scattering.

[0018] In a second aspect, the present invention provides a device for detecting an analyte comprising a substrate and a passivation layer, wherein the passivation is polymeric and comprises nanoholes with a cross-sectional dimension of less than 200 nm.

[0019] In any embodiment, the passivation layer may be configured such that it does not increase the scattering background above the substrate scattering background.

[0020] The device provided herein may further comprise a blocking element configured within the nanoholes of the passivation layer.

[0021] In a third aspect, the present invention provides a method of manufacturing the device according to the second aspect of the invention disclosed herein, the method comprising the steps of:

[0022] (a) depositing at least one blocking element onto the surface of a substrate such that a first part of the substrate is covered by the blocking element and a second part of the substrate is exposed;

[0023] (b) depositing a passivation layer onto the second part of the substrate;

[0024] (c) removing the blocking element from the surface of the substrate to provide one or more nanoholes exposing the surface of the substrate.

[0025] In some embodiments, the blocking elements may be deposited with an average nearest neighbour distance of 1.0-1.1 , 1.1-1.5, 1.5-2.0, 2-10, 10-100 or more times the cross-sectional dimension of the blocking element. The blocking element according to the methods disclosed herein may be a spherical particle, a nanoparticle with flat facets, or a nanocube. The blocking element may have a cross- sectional dimension of <10 nm (less than 10 nm), 10-50 nm, 50-100 nm, 100-200 nm or 200- 1000 nm.

[0026] Step (c) of the method disclosed in the third aspect herein may comprise removing the blocking element from the surface of the substrate by sonication, dissolution, photodegradation, illumination, use of magnets, photocleavage, or chemical cleavage.

[0027] In any embodiment, it is possible to adjust the diameter and / or density of one or more nanoholes, wherein the diameter of the nanohole may be adjusted by changing the shape and / or cross-sectional dimension of the blocking element, and, wherein the density of the nanoholes may be adjusted by the changing the surface density of the blocking elements. The diameter of the nanohole may be adjusted by changing the shape of the blocking element. The diameter of the nanohole may be adjusted by changing the cross-sectional dimension of the blocking element. The density of the nanoholes may be adjusted by the changing the surface density of the blocking elements.

[0028] The size or density of the one or more nanoholes may be adjusted according to the analyte concentration to be tested and / or the point spread function (PSF) of the analyte. The size of the one or more nanoholes may be adjusted according to the analyte concentration to be tested. The size of the one or more nanoholes may be adjusted according to the point spread function (PSF) of the analyte. The density of the one or more nanoholes may be adjusted according to the analyte concentration to be tested. The density of the one or more nanoholes may be adjusted according to the point spread function (PSF) of the analyte.

[0029] In a fourth aspect, the present invention provides a system for measuring a property of an analyte, the system comprising:

[0030] (I) a device for detecting an analyte according to the second aspect of the invention disclosed herein;

[0031] (II) an illumination source for illuminating the surface of the device;

[0032] (III) a detector of scattered light for detecting a signal from the analyte.

[0033] In any embodiment, it is provided that the surface area of the nanoholes to the surface area of the passivation layer is at a ratio of between 1 : 10000 and 1 :1. The methods, devices and systems disclosed herein allow for the measuring of a property of the analyte to include (i) quantifying the mass of the analyte and / or (ii) measuring or quantifying a change in the mass of the analyte.

[0034] In any embodiment, it is provided that the analyte binds to the surface of the substrate exposed by the nanoholes. The surface exposed by the nanoholes may comprise at least one substance for binding to the analyte. The surface exposed by the nanoholes may comprise at least one substance for binding to the analyte, and optionally, the substance may fill or partially fill the nanohole.

[0035] In any embodiment, the surface exposed by the nanoholes may be coated, derivatised or modified to increase analyte binding. The surface exposed by the nanoholes may be coated by a substance comprising amino groups, carboxyl groups, thiol groups, His-tags, N- hydroxysuccinimide esters, sulfo-NHS esters, imidoesters, maleimide, haloacetyl, pyridyl disulfide, succinimide, hydrazide, diazirine, aryl azide, benziphenones, azides, alkynes, or biotin.

[0036] Increased analyte binding may be due to covalent binding using PEG-mixtures coated, derivatised or modified onto the surface of the substrate exposed by the nanoholes.

[0037] In a fifth aspect, the present invention provides a device for detecting an analyte, the device comprising: an optical system having a point spread function, a substrate, a passivation layer provided on the substrate, and wherein a plurality of nanoholes is distributed within the passivation layer.

[0038] The device reduces the analyte binding to the substrate by the provision of a passivation layer. The device is configured to measure signal given by individual analyte particles i.e. single molecule measurement.

[0039] The passivation layer may be a polymeric brush passivation layer. The separation of the nanoholes may exceed the point spread function of the optical system.

[0040] For larger holes, in the region of 100nm, with a separation that exceeds the point spread function of the optical system.

[0041] For smaller holes, in the region of 30nm, the separation can be slightly smaller than the point spread function diameter. The smaller hole implementation better exploits the entire surface of the passivation layer. In a sixth aspect, the present invention provides a method for measuring a property of an analyte, the method comprising the steps of:

[0042] (i) providing a device according to the fifth aspect disclosed herein;

[0043] (ii) adding a solution comprising an analyte to the substrate such that the analyte is received by the nanoholes;

[0044] (iii) illuminating the surface of the device and detecting a signal from the analyte using a detector; and

[0045] (iv) measuring at least one property of the analyte using the detected signal.

[0046] The methods, devices and systems disclosed herein provide that the detected signal can comprise light interference from the analyte and the substrate. The detected signal can also be pure light scattering from the analyte.

[0047] The methods disclosed herein may further comprise the step of collecting one or more images of the analyte. The images collected can then be used to determine a point spread function (PSF) contrast of each of the analyte within a field of view (FoV) of said image.

[0048] In any embodiment, the dimension of the nanohole can be smaller than the point spread function (PSF) area of the analyte. The nanoholes may be arranged such that the PSF area of the analyte comprises a plurality of nanoholes. The nanoholes may have a cross-sectional dimension of less than 10 nm, 10-20 nm, 20-50 nm, 50-100, or 100-180 nm.

[0049] In any embodiment related to the methods, devices and systems disclosed herein, the analyte sample may be a biomolecule sample, polymeric sample, or inorganic sample. The analyte sample may include one type of analyte, or may include multiple different analyte types. The analyte sample may have a concentration range selected from: 0.05-0.20 pM, 0.2-1.0 pM, 1- 10 pM, 10-100 pM, or 0.1-1.0 mM or higher.

[0050] In embodiments, the property measured may be a low-affinity interaction between the same or different analytes.

[0051] In any embodiment, the analyte property may be measured from a plurality of nanoholes or from individual nanoholes. For the measuring of a property of an analyte to be possible, at least one nanohole should comprise an analyte.

[0052] In any embodiment of the disclosed methods, devices and systems there is provided a passivation layer comprising 1 to 5, 5 to 10, 10 to 20, 20 to 50, or 50 or more nanoholes per pm2. These nanoholes may then be distributed over the passivation layer randomly or in a geometric pattern / arrangement.

[0053] In any embodiment, the passivation layer may be polymeric and optionally provided onto the surface of the substrate by polymer brushes, plasma polymer coatings, hydrophobic coating with tween-20, zwitterion polymer brushes, or polyelectrolyte multilayers.

[0054] In any embodiment, the passivation layer may be polymeric and comprises nanoholes with a cross-sectional dimension of less than 200 nm.

[0055] In any embodiment, the passivation may be selected from: poly-L-lysine-g-poly(ethylene glycol) (PLL-g-PEG), methyl terminated PEG (mPEG).

[0056] In any embodiment, the passivation layer has a thickness in the ranges of 1-10 nm, 10-20 nm, or 20-100 nm.

[0057] The substrate of the methods, devices and systems disclosed herein may be transparent and the transmittance of said substrate may be 1-35 %, 35-65 %, 65-80 %, or 80-100 % for at least one wavelength of the illumination device. Preferably, the substrate can be selected from a plastic slide or a glass slide. However, the substrate material can be selected from: polymeric, glass, quartz glass, fused silica, borosilicate glass, BK7, sapphire, CaF2, SiO2, TiO2, mica, graphene, germanium, ZnSe, KBr, KRS-5, silicon, or transparent conductive oxides as indium- doped tin oxide, fluorine-doped tin oxide, aluminium-doped zinc oxide, zinc oxide, and cadmium oxide.

[0058] FIGURES

[0059] Figure 1A includes representations of both side-view and top-view of a device 100 for detecting an analyte according to the present invention. The device 100 comprises a substrate 101 that is overlaid with a passivation layer 103 comprising a plurality of nanoholes 105. The passivation layer is a coating which reduces the binding of the analyte. In use, a solution including analyte particles 107 is introduced into the device 100 such that the analyte particles 107 come into contact with the passivated surface of the device 100. The analyte particles 107 can be received by the nanoholes 105 or the analyte particles 107 will rebound from the passivation layer 103. The passivation layer 103 ensures that the analyte particles 107 do not bind to the surface between the nanoholes 105. The nanoholes therefore dictate the separation of the analyte particles 107. An illumination source 113 provides the light, which is scattered by analyte particle 107 and this scattered light 115 is received by a detector 117. The optical system comprising the illumination source 113 and the detector 117 has a Point Spread Function that is inherent to the optical components. The light scattered from each analyte particle 107 is a point source of light. However, it is spread due to the Point Spread Function of the optical system. The nanoholes 105 are spaced such that the scattered light 115 from the analyte particle 107 in each nanohole does not overlap with the scattered light 115 from the adjacent analyte particles in adjacent nanoholes 105. With reference to the top view, the separation of the nanoholes 105 is random, rather than geometrical. The critical aspect of the arrangement of the nanoholes 105 is that the scattered light from the analyte particle in each nanohole does not interfere with the signal from adjacent nanoholes 105, or the probability of this interference is significantly reduced.

[0060] The density of holes is given by the hole area / surface area ratio and hole size. When there is a need to drop landing rate by factor of 1:1000 - 1:10, the same hole area / surface area ratios are required. The minimum size to fit protein is 5 nm, meanwhile, maximum size is 100 nm which is roughly an area of1 / io of PSF area. A 5 nm hole have 79 nm area to achieve 1:10 ratio, we need to have 1266 holes per pm2. To achieve 1:1000 ratio, 13 holes per pm2are required. With a 100 nm hole, it is 10 holes per pm2although 1 hole per pm2will work. Therefore, the most usable range is 10-1266 per pm2.

[0061] Figure 1B depicts measurement principle involving incident and reflected light 120 and scattered light 115. Figure 1 B shows (I) standard measurement at high concentration and (II) nanohole measurement at high concentration. (I) shows that the absence of a passivated surface comprising nanoholes results in the overlapping of point spread functions 121 within the detector field of view 123. (II) shows that a passivation layer 103 comprising nanoholes 105 leads to individual point spread functions 129 that do not overlap.

[0062] Figure 1C shows a representation of the side-view of the passivated device 100. The device 100 comprises a substrate 101 with a passivation layer 103. The nanoholes 105 within the passivation layer 103 are filled with an analyte binding substance 135. The analyte particles 107 can bind to the analyte binding substance 135 in order to be localised to the nanohole 105. Those analyte particles 107 that are not bound to the analyte binding substance 135 will remain in solution as they cannot bind to the passivation layer 103.

[0063] Figure 1D shows a representation of a side-view of the passivated device 100. A plurality of blocking elements 141 are added to the surface of the substrate 101 in order to define the intended locations of the nanoholes 105. The passivation has then been implemented via the provision of a polymer brush 103a which covers the substrate 101 except in the locations of the blocking elements 141. The blocking elements 141 can then be removed to reveal the nanoholes.

[0064] Figure 1 E shows a substrate 101 with a polymer brush passivation layer 103a and a polymeric brush binding substance 106a within the nanoholes 105 in the passivated layer 103a.

[0065] Figure 1 F illustrates an alternative morphology of nanoholes 105 in which a substrate 101 is provided with a polymer brush passivation layer 103a and a biomolecule binding substance 106b within the nanoholes 105 in the passivation layer 103a.

[0066] Figure 1G illustrates different arrangements of nanoholes 105, showing the top view of a passivation layer 103 with nanoholes 105, wherein the nanoholes 105 are spaced via (i) rectangular spacing, (ii) triangular spacing, or (iii) random spacing. The average nearest neighbour distance X of the nanoholes 105 is illustrated. The ratio of hole area in field of the view I area of field of the view defines the drop in number of counts detected at certain concentration, which lowers the probability of overlapping of point spread functions.

[0067] Random spacing is efficient in configurations where the distance between holes is less than Point Spread Function diameter and allows exploiting entire field of the view in the experiment. The provision of a large number of small holes will decrease the adhesion of the surface so only around 1 % of molecules will bind at a certain timepoint and location. The advantage of geometric configuration is in cases where distance between holes is more than Point Spread Function diameter, where it ensures that the minimum distance between the nanoholes 105 can be aligned exactly with the Point Spread Function of the optical system.

[0068] Figure 1 H illustrates different blocking element styles and arrangements. The blocking element 141 can be a spherical blocking element 141a, a cubical blocking element 141 b, or a flat facet-containing blocking element 141c.

[0069] Figure 11 shows closely packed blocking elements 141 on the device stopping the passivation of the substrate’s surface. In the case of spherical blocking elements, the nanoholes are arranged in triangular arrangement. In the case flat facet containing blocking elements, this can provide a larger nanohole that has a lateral extent spanning several blocking elements 141.

[0070] Figure 1J shows sparsely distributed blocking elements 141 with easily diffusing passivation brushes 103a around the blocking elements 141. Figure 2. Comparing empty nanoholes with covalent capture by substance-increasing binding, a., b. Principle of nanohole measurement for low affinity interaction of dimerization of CRP pentamer, c. histogram showing the two individual peaks of monomer and dimer of CRP pentamer that can be used for evaluation of dissociation constant 2.6 pM . d. Nanoholes filled by maleimide-PEG brush MW5000 with size 2-4 nm that increases analyte binding, e. Frame from measurement, f. Analysed spectra at high micromolar concentration.

[0071] Figure 3. Comparison of standard mass photometry, mass photometry using fully passivating surface and nanocube lithography of passivated surfaces for high concentration mass photometry, a, Principle of standard mass photometry including an MP image of 10 nM monoclonal SARS-CoV-2 spike antibody on a standard aminated microscope coverslip providing a substrate 101. b, Equivalent snapshot at 1 pM analyte concentration, c, Density map of landing events for a 30-second recording at 10 nM antibody concentration, d, Schematic of high-density covalent PEGylation and resulting passivation mechanism - cloudpoint PEGylation of an animated coverslip as substrate 101 is shown, e, Corresponding MP image at 1 pM antibody concentration, f, Resulting landing density map. g, Principle of nanoparticle-assisted nanopatterning of the PEGylated glass surface providing the substrate 101 , with the nanohole locations defined by Au NC blocking elements 141 and a polymer brush passivation layer 103a being provided. Following sonication, the Au NC blocking elements are removed providing nanoholes 105 into which analyte particle 107 can move. Any analyte particles 107 that come into contact with the polymer brush passivation layer 103a are repelled and remain in solution. The mechanism of measurement on such a partially passivated surface is also illustrated, h, Corresponding MP image at 1 pM antibody concentration, i, Resulting landing density map including close-ups of regions with high landing density.

[0072] Figure 4. Application of nanocube lithography, a. Nanocube blocking elements on surface, before and after removal, b. comparison of landing rates of antibodies on glass, nanocube- nanohole-PEG and fully passivating-PEG. c. comparison of spectra at concentration of 10 and 1000 nM of glass and 1000 nM on nanocube-nanohole-PEG.

[0073] Figure 5. Passivation tunability with silica nanospheres (SNPs). a, Schematic and corresponding raw cover glass images during 1 seconds and 10 minutes of binding of 40 pg cm-2of 100 nm SNPs. The relative reflectivity ranges on the X-axis of the 1s plot correspond to counts x104values in the range 0.14 to 1.41 and for the 10 minute plot correspond to counts x104values in the range 0.47 to 3.21. b, Comparison of raw cover glass images with and without the PEG passivation layer. The relative reflectivity ranges on the X-axis of the glass plot correspond to counts x104values in the range 2.48 to 4.13 and for the nanohole-PEG plot correspond to counts x104values in the range 2.56 to 4.46 c, Landing density map for antibodies at 1 pM concentration obtained with 40 pg cm-2SNPs. d-f, MP images of antibody samples at 100 nM and 1000 nM concentrations using different SNP concentrations and sizes, g, Comparison of antibody mass distributions on standard aminated microscope coverslip and nanopatterned PEG obtained with 40 pg cm-2SNPs. h, Resulting landing rates for silica nanospheres with 100 and 50 nm in diameter. The dashed lines indicate landing rates for PEG without nanoholes.

[0074] Figure 6. Nanopatterned PEG performance for high concentration mass photometry, a, b Comparison of MP images and resulting mass histograms of C-reactive protein at 50 nM on standard cover glass and at 500 nM on nanopatterned PEG. c, Pentamerdecamer ratio and associated KD as a function of monomer concentration, d, e Comparison of MP images and resulting mass histograms of 1 :1 FcRn: Herceptin mixtures on standard cover glass and on nanopatterned PEG at indicated dilutions and pH. f, Resulting complex fraction and KD as a function of monomer concentration.

[0075] Figure 7. Mass histogram of Sars-CoV-2 antibodies measured at 4 individual nanoholes prepared using blocking elements of Au 100 nm nanocubes, subsequent PEGylation and sonication. Peak for Hole 1 (1 min) = 149 kDa, o 23 kDa, 216 counts (81%). Peak for Hole 2 (1 min) = 145 kDa, o 20 kDa, 142 counts (80%). Peak for Hole 3 (1 min) = 153 kDa, o 21 kDa, 161 counts (87%). Peak for All (5 s) = 149 kDa, a 123 kDa, 212 counts (77%).

[0076] Figure 8. illustrates the concept of membrane protein measurements in detergents, a, Principle of mass photometry for soluble proteins in the water-based buffer. Protein landing on a glass substrate 101 generate a ratiometric signal in the form of point spread functions at the field of view, which contrast linearly scales with protein mass, (a) Incident and reflected light and (b) scattered light shown, b, When insoluble membrane proteins are rapidly diluted into the water-based buffer, proteins denaturate and cannot be measured, c, The resulting mass histogram shows various masses, d, Soluble proteins in detergent cannot be measured thanks to increased noise caused by micelles landing on a glass substrate 101 , which obscure signals from proteins, e, Membrane proteins have a more significant mass. Therefore, it is detectable on top of the noise, f, A histogram of membrane proteins show two symmetrical noise peaks around 220 kDa caused by micelles landing and one peak at 369 kDa of the membrane proteins; however, the peak is very broad, g, Passivation coating with nanoholes generated by its pre-illumination by laser (2m) 803 allows for passivating micelles from the surface while the soluble proteins are landing on the passivated substrates surface with nanoholes caused by illumination, h, The same surface is used for the measurement of membrane proteins with diminished signal from micelles and clear binding events from proteins, i, Histogram showing one clear peak at 323 kDa belonging to a membrane protein.

[0077] Figure 9. Reducing the noise of detergents by surface passivation, a-c, Ratiometric images of different detergents of GDN, DDM, and Tween-20 on glass substrates, d-f, Ratiometric images of different detergents on PEGylated substrates, dramatically reducing the noise, g-i, quantification of noise mass equivalent of different detergents measured on standard and passivated substrates. Data shows in all the cases that the noise was diminished by 5-1 Ox. From this data the conclusion can be drawn that the developed method is detergent independent.

[0078] Figure 10. Protein- protein interactions observed in ammonium acetate buffer with 2x CMC GDN detergent, a, Comparison of ratiometric frames of ammonia transporter membrane protein rapidly diluted from detergent into PBS on a glass substrate and measured directly in detergent using a passivated surface activated by pre-illumination. b, Comparison of histograms acquired on glass substrate and passivated substrate surface, c, Comparison of ratiometric frames of p-Barrel Assembly Machinery Complex of rapid dilution on glass substrate and equilibrium measurement on passivated substrate surface, d, Comparison of histograms acquired on glass substrate and passivated substrate.

[0079] Figure 11. Functionalized nanoholes, a, Principle of measurement on nanohole surface with nanoholes filled by Azide-PEG polymer, which are functionalized by NHS-DBCO linker, b, Ratiometric image of MSCL membrane protein in GDN detergent binding the surface, c, Mass spectra of MSCL protein, d, Ratiometric image of BacA protein in GDN detergent, c, corresponding mass spectra.

[0080] DETAILED DESCRIPTION OF THE INVENTION

[0081] In a first aspect, the present invention provides a method for measuring a property of an analyte, the method comprising the steps of:

[0082] (i) providing a device comprising a substrate and a passivation layer provided on a surface of the substrate, wherein the passivation layer comprises nanoholes with a cross- sectional dimension of less than 200 nm;

[0083] (ii) adding a solution comprising an analyte to the substrate such that the analyte is received by the nanoholes; (iii) illuminating the surface of the device and detecting a signal from the analyte using a detector; and

[0084] (iv) measuring at least one property of the analyte using the detected signal.

[0085] Also provided herein is a method for measuring a property of an analyte, the method comprising the steps of:

[0086] (i) providing a device comprising a substrate and a passivation layer provided on a surface of the substrate, wherein the passivation layer comprises nanoholes with a cross- sectional dimension of less than 200 nm;

[0087] (ii) adding a solution comprising an analyte to the substrate such that the analyte is received by the nanoholes;

[0088] (iii) illuminating the surface of the device and detecting a signal from the analyte using a detector; and

[0089] (iv) measuring at least one property of the analyte using the detected signal, including quantifying the mass of the analyte and / or measuring or quantifying a change in mass of the analyte.

[0090] Further provided herein is a method for measuring a property of an analyte, the method comprising the steps of:

[0091] (i) providing a device comprising a substrate and a passivation layer provided on a surface of the substrate, wherein the passivation layer comprises nanoholes with a cross- sectional dimension of less than 200 nm;

[0092] (ii) adding a solution comprising an analyte to the substrate such that the analyte is received by the nanoholes;

[0093] (iii) illuminating the surface of the device and detecting a signal from the analyte using a detector;

[0094] (iv) measuring at least one property of the analyte using the detected signal; and

[0095] (v) collecting one or more images of the analyte.

[0096] The methods disclosed herein may further comprise the steps of determining a point spread function (PSF) contrast of each of the analyte within a field of view (FoV) of one or more images collected.

[0097] In a second aspect, the present invention provides a device for detecting an analyte comprising a substrate and a passivation layer, wherein the passivation is polymeric and comprises nanoholes with a cross-sectional dimension of less than 200 nm. Further provided herein is a device for detecting an analyte comprising a substrate and a passivation layer, wherein the passivation layer is polymeric and has a thickness in the range of 1 to 10 nm, and wherein the passivation layer comprises nanoholes with a cross-sectional dimension of less than 200 nm.

[0098] Also provided herein is a device for detecting an analyte comprising a substrate and a passivation layer, wherein the passivation is polymeric and comprises one or more nanoholes with a cross-sectional dimension of less than 200 nm.

[0099] In any embodiments, the nanoholes may be distributed less than 100 nm apart.

[0100] In a third aspect, the present invention provides a method of manufacturing the device according to the second aspect of the invention disclosed herein, the method comprising the steps of:

[0101] (a) depositing at least one blocking element onto the surface of a substrate such that a first part of the substrate is covered by the blocking element and a second part of the substrate is exposed;

[0102] (b) depositing a passivation layer onto the second part of the substrate;

[0103] (c) removing the blocking element from the surface of the substrate to provide one or more nanoholes exposing the surface of the substrate.

[0104] The methods and devices disclosed herein may comprise a passivation layer which is transparent. The substrate provided in the methods and devices herein may also be transparent, preferably the substrate being a glass slide or a plastic slide.

[0105] In a fourth aspect, the present invention provides a system for measuring a property of an analyte, the system comprising:

[0106] (I) a device for detecting an analyte according to the second aspect of the invention disclosed herein;

[0107] (II) an illumination source for illuminating the surface of the device;

[0108] (III) a detector of scattered light for detecting a signal from the analyte.

[0109] In a fifth aspect, the present invention provides a device for detecting an analyte, the device comprising: an optical system having a point spread function, a substrate, a passivation layer provided on the substrate, and a plurality of nanoholes distributed within the passivation layer.

[0110] The point spread function of the device becomes apparent when the analyte binds within the nanohole and rather than recording the emitted signal as a point source, it is instead spread as a function of the optical setup. The distribution of the nanoholes is selected such that the probability of intersection of recorded signals from adjacent nanoholes is minimized and in the same time the most of the field of the view is utilized for the signal recording.

[0111] The surface reflectivity under measurement conditions of coated substrate with the passivation layer and provided with the nanoholes may be of the same order of magnitude as the substrate.

[0112] Alternatively, or additionally, the variations of surface reflectivity within the length scale of 200 nm - 20 pm under measurement conditions of coated substrate with the passivation layer and provided with the nanoholes may be of the same order of magnitude as the substrate.

[0113] Increasing the surface reflectivity and / or variations of the substrate reflectivity could lead to an increase of photons seen by the imaging device, for example a mass photometer, resulting in an oversaturated camera and an unclear image.

[0114] Variations of the substrate reflectivity can be estimated for example as (i) arithmetic average of absolute deviations from the mean, (ii) Root Mean Square (square root of the arithmetic mean of the squares of the values), (iii) average difference between the highest peak and lowest valley in multiple sampling lengths.

[0115] This alignment of optical properties before and after the coating process differs considerably from other coating procedures which intentionally change the optical properties of the surface. In contrast with, for example, the provision of gold substrate on a PDMS substrate, which changes the reflectivity by several orders of magnitude, the intention of the present invention is to maintain the optical properties of the substrate as closely as practically possible.

[0116] In a sixth aspect, the present invention provides a method for measuring a property of an analyte, the method comprising the steps of:

[0117] (i) providing a device according to the fifth aspect disclosed herein; (ii) adding a solution comprising an analyte to the substrate such that the analyte is received by the nanoholes;

[0118] (iii) illuminating the surface of the device and detecting a signal from the analyte using a detector; and

[0119] (iv) measuring at least one property of the analyte using the detected signal.

[0120] The substrate of the methods, systems or devices disclosed herein, can comprise any suitable material.

[0121] A substrate is suitably solid (i.e., not a gel or a liquid). Suitably the substrate allows transmission of ultraviolet light (which may be defined herein as having wavelengths in the range from 10nm to 380 nm); visible light (which may be defined herein as having wavelengths in the range from 380 nm to 740 nm); and / or infrared light (which may be defined herein as having wavelengths in the range from 740 nm to 300 pm). Suitably, the substrate allows transmission of light in the visible light spectrum. Suitably, the substrate is substantially transparent. Suitably, the substrate does not contain metal.

[0122] The substrate of the present invention can be transparent and have a transmittance of between 1-35 %, 35-65 %, 65-80 %, or 80-100 % for at least one wavelength of the device.

[0123] A substrate may be of any suitable material, including for example but without limitation glass, glass derivatives, crystal, quartz, fused silica, borosilicate glass, BK7, sapphire, CaF2, SiC>2, TiC>2, mica, graphene, germanium, ZnSe, KBr, KRS-5, diamond, plastic, polymeric material (e.g. cyclic olefin copolymer, polyvinyl, polyethylene (PE) including for example polyethylene terephthalate (PET) and high-density polyethylene (HDPE) and low-density polyethylene (LDPE), polyacrylate (acrylic), polystyrene (PS) including high impact polystyrene (HIPS), silicone, polyester (for example polylactic acid (PLA) or polylactic coglycolic acid (PGLA)), polyurethane, polypropylene (PP), polyamide (nylon), Acrylonitrile butadiene styrene (ABS), Polyethylene / Acrylonitrile Butadiene Styrene (PE / ABS), bakelite, rubber, latex, polycarbonate (PC), Polycarbonate / Acrylonitrile Butadiene Styrene (PC / ABS) and polyvinyl chloride including for example polyvinylidene chloride (PVDC), or a transparent conductive oxide such as indium-doped tin oxide, fluorine-doped tin oxide, aluminium-doped zinc oxide, zinc oxide, and cadmium oxide.

[0124] Preferably, the substrate may be of any suitable material selected from: polymeric, glass, quartz glass, fused silica, borosilicate glass, BK7, sapphire, CaF2, SiO2, TiO2, mica, graphene, germanium, ZnSe, KBr, KRS-5, silicon, or transparent conductive oxides as indium- doped tin oxide, fluorine-doped tin oxide, aluminium-doped zinc oxide, zinc oxide, and cadmium oxide.

[0125] A substrate may have any suitable geometry. For example, the substrate may comprise a flat plate, such as a coverslip or slide, or may be a well, plate, channel, container, flow cell, flow chamber, microfluidic cell or chamber, or slide. The substrate may be part of a larger geometry or device. A suitable substrate may be a glass coverslip or a plastic coverslip, wherein the plastic is as described above.

[0126] A substrate preferably forms part of a sample holder. Said sample holder may be an element of a light scattering microscope. The sample holder may be a high surface-to-volume chamber.

[0127] The substrate comprises a surface. A surface for use in the present invention may be any suitable surface which may be passivated and is compatible with the chosen detection method. A suitable surface may be capable of having bound thereto or immobilised thereon a passivating agent.

[0128] Preferably, the surface is planar or substantially planar. The surface may be curved or include some curvature, for example a concave depression or convex structure on a substantially planar surface.

[0129] A passivation layer is provided on a surface of the substrate. Passivation is the process of treating or coating a surface to reduce the chemical reactivity, thus decreasing the number of binding events. Those skilled in the art will be aware of suitable passivating agents, examples of which include BSA, polyvinylphosphonic acid (PVPA), Dichlorodimethylsilane (DDS) and / or polyethylene glycol (PEG). The passivation prevents analytes such as proteins from non- specifically interacting with the surface of the substrate.

[0130] Surface passivation may be achieved by coating or treating the substrate, such that a layer is formed on the surface. A passivation layer may be added to a surface of the substrate. The material used in surface passivation may be any suitable molecule, for example, polymer brushes (including PEG), zwitterionic materials (including polyelectrolytes), surfactants and detergents (including tween-20), lipids (forming lipid bilayers), proteins (including BSA), plasma polymers (including PTFE). The passivation layer may comprise polyethylene glycol (PEG), for example, poly-L-lysine-g- polyethylene glycol (PLL-g-PEG), a covalently linked methyl terminated PEG (e.g., mPEG- 5000-SVA).

[0131] Passivation of the substrate may occur via PEGylation (e.g. cloud-point PEGylation), polymer brushes in general, plasma polymer coatings, hydrophobic coating with tween-20TM, zwitterionic polymer brushes, or polyelectrolyte multilayers.

[0132] PEGylation is the process of attaching the strands of the polymer PEG to molecules, most typically peptides, proteins, and antibody fragments. PEGylation may further be a process of synthesizing polymer brushes directly from the surface of the substrate.

[0133] The passivation may be provided by polymer brushes. The polymer brush passivation layer may be a zwitterion polymer brush, poly-L-lysine-g-poly(ethylene glycol) (PLL-g-PEG) or methyl terminated PEG (mPEG). Cross-linked polymer brushes may be used as the passivation layer, as long as weakly cross-linked polymer brushes are used i.e. a polymer brush where the chains are connected by relatively weak crosslinks, often ionic or hydrogen bonds, rather than strong covalent bonds.

[0134] Bulk polymers, such as polystyrene, polyethylene and polypropylene, are not effective passivation materials, moreover result in high scattering signals, making it more difficult to measure properties of the analyte.

[0135] Polymer brushes are special macromolecular structures with polymer chains densely tethered to another polymer chain (one-dimensional) or the surface of a planar (two-dimensional), spherical or cylindrical (three-dimensional) solid via a stable covalent or non-covalent bond linkage. Polymer brushes are functional and useful structures which can be formed on different scaffolds and can be used to coat a variety of surfaces. They can easily coat a surface with materials such as nanoparticles, which are increasingly important for bioengineering and medicine.

[0136] The individual molecules to form polymer brush may have mass of 0.3-100 kDa, preferably 2- 10 kDa. Contour length of such molecules may be 2 - 1 pm, preferably 10-100 nm. The passivation layer composed of polymer molecules is thinner as molecules are not fully stretched, it may have thickness 1 - 500 nm, preferably 2-50 nm. The passivation layer may be transparent. When a transparent passivation is used, the surface of the substrate is almost indistinguishable from the substrate in iSCAT / mass photometry, thereby making highly sensitive measurements of single molecules possible by minimizing the background. It may be preferred that the passivation layer has a substantially similar refractive index to either the substrate and / or the sample, as discussed further below.

[0137] The methods or devices disclosed herein may comprise a passivation layer which produces little or no reflection or scattering of the incident radiation at the boundary between the passivation layer and the sample. This can be achieved by forming the passivation layer from a substance with a refractive index that substantially matches the refractive index of the sample comprising the analyte, while being a different material to that of the sample. The passivation layer can then be ‘invisible’ to any detection apparatus due to little or no reflection at such boundary.

[0138] Mass photometry (MP) is a means for detecting and measuring the mass of single analytes and the complexes they form in solution. MP detects single molecules by their light scattering as they bind to or interact with a surface. Each binding or interaction event leads to a change in refractive index at the surface / sample interface, which effectively alters the local light scattering and can be detected with high accuracy by taking advantage of optimized interference between scattered and reflected light. The magnitude of the signal change can be converted into a molecular mass, for polypeptides with ~2% mass accuracy and up to 20 kDa mass resolution by calibration with molecules of known mass. The scattering signal is thus directly proportional to the molecule’s mass; making it possible to weigh single molecules with light.

[0139] In a standard mass photometry set-up, the device generally detects analytes at the boundary between the sample and the substrate, at the surface. The analyte may bind to / interact with the surface. The binding / interaction may permit the analyte to adsorb to the surface, which can be described as the adhesion of analytes onto the surface. The interaction may be specific or non-specific. Specific interactions can involve functionalising the surface, for example, as described further below.

[0140] The present invention works in the same way, i.e. , the only analytes detected are interacting with or binding to the (non-passivated) surface. Analytes that reside at the boundary between the passivation layer and the sample are not detected at all. The addition of the passivation layer, formed from a substance with a refractive index that substantially matches the refractive index of the sample comprising the analyte, can create a ‘mask’ over the substrate to reduce the area over which the analytes may be detected.

[0141] Due to the passivation layer, analytes within the sample can only bind to / interact with the substrate via the surface at the location of the nanohole. This means that, across the entire detected area, fewer binding events may occur. The proportional area of the nanoholes can be used to reduce the number of binding events accordingly. For example, in a standard MP experiment there are approximately 1 ,000 binding events occurring within 60 seconds on a ~30 pm2area of glass, which represents the limit at which maximum measurement precision can currently be achieved.

[0142] The reduction of substrate surface area wherein the sample analyte can bind, i.e., surface area of the substrate exposed by each nanohole, aids in reducing the background noise seen during optical detection without the need to reduce the concentration of the sample, for example via dilution. Less background noise during detection can then lead to a more accurate and defined reading of the analyte properties, as well as the possibility of conducting said detection at single molecule level.

[0143] The analyte may bind to / interact with the surface of the substrate which is exposed and nonpassivated. The binding of said analyte will occur prior to or during detection. The surface exposed by the nanohole can be functionalised by a substance that increases binding / interaction or provides specific analyte binding, such as a capture agent.

[0144] One or more analyte molecules may enter a single nanohole. This landing event changes local reflectivity of substrate. This change is small with respect to background given by scattering by glass. To quantify this change of reflectivity at that certain timepoint background needs to be removed by measuring reflectivity before and after this binding event, so called averaging window. If averaging window is too short, shot noise prevents precise quantification, if it is too long, then the slow changes of background given by measurement setup became more pronounced, which again reduces the measurement precision. In other words, typical measurement of single molecule is performed with averaging window ranging from 1-1000 ms, and the analyte molecule can only be seen on field of the view only in this averaging window around the time event of its binding to glass. This results in that there is high probability that only a single analyte molecule is being detected in a single nanohole at any one time point. Also, by providing a limited non-passivated surface of the substrate (in the form of nanoholes) reduces the landing rate of analyte molecules, in turn, significantly increasing probability that the signals of individual molecules will not overlap and single molecule measurements being possible. The ability to measure the properties of a single analyte molecule differs to other known techniques where an average signal is measured across the whole surface of the substrate.

[0145] The nanoholes expose areas of the surface of the substrate. The surface may be functionalised. The exposed surface may be, activated, coated, treated or derivatised to alter the chemistry of the surface. Functionalisation of the surface enables the determination of a particular characteristic of the analyte. Functionalisation of the surface may be to permit various interactions, such as chemical interactions (e.g., covalent bonding) or physical interactions (e.g., adsorption). For example, ligands that bind to the analyte may be present on the surface, e.g. antibodies that bind specifically to the analyte, or NHS ester that can covalently bind any biomolecule. The surface of the substrate may be treated with a material which allows adsorption of the analyte sample or allows the analyte to ionically bond prior to detection.

[0146] If the surface is coated, derivatised or modified, thin surface alterations may be desirable. Altered surface layers that are too thick can change the light scattering properties of the surface. Alteration of only the outermost few molecular layers (3-10 nm) may be desirable.

[0147] Any suitable surface coating may be applied, in order to change the chemistry of the surface. The surface may be altered to increase hydrophobicity or increase hydrophilicity. Suitable methods and coatings will be known and available to persons skilled in the art, including for example and without limitation, functionalised PEG, silanes, carboxy-silanes, amines, aminosilane, trimethylchlorosilane, aldehyde modification with amine modification reagents such as APTES, epoxy modification using for example GOPTS, carboxylate modification using for example EDC, NHS, HOBt, TBTLI, PAMAM; diazo using for example NaNO2, supramolecules for example calixarenes, or to capture by click chemistry, TCO-tetrazine, DBCO-azide. When the surface is coated, derivatised or modified the material utilised may fill or partially fill the nanohole.

[0148] A functionalised polymer brush may be added within the nanoholes on the surface of the substrate. The analyte may then bind to / interact with the functionalised polymer brush situated within the nanohole. This functionalised polymer brush added to the nanoholes may be the same or different to the polymer brush used to passivate the surface of the substrate. The polymer brush passivation layer will passivate against the functionalised polymer brush, regardless as to whether or not the same or a different polymer brush is used. When the analyte binds to the functionalised polymer brush deposited within the nanoholes, the analyte sits on top of the functionalised polymer brush for detection.

[0149] Suitably, changing the surface chemistry of the surface does not substantially affect the light transmission of the surface.

[0150] Analyte molecules can only interact / bind to the surface where such is exposed by the nanoholes in the passivation layer. In general, analytes approaching the passivated surface (outside of the nanoholes) are bounced away, which significantly reduces the landing rate and results in the observation of individual PSFs even at 1000 nM concentration.

[0151] The interaction between the analyte and the surface, whether functionalised or not, may be permanent or reversible. If reversible, the analyte may be able to dissociate on its own over time, or it may require chemical washing or other methods for the analyte to be removed from the surface.

[0152] The cross-sectional dimension or diameter of the nanoholes may be 200 nm or less. The diameter of the nanoholes may be between 1 and 200 nm, for example, the diameter of the nanoholes may be 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120 ,130, 140, 150, 160, 170, 180, 190 or 200 nm. Suitably, the nanoholes may have a diameter of 180-100 nm or less, 100-50 nm or less, 50-20 nm or less, 20-10 nm or less.

[0153] The terms “cross-sectional dimension”, “dimension” and “diameter” are used interchangeably herein when referring to nanohole size.

[0154] The nanoholes may have a constant cross-section across the depth of the passivation layer. The depth of the nanoholes will be defined by the depth of the passivation layer. For example, if the passivation layer is 1 nm in depth, the corresponding nanoholes within the passivation layer will also have a depth of 1 nm.

[0155] Nanoholes may comprise analyte-binding substances with a thickness lower, comparable, or higher than nanohole depth

[0156] The nanoholes may be cylindrical, cuboidal, pyramidal, polyhedral, or any other shape. The passivation layer may comprise between 1 nanohole in the field of the view, up to nanoholes covering half of the passivation surface area. Preferably, from 1 nanohole per point spread function area, up to 1 / 10 of the passivation surface area.

[0157] The nanoholes may be distributed randomly across the surface of the substrate or distributed in a geometric arrangement. The specific arrangement of nanoholes can be created e.g., using E-UV / X-ray lithography. The nanoholes may be distributed in a geometric arrangement. The geometric arrangement may be, but not limited to, a triangular grid or a quadrilateral arrangement, e.g. a square grid, or a rectangular grid.

[0158] The nanohole dimensions are selected to be within the diffraction limit of the detection apparatus, < 200 nm cross-sectional dimension. Preferably, the nanoholes have a cross- sectional dimension of less than 200 nm, and may have a cross-sectional dimension of between 180-100 nm, 100-50 nm, 50-20 nm, 20-10 nm, or less than 10 nm.

[0159] The diameter and / or density of one or more nanoholes can be adjusted. The diameter of a nanohole can be adjusted by changing the shape and / or cross-sectional dimension of the blocking element used. The density of a nanohole can be adjusted by changing the surface density of the blocking element.

[0160] The size or density of one or more nanoholes may also be adjusted according to the concentration of the analyte to be tested and / or the point spread function (PSF) of said analyte.

[0161] When using concentrated samples of analyte, having large nanoholes positioned microns apart i.e. , having a diameter greater than 200 nm results in the signal density of each nanohole being high, leading to the problem of too many counts within individual holes and the concentration of the sample needing to thus be reduced to combat this. Therefore, the nanoholes of the present invention may have a diameter smaller than 200 nm.

[0162] The number of nanoholes within the passivation layer is, in part, determined by the dimension of said nanohole. However, a more important parameter to consider is the ratio of the area of the nanoholes to the passivation surface area. This can range from 1 :10000 up to 1 :1 , preferably from 1 :1000 up to 1 :10. Ratios in the region of 1 :10 up to 1:1000 are optimal for high nanomolar to low micromolar concentrations, whilst ratios in the region of 1 :1000 provide data for 10 micromolar concentrations, but with higher noise. Ratios as high as 1 :10000 may be applicable in the case of nanoholes filled with analyte binding substances. It is possible that both the size and density of the nanoholes can be altered depending on the concentration of analyte being added. The tunability of the present invention allows for different degrees of passivation to be achieved, thereby resulting in the same landing frequency being achieved for different analyte concentrations.

[0163] As the nanoholes are spaced apart within the passivation layer, the presence of an analyte in one nanohole may not affect the analysis of a further analyte present in a neighbouring nanohole. Additionally, as the landing analytes to the surface of the substrate can only occur in well-defined regions (i.e., the areas of the substrate surface exposed by the nanoholes), the detection and analysis of bound / interacting analytes can be simplified, as only the surface exposed by nanohole regions on the substrate require analysis. Again, this helps in reducing the background noise generated during detection which can lead to a clearer and more defined detection.

[0164] Suitably the nanoholes may be arranged at the following densities: for nanoholes with single hole area up to 100 nm2: 20-50000 per pm2; nanoholes up to 500 nm2: 15-10000 per pm2; nanoholes up to 2500 nm2: 10-200 per pm2; nanoholes up to 10000 nm2: 5-50 per pm2. Suitably the nanoholes may be at a density of 3, 5, 10, 15, 20, 25, 30, 35 or 50 or more nanoholes per pm2.

[0165] The area available for binding / interaction events to occur can be reduced to a detectable level to allow a greater range of sample concentrations to be analysed. Nanoholes can be sized far below the diffraction limit, i.e., are much smaller than the point spread function (PSF), which allows the reduction of the observed analyte landing events in that particular spot and, therefore, allows measurement of high concentration.

[0166] The nanohole size may be smaller than PSFs to avoid overlapping of events; any nanoholes larger than PSF would permit too many binding / interaction events to occur and not solve the problem of measurements at high concentrations.

[0167] There can be multiple nanoholes per point spread function (PSF) area. It is preferable to have a plurality, i.e., multiple nanoholes per PSF area, as this allows the entire area of field of view to be utilized.

[0168] It is preferred that the nanoholes are distributed across the whole of the surface of the substrate and not contained to only one area of the substrate’s surface. Having the nanoholes spaced across the field of the view reduces the chance of overlapping signals For example, if there are 10 smaller activated areas (each 1000 nm2) within one PSF area, there is a higher probability that two analytes will land in different activated areas compared with a single bigger spot with an area of 10000 nm2where it would be more likely that two analytes land in the same activated area. If the two analytes land in different areas, this would create an oval "PSF" composed of two PSF functions. Such an event can be filtered by radial symmetry as a non-valid event, which is standard processing for mass photometry. In the case of a single bigger activated area, two analytes landing simultaneously results in one PSF with double mass.

[0169] All existing approaches for patterning passivated surfaces by particles are intentionally using microspheres to fabricate larger activated areas around microns apart, i.e., spheres with diameter >100 nm, and work with microscale activated areas, i.e., »100 nm. Such configurations will not work for mass photometry ideally because of too low amount of events from sparsely (microns apart) distributed small < 200 nm nanoholes, and enlarging nanoholes of more than 200 nm will lead to overlapping events.

[0170] Standard mass photometry measurements are accompanied by the exponential decrease in the number of detected events in time because analyte molecules land on the surface, naturally causing their concentration to decrease. Exponential decay can impair measurements of interactions in situ when different analytes are mixed in the well, relate concentration with landing rate, or simply do not allow measurements over a longer time at optimum landing rate. In the case of a passivated surface as described herein, such a decrease is not observed because only a small part of the analytes approaching the substrate are adsorbed on the surface exposed by the nanoholes; depletion of analyte from solution is therefore negligible, and landing rate is close to constant. This allows for longer measurement time at constant conditions and, thereby, detecting more counts of minor species in the sample.

[0171] The passivation layer and nanoholes may be formed by any suitable method, such as those disclosed https: / / doi.org / 10.1039 / c2cs15225h. For example, the nanoholes provided on the device may be formed by placing a plurality of blocking elements onto a surface of the substrate so that a first part of the surface is covered. Once the blocking elements are in position, the passivation layer is provided onto the same surface of the substrate, wherein the passivation layer covers a second part of the surface. Thus, the passivation layer covers the surface of the substrate which is not already covered by the blocking elements. The blocking elements on the first part of the surface are then removed, leaving behind a plurality of nanoholes within the passivation layer. The nanoholes may be formed using a photocleavable PEG brush.

[0172] The nanoholes may be formed by illuminating the surface of a passivated substrate. Illumination may be carried out using a laser. For example laser working at wavelengths ultraviolet to visible light, with the power of 100 mW to 1W.

[0173] Illuminating the passivated substrate leads to the degradation (e.g. photodegradation) of the passivation layer at the points of illumination, thus creating nanoholes in the passivation layer. This process is selective, when only certain spots of surface are illuminated, the hole size may be then by diffraction limited focused spot, that is for high numerical aperture objective and 400 nm laser in the range around 150 nm at 50% of laser power. Another approach is to illuminate entire field of the view, if inhomogeneities in the surface are present, these may induce selective brush degradation and random distribution of nanoholes.

[0174] Illumination of the passivated surface may be performed by long exposure of a laser for between 2 and 10 minutes.

[0175] Illumination of the passivated surface may be performed within the field of view intended for measuring the analyte properties.

[0176] The dimensions of the nanoholes can be changed, in the case of nanoparticle lithography by change nanoparticle size and shape. In the case of spherical (curved) blocking elements it is in addition changing pH or salt of solution for PEGylation, which changes the brush conformation (hydrodynamic radius), and therefore the area which is masked by nanoparticle (globular molecule cannot penetrate under the nanoparticle). In the case of spherical (curve) blocking element it is also PEG chain length, that changes the hydrodynamic radius.

[0177] Nanohole density can be tailored by nanoparticle concentration during their landing on the surface.

[0178] The analyte may bind to the surface of the substrate exposed by the nanoholes. This surface may comprise one, at least one, or more than one substance for binding to the analyte. The surface exposed by the nanohole can be coated, derivatised or modified to increase analyte binding. The surface exposed by the nanohole may be coated by a substance selected from: Amino groups, Carboxyl groups, Thiol groups, His-tags, Succimide Esters, N-hydroxysuccinimide esters, Sulfo-NHS Esters, Imidoesters, Pentafluorophenyl ester, Hydroxymethyl phosphine, Carbodiimide, Maleimide, Haloacetyl, Pyridyl disulfide, Thiosulfonate, Vinyl sulfone Aldehydes, Ketones, Hydrazide, Alkoxyamine Diazirine, Aryl azide, Benzophenones, Isocyanate, Isothiocyanates, Azides, Alkynes, Phosphine, p-nitrophenyl carbonates, Acrylates, Diazonium compounds, Epoxides, Aziridines, Tetranitromethane, Acyl Azides, Avidin, Biotin, TCO-tetrazine, DBCO-azide, antibodies or nanobodies.

[0179] Preferably, the surface exposed by the nanoholes is coated by a substance comprising amino groups, carboxyl groups, thiol groups, His-tags, N-hydroxysuccinimide esters, sulfo-NHS esters, imidoesters, maleimide, haloacetyl, pyridyl disulfide, succinimide, hydrazide, diazirine, aryl azide, benziphenones, azides, alkynes, or biotin.

[0180] The blocking elements may be formed from polymer nanoparticles, such as PMMA, polystyrene, polyethylene, polypropylene, cellulose, that can be dissolved in organic solvents instead of ultrasonication the nanoparticles off.

[0181] Nanosphere or nanoparticle lithography (NSL) is an inexpensive, simple to implement, high throughput nanofabrication technique capable of producing an unexpectedly large variety of nanoparticle structures and well-ordered 2D nanoparticle arrays. NSL may be used to provide blocking elements onto the surface of the substrate prior to deposition of the passivation layer. Nanoparticle lithography can be a vacuum process employing a gas aggregation source of nanoparticles, which would, together with plasma polymerisation of PEG, provide a fully dry approach for large-scale PEG nanopatterning. On the other hand, a wet chemistry approach for nanoparticle lithography can be employed; so-called colloidal lithography can be used for PEG brush patterning by masking the surface by particles during initiator deposition, vapour deposition of organosilanes, plasma etching the polymer brushes, ATRP initiators and PLL-g- PEG deposition. Colloidal lithography can utilise spheres that are sparsely distributed, close- packed, aligned by capillary forces, or EUV lithography.

[0182] The blocking element can be a nanoparticle of any shape, wherein said nanoparticle may be inorganic or organic. The blocking element may be cuboidal, pyramidal, polyhedral, or another shape which comprises flat facets.

[0183] The blocking elements may also be nanospheres or microspheres, such as silica microspheres, which naturally disperse in water and bind to the surface of a substrate. Microspheres have a smaller surface area capable of coming into contact with the substrates surface compared with blocking elements comprising flat facets. Therefore, to make a 100 nm hole in 3 nm PEG film, a 1 pm microsphere would need to be used as the blocking element. A 100 nm nanosphere would make a 30 nm circular nanohole in diameter. Suitably, the nanoholes may be formed using spherical particles of between 5nm and 5 pm.

[0184] Preferably, the blocking element may be a spherical particle, a nanoparticle with at least one flat facet (i.e. flat facets), or a nanocube.

[0185] The blocking element can have a cross-sectional dimension of between 1000nm and 10 nm, or less. The cross-sectional dimension of a blocking element may be selected from: less than 10 nm, 10-50 nm, 50-100 nm, 100-200 nm, or 200-1000 nm.

[0186] It is possible to increase the blocking element density by their functionalisation or precise positioning by capillary forces. This will allow more of the surface to be exposed during measurements of analyte interacting / binding to the surface.

[0187] The blocking elements can be removed from the surface by any suitable means, depending on their manner of initial attachment. For example, the blocking elements may be removed by sonication, dissolution, photodegradation, illumination, use of magnets, photocleavage, or chemical cleavage.

[0188] If the nanoparticles are magnetic (MNPs), they can be removed using magnetic fields. If the nanoparticle is attached to the surface using a cleavable linker, the appropriate cleaving agent can be used to release the nanoparticle, for example light to release a photolabile linker, enzymes can be used for enzymatically cleavable linkers, and other forms of linkers, such as disulphide linkers, can be cleaved chemically using an appropriate reagent.

[0189] Alternatively, or additionally, removal of the blocking elements can be conducted by destroying or dissolving nanoparticles on the surface, such as e.g., PMMA nanoparticles dissolved in acetone and polystyrene in toluene.

[0190] Another way to remove the blocking elements is by illumination e.g., heated gold particles leave the surface upon illumination. Photodegradation / photo dissolution may also be carried out to remove blocking elements. This method includes irradiating polymer nanoparticles (i.e. , blocking elements) with UV light to break the chains, or disassemble non-covalently crosslinked polymer.

[0191] The nanoholes may be provided by depositing inorganic or organic film to the surface of the substrate, and heating the film above its melting temperature to form separated nanoislands on the surface. The passivation layer may then be provided onto the remaining surface of the substrate, and the nanoislands removed via any appropriate method as detailed here.

[0192] The device according to the present invention may be provided with the blocking elements still provided on the first part of the substrates surface, or the device may be provided with the blocking elements removed and the nanoholes present.

[0193] If the device is provided with the blocking elements still present on the surface of the substrate, the user of such device will need to remove said blocking elements using an appropriate means before or whilst adding a sample.

[0194] The analyte may also be described as an object or a molecule. The analyte is the entity of interest, for which it is desired to detect or determine particular characteristics or properties, such as mass, concentration or simply determining the presence of a particular analyte using such characteristics.

[0195] The analyte may be a biological molecule or a biomolecule, or a chemical molecule. The analyte may be a polymer. The analyte may be an inorganic molecule. The analyte sample may comprise a biomolecule sample, a polymeric sample, or an inorganic sample. The analyte sample may comprise one or more of the different analyte types listed herein.

[0196] The analyte may be a protein, a peptide, a polypeptide, a lipoprotein, a glycoprotein, a lipid, a carbohydrate, an organic polymer, a protein complex, an antibody or an antibody fragment thereof, an enzyme or it may be a nucleic acid molecule such as DNA, RNA, a polysaccharide, or it may be a virus, or a viral vector such as an adenovirus and / or a lentivirus, a virus-like particle, or a small molecule, an exosome, a vesicle, an assembly complex, a nanoparticle, a compound, an ion or a quantum dot.

[0197] The analyte may be a membrane-bound protein. The analyte may be an integral membrane protein, for example an ion channel or ion gate, a glycoprotein, a gap junction protein, a transmembrane protein, a G protein-coupled receptor (GPCR), an ammonia transporter membrane protein, a p-barrel membrane protein. The analyte may be a peripheral membrane protein, for example actin, spectrin. The analyte may be a lipid-anchored protein, for example a G Pl-anchored protein.

[0198] The analyte may be a single molecule, mixtures of molecules, a macromolecule, a supermolecule, or an association of molecules, macromolecules (such as polymers) and supermolecules. Examples of suitable macromolecules may include, but are not limited to, nucleic acid molecules, either natural nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or artificial nucleic acids such as peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Associations of molecules can include assemblies such as virus-like particles where envelope or capsid proteins are associated.

[0199] The analyte may be multi-molecular complexes comprising agglomerations of components, including proteins, such as monomer, dimer and trimer species, or other higher order agglomerations.

[0200] With iSCAT and MP techniques, it is not required to label or tag the analyte prior to detection.

[0201] The analyte may have a mass of 6,000 kDa or less, for example 5 kDa to 5000 kDa.

[0202] The analyte is suitably provided in a solution or suspension. The analyte is thus present in a fluid. Suitably, a sample of the analyte in solution / suspension is provided for detection. A sample may be described as a representative portion or part of a whole, which has been taken for the purpose of determining the characteristics of a larger entity from which the portion or part is taken.

[0203] The sample may be any suitable sample, but preferably contains or is suspected of containing, an analyte.

[0204] The sample is preferably liquid, such as a solution or suspension. The solution or suspension may contain any suitable solvent, particularly water. The sample may contain an organic solvent. In particular, the solvent, solution or suspension may have a higher refractive index than water.

[0205] The solution or suspension may be water or a buffer. The buffer may be a water-based buffer. The buffer may be selected from phosphate-buffered saline (PBS), an acetate buffer (e.g. ammonium acetate), a citrate buffer (e.g. sodium citrate), Tris buffer, HEPES, borate. The solution or suspension may include a detergent. The detergent may be selected from lauryl maltose neopentyl glycol (LMNG), glycol-diosgenin (GDN), n-dodecyl-p-D-maltoside (DDM), n-Decyl-p-D-maltopyranoside DM, n-Octyl-p-D-glucopyranoside OG, n-Nonyl-p-D- glucopyranoside NG, Brij-35, Tween-20.

[0206] Measurements of mass photometry when providing analyte in detergent can be problematic as the detergent micelles landing on the substrate generate a high level of noise, obscuring signals from the analyte. The use of a device as disclosed herein, wherein the substrate is passivated and the passivation layer comprises nanoholes, reduces the noise from the detergents by passivating micelles off the surface of the substrate while allowing the analyte to bind within the nanoholes. Without the detergent micelles present, there is a reduction in noise and the analyte signals can be seen more clearly.

[0207] The detergent micelles are larger in size than the dimensions of the nanoholes meaning the detergent micelles cannot fit within the nanoholes. This means the detergent micelles cannot bind to the substrate and flow off the passivated surface. The absence of detergent micelles reduces the background noise seen when measuring the analytes properties with mass photometry.

[0208] A solution comprising a detergent may be used when the properties of a membrane-bound protein are being measured. Membrane-bound proteins require stabilisation when they are not bound to a membrane in order to prevent denaturation. Membrane proteins are not stable and aggregate or misfold in a buffer without or with low concentration of detergents. Therefore, trying to perform mass photometry measurements on these proteins can be problematic. Membrane-bound proteins can be insoluble when diluted in a water-based buffer, leading to protein denaturation and the inability to measure the proteins properties. It has been found that exploiting detergents above its critical micelle concentration (CMC) can stabilise the proteins when they are not bound to a membrane, and the use of detergents is a far less laborious exercise compared to the use of pepti-discs or nano-discs. Therefore, the use of a buffer solution comprising a detergent can stabilise the membrane-bound proteins, making it far easier to conduct mass photometry measurements.

[0209] Membrane proteins may be measured in buffer containing detergent at a concentration of between 0.5 to 5 times the critical micelle concentration. The concentration equal to or above 1 CMC is beneficial to maintain protein stability. Meanwhile, the concentration equal to or below 2 CMC is beneficial to maintain low noise. The optimum concentration for MP measurement is then 1-2 CMC.

[0210] CMC is defined as the concentration of surfactants above which micelles form and all additional surfactants added to the system will form micelles e.g. LMNG has a CMC of : (H2O) ~ 0.01 mM, (0.001%); DDM has a CMC of : (H2O) ~ 0.17 mM, (0.0087%), GDN has a CMC of : (H2O) ~ 18 pM (0.0021%), Tween-20 has a CMC in water of 0.06 - 0.07%. The best measurement data is obtained between 1-2CMC, but usable measurements are obtainable between 0.5-5CMC.

[0211] The sample may be a biological sample, such as a medical or veterinary sample. Such biological sample may be a bodily fluid of any appropriate type or may be a suspension of a tissue of any appropriate type. The sample may be an environmental sample. Such environmental sample may be a water sample, or a sample taken from an environment and suspended in a solvent. The sample may be an industrial sample, such as a sample from a production process.

[0212] The sample can contain the analyte at any concentration and does not require dilution prior to the detection steps. The concentration may be above 10 nM (total protein), for example 10 pM for 100 kDa analytes. The analyte sample may have a concentration range selected from: 0.05-0.20 pM, 0.2-1.0 pM, 1-10 pM, 10-100 pM, or 0.1-1.0 mM.

[0213] Due to the nanoholes exposing the surface of the device, only a small proportion of the available analyte is able to bind to the surface of the device. This helps achieve clear detection with little to no background noise, making it possible to conduct detection at single-molecule level.

[0214] The device may be contacted with the sample to allow the analyte to contact the device and interact with / bind to the surface exposed by the nanoholes.

[0215] Any suitable means and methods may be used to contact the sample and the surface. Contacting the sample and the surface includes incubating exposing, mixing, or delivering, a sample to the surface. Contacting may require in certain embodiments agitating, vortexing, pipetting etc. Contact may be performed for a sufficient time to allow binding of analyte present in the sample to the surface. The contact time can be of any suitable length, depending on the sample of interest and other parameters such as incubation conditions (e.g., temperature). Contact time may be at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours or longer. A person skilled in the art will be able to adjust the contact time and conditions accordingly.

[0216] The device according to the present can be used to detect and / or measure properties and / or characteristics of an analyte. The detection step involves the detection of analyte bound to or interacting with the surface of the device. Such surface is only exposed by the nanoholes in the passivation layer. The device according to the present invention can be used to detect properties of an analyte and / or can be used to measure properties of an analyte. The properties of the analyte which can be detected and / or measured by said device include molecular mass, concentration, oligomerisation status, modification status, molecule charge, mass dispersity, molecule conformation, molecule surface free energy (hydrophobicity), biospecificity, protein-protein or protein-DNA interaction.

[0217] In order for the devices disclosed herein to be able to detect and / or measure properties and / or characteristics of an analyte, there must be at least one nanohole within the passivation layer which comprises an analyte.

[0218] The present invention provides the ability of measuring or quantifying the mass of an analyte. Also provided is the ability of measuring or quantifying a change in the mass of an analyte.

[0219] A key application of the methods and devices provide herein, is for the measurement of low- affinity interactions between analytes (i.e. between the same or different analytes), in other words, interactions with higher dissociation constants Kd, as these require higher concentrations of analyte to be used, for example a sample with a protein concentration of at least 10 nM, preferably above 50 nm.

[0220] Detection of a binding / interaction event may be performed using a light scattering microscope, for example an iSCAT with a spatial filter or a mass photometer, for example as described herein. iSCAT comprises determining interference between light scattered by an analyte in a sample and light reflected from the analyte location. The interference is dependent on the scattering amplitude of the analyte and is measured as an iSCAT signal. Detection is performed by capturing an image of the surface. Multiple images, or frames, may be combined to provide a film. A video or film may comprise any number of frames, preferably it may comprise 1 ,000 to 48,000 frames. It may comprise up to 60,000 frames or up to 100,000 frames.

[0221] A method of the invention may further comprise comparison of the iSCAT contrast with a calibration or standard curve to determine the mass or concentration of the analyte of interest.

[0222] A method of the present invention may also comprise one or more image processing steps, including for example and without limitation removal of the background on the image, and improving the image quality.

[0223] It is possible to measure at least one property of the analyte interacting with the surface exposed by the one or more nanoholes. The property of the analyte may be measured from individual nanoholes or a plurality of nanoholes.

[0224] The ratio of scattering contribution of one or more nanoholes to intensity of reflected light by the substrate may be: 1-0.1 :1 , 0.1-0.01 :1 , 0.01-0.001 :1 , 0.001-0.0001 :1 or less.

[0225] The detected signal may correspond to a point spread function (PSF) area within a field of view (FoV).

[0226] The detected signal may correspond to a point spread function (PSF) contrast. This PSF contrast relates to the area outlined by the first minima / maxima of intensity of PSF with positive / negative contrast.

[0227] The detected signal may comprise light interference from the analyte and the substrate. The detected signal can be pure light scattering from the analyte.

[0228] The point spread function (PSF) is the response of a focused optical imaging system to a point source or point object. A more general term for the PSF is the system's impulse response; the PSF is the impulse response or impulse response function (IRF) of a focused optical imaging system. The PSF in many contexts can be thought of as the extended blob in an image that represents a single point object, which is considered as a spatial impulse.

[0229] The image of an object in a microscope as a non-coherent imaging system can be computed by expressing the object-plane field as a weighted sum of 2D impulse functions, and then expressing the image plane field as a weighted sum of the images of these impulse functions. The images of the individual object-plane impulse functions are called point spread functions (PSF), reflecting the fact that a mathematical point of light in the object plane is spread out to form a finite area in the image plane. PSFs can be considered impulse response functions for imaging systems.

[0230] When the object is divided into discrete point objects of varying intensity, the image is computed as a sum of the PSF of each point. As the PSF is typically determined entirely by the imaging system (that is, microscope or telescope), the entire image can be described by knowing the optical properties of the system.

[0231] Field of view (FoV) describes the viewable area that can be imaged by a lens system. This is the portion of the object that fills the camera’s sensor. This can be described by the physical area which can be imaged, such as a horizontal or vertical field of view.

[0232] The dimension of the nanohole may be smaller than the PSF area, and each PSF area may comprise a plurality of nanoholes. The nanoholes may be arranged such that the PSF area of the analyte comprises a plurality of nanoholes.

[0233] The method may comprise use of a suitable light scattering microscope, for example an interferometric scattering microscope (iSCAT) comprising a spatial filter or a mass photometer. Any suitable apparatus may be used, including the arrangement described below.

[0234] A suitable microscope or photometer may comprise: a sample holder for holding a surface in a sample location; an illumination source arranged to provide illuminating light; a detector; and an optical system being arranged to direct illuminating light onto the sample location and being arranged to collect output light in reflection, the output light comprising both light scattered from the sample location and illuminating light reflected from the sample location, and direct the output light to the detector.

[0235] A microscope may further comprise a spatial filter positioned to filter the output light; the spatial filter being arranged to pass output light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures. Such a spatial filter advantageously maximises image contrast, as described in PCT / GB2017 / 052070, and also in Cole et al (ACS Photonics, 2017, 4(2), pp 211-216).

[0236] The device according to the present invention may comprise a sample holder for holding a sample in a sample location, an illumination source arranged to provide illuminating light, a detector, an optical system being arranged to direct illuminating light onto the sample location and being arranged to collect output light in reflection, and a spatial filter.

[0237] The output light may comprise both light scattered from the sample location and illuminating light reflected from the sample location.

[0238] The optical system may direct the output light to the detector.

[0239] The spatial filter may be positioned to filter the output light, the spatial filter being arranged to pass output light but with a reduction in intensity that is greater within a predetermined numerical aperture than at larger numerical apertures.

[0240] The device may be an existing commercial microscope which has been adapted by inclusion of a spatial filter. Such adaptations can be performed simply and cheaply, allowing the present invention to be implemented in an extremely cost-effective manner.

[0241] In the methods and devices described herein, the light used can be selected from: ultraviolet light (which may be defined herein as having wavelengths in the range from 10 nm to 380 nm); visible light (which may be defined herein as having wavelengths in the range from 380 nm to 740 nm); infrared light (which may be defined herein as having wavelengths in the range from 740 nm to 300 pm). The light may be a mixture of wavelengths. Herein, the terms ‘optical’ and ‘optics’ are used to refer generally to the light to which the methods are applied.

[0242] The illumination source can be arranged to provide illuminating light. The illuminating light may be coherent light. For example, the illumination source may be a laser. The wavelength of the illuminating light may be selected in dependence on the nature of the sample analyte and / or the properties to be examined.

[0243] Optionally, the illumination light may be modulated spatially, to remove speckle patterns that arise from the coherent nature of the illumination and laser noise, for example as detailed in Kukura et al. , “High-speed nanoscopic tracking of the position and orientation of a single virus”, Nature Methods 2009 6:923-935.

[0244] The detector of the device can receive output light which is reflected from the sample location. Typically, the device may operate in a wide-field mode, in which case the detector may be an image sensor that captures an image of the sample analyte. The device may alternatively operate in a confocal mode, in which case the detector may be an image sensor or may be a point-like detector, such as a photo-diode, in which case a scanning arrangement may be used to scan a region of the sample to build up an image. Examples of image sensors that may be employed as the detector include a CMOS (complementary metal-oxide semiconductor) image sensor or a CCD (charge-coupled device).

[0245] The methods as described herein utilise electrochemical measurement to detect a signal of an analyte within the one or more nanoholes. In some embodiments, the nanohole is provided with an electrode. An external voltage source may be provided to connect and supply current to the electrode.

[0246] The methods as described herein can utilise electrochemical measurement to detect the presence of a target analyte via immobilisation to the electrode provided within the one or more nanoholes. These measurements are thus a measurement of the electrical signal change across an electrode generated by the binding / interaction of the analyte to the surface of the electrode. The is binding / interaction may be non-specific adsorption of the analyte onto the surface of the electrode. In other examples, a sandwich complex may be formed on the electrode. For example, a first biological component may be immobilised onto the surface of the electrode. A target analyte and a second biological component may form a complex in conjunction with a catalyst, such as an enzyme. The complex may then contact the first biological component to form a sandwich assay on the electrode surface. The formation of a sandwich complex containing the target analyte can be detected using electrochemical techniques.

[0247] One of the most sensitive electrochemical techniques is electrochemical impedance spectroscopy (EIS), which effectively separates a number of complex processes occurring at an electrochemical interface by measuring at a range of frequencies, thus separating processes by the characteristic timescales they occur on. In EIS, the parameters measured include various resistances and capacitances relating to the electrode and secondary processes. Of particular use to immunoassays, are the processes related to electron transfer at the modified electrode surface; an increase in bound species (such as antibodies) will affect these processes. The main surface specific parameters that can be measured are the charge transfer resistance (ROT) and the double layer capacitance (CDL), of which the ROT gives the most direct measurement of surface blocking. However, low concentrations of surface bound species will have a minimal effect on these. The methods described herein utilise electrochemical measurement, for example faradaic impedance measurement or non-faradaic impedance measurement and thus use AC current and may, therefore, be practiced without the use of a redox probe reagent.

[0248] The electrochemical measurement technique can be electrochemical impedance spectroscopy, differential pulse voltammetry, square wave voltammetry, cyclic voltammetry, chronoamperometry, open circuit potential or chronopotentiometry.

[0249] EXAMPLES

[0250] Example 1. Nanopatterning of PEG-brushes by nanocube lithography for activation of protein landing

[0251] The challenge of measurement at high concentrations

[0252] Mass photometry measurement is performed by putting a droplet with analyte on a glass coverslip; part of incident light is reflected from the glass-analyte interface, and landed molecules scatter another part; both reflected and scattered light interfere at the detector and after rolling average ratiometric processing results in airy disk-shaped PSFs as is shown in Figure 3 for antibodies measured on standard aminated coverslip, which is commonly used improve antibodies binding. At 10 nM, individual PSFs are distinguishable and can be fitted; thereby, their contrast, which is related to the mass of a landed molecule, can be estimated. However, increased concentration around 100 nM leads at first to overlapping of PSFs, which makes the contrast estimation difficult and at 1000 nM, individual events cannot be observed thanks to overcrowding FoV, as shown in Figure 3. Figure 3 shows the landing density of all events detected at a concentration of 10 nM on standard aminated coverslip; landing events occurred at random places and are homogeneously distributed throughout the FoV.

[0253] High-performance passivation of glass surfaces using cloud point PEGylation

[0254] Figure 3 shows schematics and the principle of glass slide passivation by cloud point PEGylation using mPEG-MW5000-SVA polymer brush that prevents molecules from landing. Nearly perfect passivation can be seen on a snapshot of measurement taken at 1000 nM, see Figure 3, where no PSFs were detected. Figure 3 shows the landing density of measurement at 1000 nM concentration on passivated surface; in total, only 33 events were observed during 30 s measurement, which is much less than 2745 events measured on standard coverslip at 100-fold higher concentration. In other words, only one molecule from almost 10,000 can land on the coverslip and be detected.

[0255] Nanoparticle-assisted partial nano-activation of passivated surfaces However, on fully passivating coating, molecules cannot be detected. Therefore, we introduced the nanoscopic holes in the PEG coating to allow for molecule binding. At first, Au nanocubes with an edge size of 100 nm were landed on a standard aminated coverslip; then, cloud point PEGylation was performed. In the next step, nanocubes were sonicated away, and only PEG coating with nanoholes (nanohole-PEG) was left on the glass substrate. On such a surface, analyte molecules can land only in the nanoholes; meanwhile, molecules approaching the surface out of the holes are bounced away, which significantly reduces the landing rate on the surface and results in the observation of individual PSFs even at 1000 nM concentration, see Figure 3. Figure 3 shows the landing density at the same concentration and on the same surface. In this case, landing density is negligible on the majority of the FoV; however, we can observe isolated spots with much higher landing density. The super resolution image shows that the landing events in those particular spots result in a square-like landing density corresponding to the surface masked by Au nanocubes. The number of landing events, in this case, is comparable to measurement at 10 nM on a standard coverslip, which means that approximately one of 100 molecules approaching the coverslip landed into nanoholes and was detected. It is worth mentioning that the nanohole size needs to be smaller than PSF's to avoid overlapping of events; the holes larger than PSF would behave only as standard glass in that particular area and not solve the problem of measurements at high concentrations.

[0256] Figure 4a shows a comparison of the native image of the surface with 100 nm nanocubes masking the surface before surface PEGylation and a native image of the nanohole-nanocube- PEG surface after the removal of those nanocubes; such a surface is indistinguishable from glass. Figure 4b shows the comparison of landing rates of antibodies measured on glass, PEG, and nanocube-nanohole-PEG; we can see that nanocube surface activation leads to a similar number of events for 100-1000x lower concentrations in comparison to glass. Figure 4c shows a comparison of spectra measured on glass, PEG, and nanocube-nanohole-PEG. At 10 nM on glass, we see two peaks, one at 148 kDa, which denotes antibody binding, and at -147 for antibody unbinding from glass as antibodies binding to glass is weak; at 1000 nM on glass, the peaks around 500 kDa are caused by overcrowding of FoV, at such conditions the correct mass of antibodies cannot be estimated. However, by exploiting nanocube- nanohole-PEG surface, it is possible to detect antibodies even at 1000 nM concentrations. The unbinding peak of antibodies at -131 kDa was diminished by using primary amino groups on the surface, which increased their binding to the surface. In other words, nanohole-PEG allows for measurement at high concentrations that are inaccessible to standard mass photometry. Example 2. Optimisation of protein landing rate by nanoparticle density

[0257] Achieving high surface coverage by nanoholes

[0258] It can be seen in Figure 3 that not the whole surface is exploited during measurements on nanohole-PEG. This could be improved by increasing nanocube density by their functionalisation or precise positioning by capillary forces. An alternative approach is to use silica spheres, which naturally disperse in water and bind homogeneously to aminated coverslip. The landing of 100 nm nanospheres is shown in two native images (not processed ratiometrically) of M P measurement in Figure 5. Laser intensity needed to be decreased during this measurement because 100 nm nanoparticles scatter too much and oversaturate the camera. After 1s of landing, individual PSFs belonging to individual nanoparticles can be observed, and after 10 minutes of landing, PSFs overlap and fill the entire FoV. In other words, such a surface will compose multiple nanoholes per PSF area. The comparison of the native MP image of such a nanohole-PEG surface with a standard glass coverslip is shown in Figure 5b. Both images are indistinguishable; only the glass roughness can be observed. Figure 5c shows the landing density of the analyte at 1000 nM concentration on such nanohole-PEG surface; landing density is homogeneous all over the FoV and is comparable to the measurement on an aminated coverslip in Figure 5c acquired at 100-fold time low concentration. By comparison of the number of landing events on standard aminated glass and nanohole-PEG, it can be assumed that ratio of hole: surface area is 1 :37 in this case.

[0259] Tuning the number of events in FoV by nanoparticle concentration and size

[0260] The number of landing events at FoV can be tuned by both nanoparticle size and concentration, as shown in Figure 5d-f. A low mass of 10 pg of 100 nm nanoparticles used to cover a surface area of 1 cm2resulted in a movie with too low number of events at an antibody concentration of 100 nM as shown on a snapshot in Figure 5d; on the other hand, at a concentration of 1000 nM, PSFs are present in each frame. A four-fold increase of nanoparticle concentration in solution results in a higher density of PSFs for both concentrations; see Figure 5e. Further increase of hole:surface area can be achieved by using the same concentration of nanoparticles but with a smaller 50 nM diameter, see Figure 5f; twice smaller particles can mask roughly 2x lower surface area; however, each particle has 8x lower mass. The surface prepared using 50 nM nanoparticles shows an ideal landing density for 100 nM concentration; however, it results in FoV that is too crowded for 1000 nM analyte concentration.

[0261] Mass histograms comparison of analyte measured on standard aminated glass and nanohole-PEG Figure 5g shows a comparison of mass histograms of 30s antibody measurements at concentrations of 10 nM and 1000 nM on the standard aminated coverslip with measurement at 1000 nM on nanohole-PEG prepared using 40 pg of 100 nm silica nanoparticles to coat area of 1 cm2. Histogram acquired at a concentration of 10 nM on standard aminated coverslip shows the peak at 150 kDa, corresponding to antibody mass. On the other hand, the histogram of high concentration measurement at 1000 nM on the same surface does not show any counts at this mass but a broad peak ranging up to 800 kDa instead, which is caused by overcrowding FoV, which prevents reliable processing of the movie. The same 1000 nM concentration of antibodies was measured on nanohole-PEG and resulted, thanks to a reduced number of events in FoV, in the peak at 150 kDa, similar to that measured on standard aminated glass. This proves the usability of nanohole-PEG for measurements of analytes at concentrations unreachable by standard MP.

[0262] Landing rate as a function of nanoparticle concentration and size

[0263] A series of measurements were acquired to demonstrate how landing rate corresponds to analyte concentration and surface type. Surfaces were prepared using different concentrations of 50 nm and 100 nm nanoparticle solutions, and landing rates corresponding to antibody mass peaks were plotted for different analyte concentrations. The landing rate linearly grows with the overall weight of nanoparticles used to cover 1 cm2of surface for all analyte concentrations. In consistency with Figure 5, using 50 nm nanoparticles for surface fabrication leads to a higher landing rate than if the same mass of 100 nm nanoparticles is used. In other words, it is demonstrated that the landing rate is tuneable over two orders of magnitude by nanoparticle concentration and size.

[0264] Example 3. Measurement of low-affinity interactions

[0265] The main application of nanohole-PEG is a measurement of low-affinity interactions, in other words, higher dissociation constants Kd.

[0266] It is now possible to compare MP performance in the presence and absence of nanostructured surfaces. In many cases, the motivation to work at higher analyte concentrations originates from a desire to capture interactions in the high nM to pM range. C-reactive protein is predominantly found in a pentameric form with a total mass of 115 kDa and forms decamers at higher concentrations. The maximum possible analyte concentration of 50 nM on standard aminated coverslips (Figure 6) yields a single peak at 119 kDa (Figure 6) and no significant signs of decamer formation. Repeating the measurement on a nanohole-PEG surface prepared using 10 pg cm-2of 100 nm SNPs exhibits a much lower landing density even at 10x higher analyte concentration (Figure 6). The corresponding mass histogram is still dominated by the pentameric form at 111 kDa, but now exhibits a clear decamer signature at 225 kDa (Figure 6). Given that the number of both pentamers and decamers can be quantified, the associated affinity can be readily deduced from individual spectra, assuming that the measurement is taking place at equilibrium. Repeating this process across different analyte concentrations yields a KD of 2.6 ± 0.9 pM. Repeating the experiment at 0.5, 1 and 7 pM concentrations with native mass spectrometry, performed as previously described, results in a similar KD of 2.46 ± 0.14 pM; however, instead of PBS buffer, ammonium acetate is needed for desolvation with native mass spectrometry. MP experiments in ammonium acetate resulted in Kd = 3.40 ± 1.23 pM, evidencing overall good agreement.

[0267] In a further experiment, the interaction between the neonatal fragment crystallisable receptor (FcRn) and the therapeutic humanized IgG antibody trastuzumab (Herceptin) with molecular masses of 50 kDa and 148 kDa, representative of a stereotypical proteimprotein interaction, was chosen. The chosen pair is particularly advantageous because the strength of the interaction varies with pH. This interaction was previously captured at low pH with MP using a rapid dilution method. Considering that fast dilution in the gasket can reveal the interaction, we 100x diluted an equimolar mixture of both proteins in the gasket to the concentration of 25 nM. However, at pH 6, this still results in a high landing density (Figure 6) and does not reveal strong evidence of FcRn: Herceptin complexes (Figure 6), indicative of rapid complex dissociation. Repeating the experiment at 500 nM at equilibrium using a nanohole-PEG surface leads to a substantial reduction in landing rate and a clear peak at the mass of the FcRn: Herceptin complex at 200 kDa. Using the same procedure as for C-reactive protein yields KD = 0.23 ± 0.06 pM at pH 6.0 and KD = 0.78 + 0.21 pM at pH 6.5.

[0268] Example 4. Measurement using nanoholes filled with binding increasing substance

[0269] Figures 2a, and b are examples of high-concentration measurements of CRP protein, comparing overlapping of PSF on glass and individual events on nanohole PEG. Nanohole PEG allows for mass estimation, as shown in the spectra in Figure 2c. If the nanoholes contain a substance that increases binding, e.g., maleimide-PEG-MW5000-SVA, which covalently captures the -SH group naturally present in proteins, the measurement at 100 nM concentration can be performed as well. Figure 2f shows mass spectra of the protein Dynamin (MS1000), protein CRP, and antibody Herceptin, which were measured using PEG with nanoholes containing a binding substance. The above examples were carried out according to the methods provided below in Example 5 below.

[0270] Example 5. Surface fabrication

[0271] Coverslip cleaning

[0272] At first, coverslips were sonicated for 5 minutes in acetone (99.5% analytical grade), then washed with MilliQ, followed by 5 minutes of sonication in a 1 :1 MilliQ: isopropanol solution. The last sonication step was 5 minutes in MilliQ. After sonication, the coverslips were nitrogen blow-dried using the highest possible flow to minimise their exposure to dust, which is naturally present in the air.

[0273] Plasma activation

[0274] Plasma activation was used to activate the glass surface with -OH groups for following silanisation. Coverslips were inserted into oxygen plasma cleaner (Zepto-BRS 200, Diener electronic), together with the two coverslips stuck together by 20 pL MilliQ droplets pipetted in between them for introducing hydrogen to the oxygen cleaner chamber. The chamber was quickly pumped down to the pressure of 0.15 mBar. Then, the flow of oxygen to the chamber was set to reach the pressure of 0.8 mBar and coverslips were plasma cleaned for 8 minutes at 50% of the power. A power of 40% was selected as the highest that does not heat the rack with coverslips above the boiling point of acetone.

[0275] Surface amination

[0276] At first, the acetone (99.9%, HPLC grade) was heated on a magnetic stirrer to the temperature of 50°C. Then (3-Aminopropyl)triethoxysilane (APTES, 99%, Sigma-Aldrich) was added to obtain a 2% solution just before dipping plasma-activated coverslips. After plasma cleaning, the coverslips were washed by dipping in a beaker with pure acetone (99.8% HPLC grade) and quickly transferred to the beaker with 2% APTES solution on a magnetic stirrer, where they were incubated for 1 hour, while the beaker was sonicated at least one time for 1 minute during this procedure. The slides were then twice sonicated in acetone (99.8% HPLC grade) for 5 minutes and 1 minute in MilliQ, followed by nitrogen blow drying.

[0277] Nanoparticle landing

[0278] Immediately after surface amination, the 1x1 cm square reusable gaskets (CultureWell, Grace Bio Labs) were placed on the coverslips and filled with 100 pL of MilliQ deionised water. The volume of 0.5-16 pL of SiO2 nanoparticles with diameters of 50 and 100 nm (AlphaNanotech) or 100 pL of 100 nm nanocubes (Nanopartz) were pipetted into 100 pL of MilliQ in 0.2 mL PCR tube (Thermowell, Corning), and then pipetted under water level in the gasket. After 10 minutes of nanoparticle landing, the gasket was pipette-washed with MilliQ to remove the remaining nanoparticles from the solution. Drying of the surface with landed nanoparticles needs to be avoided to prevent their aggregation.

[0279] Surface PEGylation

[0280] Sodium carbonate / bicarbonate stock buffer at pH 9.5 ± 0.1 was prepared by mixing sodium carbonate with bicarbonate in a mass ratio of 44:100. Then 1.044 g of potassium sulfate was added to 10 mL of buffer to prepare 0.6M solution. The PEG buffer was pipetted onto mPEG- MW5000-SVA powder (Laysan Bio) aliquote to prepare 20% w / v PEG solution and mixed using vortex. The resulting liquid is matte white (at 0.55M potassium sulfate solution, the liquid is clear). 50 pL of PEG solution was immediately transferred to the well filled with 50 pL of PEG buffer to have 10% w / v PEG in the gasket. It is important to note that MilliQ in the gasket after nanoparticle landing needs to be replaced by PEG buffer without surface drying. Coverslips were incubated with PEG in the dark for 1 hour.

[0281] Sonication removal of nanoparticles and storage of nanohole-PEG coverslips

[0282] After PEGylation, coverslips were sonicated 2x in MilliQ for 5 minutes with the gasket on, then nitrogen blow-dried and inserted into a falcon tube with a silica gel bag. Before closing the lid, the tube was filled with nitrogen, and the lid was taped with parafilm. The tube with coverslip was then promptly put at -20 °C for long-term storage.

[0283] Example 6. Mass photometry of proteins

[0284] A mass photometer (TwoMP, Refeyn) was used to acquire all data in this study, specifically measurements of SARS-CoV-2 spike antibodies (Recombinant, Mouse MAb, Sino Biological) were performed in PBS buffer (DPBS, Gibco), low-affinity protein interaction of FcRN (recombinant human, C-His-Avi tag, host cell HEK293, Stratech) and Herceptin (Trastuzumab, 600 mg / 5 mL solution for injection, Roche) in PBS buffer at pH 5.0 (tailored by HCI addition) and finally protein CD163 oligomerisation was measured in buffer. Fast manual dilution of FcRN:Herceptin mixture was performed by placing the 24.5 pL of buffer and

[0285] The examples above and disclosure provided herein, demonstrate that a passivation surface with nanoscopic nonspecific anchors allows for mass photometry measurements and studying protein-protein interaction at a micromolar concentration of analyte, which in turn allows estimation of Kd of weak interactions as shown on the examples of FcRN and Herceptin interaction and CD163 protein oligomerisation. However, it is a powerful tool for studying any weak interactions including, for example, the investigation of phase separation proteins associated with neurodegenerative diseases. Since the nanohole-PEG exhibits constant landing rates, it may also be useful for time evolution experiments. Moreover, MP proved to be an excellent benchmark for developing passivation coatings, because it can monitor both particle adsorption and desorption from the passivating layer.

[0286] For example, if the PEG brush is not dense enough, then the analyte can land on the PEG for more than 20 ms and be desorbed after; such imperfect passivation is detected as one black dot, followed by a white dot in the FoV. Other methods cannot measure this kind of imperfect passivation.

[0287] Example 7.

[0288] Larger blocking elements distanced more than PSF diameter, for example, Au 100 nm nanocubes that were distanced more than 10x of their size, allow for the estimation of the mass histogram from individual holes; see Figure 7. It can be observed that the mass estimation in individual holes varies from 145 to 153 kDa, which is caused by the spatial inhomogeneity of the interferometric measurement and the imperfection of the microscope. Therefore, constraining detection to individual nanoscopic areas allows for a more precise estimation of analyte properties.

[0289] Example 8.

[0290] The substrate with nanoholes with a diameter of less than 200 nm was installed on a mass photometer comprising the illumination source and detector of scattered light, as shown in Figure 1a. An analyte solution, i.e., a buffer containing biomolecules, polymers, or inorganic particles, was placed on that substrate and coated with a passivation layer comprising nanoholes. Passivation coating was selected from polymer brushes, plasma polymer coatings, hydrophobic coating with tween-20, zwitterion polymer brushes, or polyelectrolyte multilayers. However, polymer-brush-based PEG coatings with thickness, hydrodynamic, or gyration diameter between 1-10 nm and the length of brush between 20-100 nm, achieved the best performance.

[0291] In the case of measurement using illumination through the substrate and interference of scattered light from protein and reflected from the substrate, it was important to ensure that (i) the substrate is transparent with transmittance much higher than 1% for at least one wavelength of the illumination device to allow illumination of the measured particles and detection of scattered light by those particles, (ii) nanoholes did not significantly impair measurement due to scattering at the hole boundaries; in other words, the ratio of scattering contribution of nanoholes to intensity of reflected light by the substrate was much lower than 1 :1. The substrate material was selected from polymeric, glass, quartz glass, fused silica, borosilicate glass, BK7, sapphire, CaF2, SiC>2, TiC>2, mica, graphene, germanium, ZnSe, KBr, KRS-5, silicon, or transparent conductive oxides as indium-doped tin oxide, fluorine-doped tin oxide, aluminium-doped zinc oxide, zinc oxide, and cadmium oxide.

[0292] Most of the analyte particles were rejected by the passivation layer because the surface area of the nanoholes to the surface area of the passivation layer was between 1 :10000 and 1 :1. Therefore, nanoholes received a smaller part of the analyte approaching the substrate, such analyte was illuminated and scattered light was collected by detector of scattered light.

[0293] Standard measurement on glass at a concentration above 10 nM, especially above 50 nM, results in a high landing rate and overlapping point spread functions at the field of view of the detector; such frames cannot be processed, as shown in Figure 1 b. If the same experiment is performed on a substrate with passivation coating comprising nanoholes, the measurement results in a lower landing rate of detected particles and, therefore, lower density of detected point spread functions, which are not overlapping. Such frames can be processed to quantify the mass of the analyte or change in the mass of the analyte. Such measurement at concentrations above 50 nM, especially above 500 nM, allowed estimation of low-affinity interactions between the same analyte, e.g. protein CRP, and different analytes, e.g. FcRN and Herceptin antibody.

[0294] Nanoholes in the passivation layer were optionally partially or fully filled with analyte-binding substances, as shown in Figure 1c; the analyte is then bound to that substance instead of the substrate exposed by the nanoholes. The analyte-binding substance was selected from molecules comprising esters, succinimide, maleimide, amino, carboxyl or click-chemistry reagents, biotin, antibodies or nanobodies. The method for nanohole filling was selected from the coating, derivatisation, modification and covalent interaction. Nanoholes were fabricated by placing blocking elements, which were then removed, as shown in Figure 1d. Alternatively, nanoholes were filled by a polymer brush binding substance that was covalently attached to the aminated glass, as shown in Figure 1e, or biomolecule binding substance, as shown in Figure 1f.

[0295] The nanohole pattern was selected from rectangular, triangular, or randomly distributed, as shown in Figure 1g. The blocking elements were selected from spherical, cubical, flat facetcontaining blocking elements, see Figure 1h. The blocking elements were closely packed, as seen in Figure 1 i, or sparsely distributed; as shown in Figure 1j, sparsely distributed blocking elements allowed brush diffusion around particles and robust passivation. The method of removal of blocking elements was selected from sonication, dissolution, photodegradation, illumination, use of magnets, photocleavage, or chemical cleavage. Nanohole activation by sonication is simple. The activation of analyte binding substance by illumination is especially beneficial, as it allows the analyte to land in the holes in the illuminated area only, and by moving the FoV so the next measurement can be initiated. In other words, instead of measurement in multiple wells, only one well can be used for repeated measurements, allowing for effective measurements of time evolution and titration experiments.

[0296] Optionally, the substrate was coated with the electrode, concretely indium tin oxide, that allowed measuring electrochemical signals from the analyte by pulse voltammetry, square wave voltammetry, cyclic voltammetry, chronoamperometry, open circuit potential or chronopotentiometry.

[0297] Example 9.

[0298] Principle and concept

[0299] Surface was prepared by the cloud-point PEGylation as described above. However, instead of putting masking elements before PEGylation, holes were manufactured by long exposure to laser for 2 to 10 minutes in the field of view intended for measurements. The underlying principle is the photodegradation of the PEG coating. The illumination was performed in the same buffer which was later used for measurements. After manufacturing of the nanoholes, the protein solution in the same buffer was added to the measurement well to fill 20 pL measurement well, and data were acquired. Figure 8 compares the mass photometry measurements on glass and pre-illuminated nanohole PEG for soluble protein Dynamin at concentration of 10 nM and membrane protein Elie at concentration of 50 nM in 50-200 mM ammonium acetate buffer with or without GDN detergent of 2xCMC concentration; The 1 CMC concentration of detergent is 18 pM. Standard measurement of soluble proteins without detergent at a concentration of 10 nM results in individual PSFs as shown in Figure 8a. Membrane proteins are not stable and aggregate or misfold in the buffer without or with low concentration of detergents, see Figure 8b. Even rapid dilution by a factor of 100 from 2xCMC buffer did not lead to observation of defined protein mass, see Figure 8c. If the detergent of 2xCMC is added to the buffer, the Dynamin protein is not observable as the signal is obscured by the signal from micelles bombarding the surface, see Figure 8d. In the case of membrane proteins, the protein is surrounded by lipids and forms a micelle around this protein, which increases the protein mass, and such events are observable in the field of view, see Figure 8e. However, the spectra show that this signal from protein interferes with the signal from empty micelles, resulting in a broad protein peak that is shifted towards larger masses. If the nanohole surface is used, the micelles are passivated off the surface. Meanwhile, the protein can bind the surface, which results in clear events in the case of both soluble and non-soluble proteins, see Figures 8g, h. In this case, spectra of membrane protein show a well-defined peak corresponding to the mass of the protein increased by the mass of the surrounding micelle, see Figure 8e. Such a type of measurement can be performed at a higher concentration range than standard mass photometry; here, the spectrum was taken at a concentration of 500 nM.

[0300] Covalent capture of membrane proteins in detergents

[0301] The surface composed of nanoholes filled with functionalized Azide-PEG-MW5000 was used for covalent capture of membrane proteins, see Figure 11. Such surface was exposed to 0.01 mM - 5mM concentration of NHS-DBCO linker (Dibenzocyclooctyne-N-hydroxysuccinimidyl ester). Enough linker to cover the entire surface, with incubation times of 1 up to 60 minutes according to selected concentration. After reaction with Azide-PEG, the surface was washed with MilliQ water, leaving nanoholes filled with PEG molecules functionalized by NHS-ester, the chemical group that allows covalent binding of primary amino acids present on the protein. When MSCL and BacA membrane proteins in 1xCMC GDN were introduced at concentration of 70 nM to such a surface, they bound to the NHS-ester groups through their primary amino acids, see Figure 11a. This principle was tested on two membrane proteins measured in detergents, the MSCL and BacA as shown in Figure 11b-e. The quantification of 1 minute movie showed oligomerisation of both membrane proteins The PSFs in Figures 4b, d are sparsely distributed even at the concentration of 70 nM, as the landing rate was reduced by passivating coating around functionalized nanoholes.

Claims

CLAIMS1. A device for detecting an analyte, the device comprising: an optical system having a point spread function, a substrate, a passivation layer provided on the substrate, and a plurality of nanoholes distributed within the passivation layer.

2. The device according to claim 1 , wherein the passivation layer is a polymeric brush.

3. The device according to claim 1 or claim 2, wherein the separation of the nanoholes is smaller than the point spread function of the optical system.

4. The device according to any one of claims 1 to 3, wherein the variations of surface reflectivity under measurement conditions within the length scale of 200 nm - 20 pm of the substrate with the passivation layer and provided with the nanoholes is of the same order of magnitude as the substrate.

5. The device according to any one of the preceding claims, wherein the surface reflectivity under measurement conditions of the coated substrate with the passivation layer and provided with the nanoholes is of the same order of magnitude as the substrate.

6. The device according to any one of the preceding claims, wherein the passivation layer comprises 10-5000 nanoholes per pm2.

7. The device according to any one of the preceding claims, wherein the nanoholes have a diameter of less than 200 nm.

8. The device according to any one of the preceding claims, wherein the thickness of the passivation layer is less than 50 nm.

9. The device according to any preceding claim, wherein the surface area of the nanoholes to the surface area of the passivation layer is at a ratio of between 1 : 1000 and 1 :10.

10. The device according to any preceding claim, wherein the surface is exposed by the nanoholes coated, derivatised or modified to increase analyte binding.

11. The device according to any preceding claim, further comprising a blocking element configured within the nanoholes of the passivation layer.

12. A method for measuring a property of an analyte, the method comprising the steps of:(i) providing a device according to any one of claims 1 to 11 ;(ii) adding a solution comprising an analyte to the substrate such that the analyte is received by the nanoholes;(iii) illuminating the surface of the device and detecting a signal from the analyte using a detector; and(iv) measuring at least one property of the analyte using the detected signal.

13. The method according to claim 12, wherein measuring a property of the analyte includes (i) quantifying the mass of the analyte and / or (ii) quantifying a change in the mass of the analyte.

14. The method according to claim 12 or claim 13, wherein the analyte binds to the surface of the substrate exposed by the nanoholes.

15. The method according to any one of claims 12 to 14, wherein the surface exposed by the nanoholes comprises at least one substance for binding to the analyte, and, optionally, wherein the substance fills or partially fills the nanohole.

16. The method according to any one of claims 12 to 15, wherein the detected signal comprises light interference from the analyte and the substrate.

17. The method according to any one of claims 12 to 16, wherein the detected signal is pure light scattering from the analyte.

18. The method according to any one of claims 9 to 17, further comprises the step of collecting one or more images of the analyte.

19. The method according to any one of claims 9 to 18, wherein the substrate is conductive and step (iii) comprises measuring an electrochemical signal of the analyte, optionally wherein the electrochemical signal is electrochemical impedance spectroscopy, differential pulse voltammetry, square wave voltammetry, cyclic voltammetry, chronoamperometry, open circuit potential or chronopotentiometry.

20. The method according to any one of claims 12 to 19, wherein the analyte sample has a concentration range selected from: 0.05-0.20 pM, 0.2-1.0 pM, 1-10 pM, 10-100 pM, or 0.1- 1.0 mM or higher.

21. The method according to any one of claims 12 to 20, wherein the property is measured from a plurality of nanoholes or form individual nanoholes.

22. A system for measuring a property of an analyte, the system comprising:(I) a device for detecting an analyte according to any of claims 1 to 11 ;(II) an illumination source for illuminating the surface of the device; and(III) a detector of scattered light for detecting a signal from the analyte.

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