Solid state nanopores for nanoparticle sensing

Abstract

The invention provides a method of forming a coated nanopore in a silicon nitride membrane on a silica substrate, comprising (i)Depositing layers of SiNx on opposing surfaces of a sheet of a silica substrate to form opposed SiNx membranes; followed by steps (ii-a) and (iii-a), or (ii-b) and (iii-b): (ii-a) Removing a portion of one of the SiNx membranes and a portion of the silica substrate to form a cavity in the silica substrate which is open on one side and bounded by the opposed membrane of SiNx on the other; (iii-a) Forming a nanopore aperture through the opposed membrane of SiNx, in the region of the cavity to form a continuous channel; or (ii-b) Forming a nanopore aperture through one of the membranes of SiNx; (iii-b) Removing a portion of the other SiNx membrane and a portion of the silica substrate in the region of the nanopore aperture to form a cavity in the silica substrate in the form of a continuous channel; wherein steps (ii-a) and (iii-a) or (ii-b) and (iii-b) are followed by steps (iv) and (v): (iv) Forming a layer of SiO2 on exposed surfaces, including the surface of the nanopore aperture formed in step (iii) and the substrate surfaces; (v) Modifying the layer formed in (iv) by chemical or physical means. Chips comprising nanopores obtainable by this method, and uses in nanomedicine and resistive pulse sensing methods, are also provided.

Description

[0001] SOLID STATE NANOPORES FOR NANOPARTICLE SENSING

[0002] Field of the Invention

[0003] The invention relates to a method of forming a coated nanopore in a silicon nitride membrane on a silica substrate, chips comprising a coated nanopore obtainable using this method, uses of the chip and a coated chip comprising a nanopore in a silicon nitride membrane.

[0004] Background of the Invention

[0005] The electrical sensing zone technique has, since its discovery by W.H. Coulter around 1950, been widely employed for size and count analysis of blood cells, bacteria, and other fine particles. Over the last decades, the application range has expanded to nanoparticles, such as liposomes, exosomes, viruses and nanobubbles, as a result of improved electronics and nanopore aperture fabrication.

[0006] Spectradyne uses a unique approach, compared with the usual electrical sensing zone devices. Instead of two electrodes they use three, two biased and one sensing electrode, with the sensing electrode based in between a fluidic voltage divider, consisting of a fluidic resistor and a nano-constriction. Particles get pushed through the nano-constriction by a pressure- driven fluid flow, with the sensing electrode measuring a voltage pulse upon particle translocation. The method, alongside their design of the nano-constriction, allows for a very high bandwidth, so that very short transitions times and a high particle throughput are possible [1 , 2].

[0007] The micro-mould used to define the microfluidic polydimethylsiloxane (PDMS) structure is fabricated using a combination of metal and epoxy features and patterned by optical and electron-beam lithography. The substrate consists of a bare silicon wafer, patterned with gold alignment marks. Electron-beam lithography and a standard liftoff process are used to define the gold metal layer for the thin regions of the mould (200 nm - 500 nm in thickness), including the fluid resistor and nano-constriction.

[0008] The chip contains a microfluidic guiding and filtering system, using several pressure-controlled ports. Depending on the applied relative pressures particles can either enter the nanoconstriction or flow to a waste port. Because of their use of microfluidic elements, Spectradyne calls their method Microfluidic Resistive Pulse Sensing (MRPS). Their newest device called ARC is also capable of detecting the fluorescence signal of probe particles, using a focused laser excitation at the nano-constriction and several detection channels. Particles in a size range from 50 nm to 2 .m can be detected with Spectradyne’s devices and they have been used for the characterisation of a multitude of biologicals samples, like extracellular vesicles (EVs) and liposomes. The detection limit of 50 nm is arguably a drawback, as many samples of interest, such as viruses, are below this value. As electrokinetic transport does not play a role, the devices are not capable of measuring the charge or more explicitly the zeta potential of the analyte particles. Their cartridges are single use only.

[0009] ReSun uses a complicated preparation method of very thin silicon nitride films on silica membranes which were themselves formed through thermal growth on silicon. Pores in silicon nitride are used in ReSun’s electrical sensing zone measurement devices. The pores are fabricated with a combination of electron beam lithography and reactive ion etching [3],

[0010] Figure Analytics uses 3D printed cartridges for the fabrication of multi-resistive pulse sensor microfluidic devices. Particles in the size range of 100 nm - 30 pm can be measured simultaneously [4, 5],

[0011] Northern Nanopores produces solid-state nanopores by automated controlled breakdown (CBD). The diameter of the fabricated pores are approximately 1-20 nm, making them predominantly suitable for molecular detection [6],

[0012] Electronic Biosystems produce glass nanopore membranes for electrical sensing zone applications. The synthesis of the glass structures is initiated by sealing the tips of electrochemically sharpened Au and Pt microwires into glass membranes at the end of a soda lime or lead glass capillary. Glass nanopore membranes are fabricated by removal of the metal sealed in the glass membranes [7],

[0013] Femtoprint showcases multi-material 3D microprinting of wafer scale chips, using the unique FEMTOPRINT® microfabrication platform for precise 3D structuring in fused silica glass (SiO2) and selective silicon nitride (SiNx) ablation, in parallel with low-pressure chemical vapor deposition (LPCVD) of SiNx. This procedure leads to a freestanding SiNxwindow, which is permanently attached to a SiO2 support chip. After parallel chip manufacturing, a single nanopore in each chip is created by focused helium-ion beam or by controlled breakdown (CBD). Compared to silicon chips, the resulting fused silica nanopore chips result in a 4-fold improvement of both the signal-to-noise ratio and the capture rate for signals from the translocation of lgG1 proteins at a recording bandwidth of 50 kHz. At a bandwidth of ~1 MHz, the noise from the fused silica nanopore chips is 3- to 6-fold reduced compared to silicon chips [8]. Izon Science have developed Tunable Resistive Pulse Sensing (TRPS), a single particle analysis characterisation technique that has been shown to directly measure the particle size distribution, concentration, and surface charge density of synthetic and biological nanoparticles, such as EVs and lipid nanoparticles (LNPs). It does this electrically rather than optically, by measuring the impedance change when an analyte / particle passes through a pore. The tunability of applied pressure and voltage allows for full control of convective and electrokinetic velocity contributions of single particles translocating the pore and is a prerequisite of measuring physicochemical particle properties. The measurement of physicochemical properties such as size, shape, concentration, surface charge, density, stability and porosity of polydisperse complex biological samples is critical to assess sample quality from production and isolation methods. TRPS has been shown to be very useful for EV and nanomedicine formulation measurements, paving the way for a range of diagnostic and therapeutic applications.

[0014] TRPS technology is currently based on thermoplastic polyurethane (TPU) membranes and pores [9], The fabrication of these pores is cumbersome and has a low reproducibility, with each pore being different

[0010] , The lower size of these pores is limited due to the pore fabrication process, limiting the lower particle detection limit to approximately 40 nm. Hence, particles smaller than 40 nm, such as some extracellular vesicles, lipid nanoparticles, or viruses cannot be detected with current TRPS instruments. Each pore requires calibration with a set of nanoparticle standards. This currently makes TRPS measurements complicated and slow. For accurate and high-throughput TRPS measurements the pore fabrication method needs to guarantee pores with well-defined size and shape. There is also the need for fabrication of disposable pores in large numbers, for TRPS to be amenable for clinical diagnostic applications.

[0015] There are a number of disclosures in the prior art of nanopores formed in SiNxmembranes on silica substrates. Balan et al in

[0011] disclose the fabrication of a 100 nm thick free-standing SiNxmembrane on a glass substrate, as a low capacitance platform for sensing applications using a variety of 2D materials, including nanopores etched by focused electron beam etching with a Transmission Electron Microscope (TEM).

[0016] Lee et al in

[0018] disclose an ultrathin (5nm) SiNxmembrane transferred onto a quartz glass substrate. Nanopores of 1.5nm diameter are drilled by TEM. DNA sequencing applications are disclosed. Application of further coatings is not disclosed.

[0017] Vreede et al in [8] disclose a manufacturing process for full wafers of fused silica substrate with 30nm SiN membranes (attached by Low Pressure Chemical Vapour Deposition), with specific comparison between silicon and silica substrates. Nanopores of around 2nm diameter are drilled by He-ion beam or by controlled breakdown. Polymer coatings for reducing capacitance are mentioned.

[0018] Chien et al in

[0012] disclose fabrication of an extremely thin (< 10 nm) membrane in amorphous silicon, suspended across a 150 nm diameter aperture in a SiNxmembrane, by TEM. Polymer coatings to further modify the silicon surface are not disclosed.

[0019] Eggenberger et al in

[0019] review nanopore coating processes including extensive comments on Atomic Layer Deposition (ALD), although not with SiC>2, and silanisation onto quartz and onto SiN nanopores with a thin layer of SiC>2.

[0020] Extracellular vesicles (EVs) are small lipid-membrane-enclosed bodies of size greater than 30 nm in diameter. EVs can be classified into several different subtypes, based on biogenesis, such as exosomes (100 nm) and apoptotic bodies (>100 nm). EVs are released by cells and microorganisms and are present in body fluids such as blood, urine, saliva, bile, or cerebrospinal fluid. One of their major roles is to transport molecular components from cell to cell, protecting their content from enzymes. EVs have been considered a recent avenue for biomedical investigation in both regenerative and pathogenic contexts, with potential in diagnostic and therapeutic applications. Their potential use in manifold applications, including drug delivery, particle loading and other theragnostic applications have been highlighted recently. Physicochemical properties of EVs are critical quality attributes that must be measured in a standardised way to assure quality, potency, stability, and batch to batch consistency of EV samples. Unfortunately, the lack of standardised methods for the characterisation of those quality attributes remains a strong regulatory gap.

[0021] Nanomedicine formulations typically represent a multifunctional and multicomponent system on various size levels, making these very different from a typical small molecule drug. This complexity is reflected in the diverse physicochemical properties possessed by a variety of carrier and nanomaterials, such as liposomes, lipid nanoparticles, extracellular vesicles, polymeric particles, micelles, metal colloids, and dendrimers, which are designed for diagnostic and therapeutic application in the pharmaceutical industry. However, the lack of substantial preclinical analyte / particle characterisation has been identified as their “rate limiting step”, slowing their regulatory approval and commercialization. As the key physicochemical properties of a nanoparticle influence its biological compatibility and thus govern the resulting outcome of nano-bio interactions, a major emphasis is focused on accurate determination of these particle properties, including size, surface charge and hydrophobicity [13-15],

[0022] Hence, precise, accurate and high-resolution nanoparticle characterisation plays a critical role not only in the development of nanomedicine formulations, which include fine tuning of nanobio interactions

[0016] , but also during FDA regulatory submission, review, and acceptance.

[0023] Recombinant adeno-associated viruses (rAAVs) are commonly used vectors for in vivo gene therapy, primarily because of their non-pathogenicity to humans, low immunogenicity, and long-term gene expression. The US Food and Drug Administration recently approved rAAV- based gene therapies for treatment of two rare monogenic diseases and rAAV-based gene therapies are currently being investigated in many clinical trials for a range of diseases from cancer to neurological disorders.

[0024] AAV particles consist of a small, approximately 25 nm icosahedral capsid. Due to their small size, the measurement of these particles with currently available electrical sensing zone methods is not possible. Chromatography- and light-scattering-based methods have progressed to facilitate rapid, high-throughput characterization of three potential critical quality attributes of rAAVs: capsid titer, content ratio, and aggregate content

[0017] ,

[0025] There remains a need for more accurate techniques than light-scattering-based methods for the characterisation and measurement of biological molecules, including AAVs.

[0026] Summary of the Invention

[0027] In accordance with a first aspect of the invention, there is provided:

[0028] A method of forming a coated nanopore in a silicon nitride membrane on a silica substrate, comprising

[0029] (i) Depositing layers of SiNxon opposing surfaces of a sheet of a silica substrate to form opposed SiNxmembranes; followed by steps (ii-a) and (iii-a), or (ii-b) and (iii-b):

[0030] (ii-a) Removing a portion of one of the SiNxmembranes and a portion of the silica substrate to form a cavity in the silica substrate which is open on one side and bounded by the opposed membrane of SiNxon the other; (iii-a) Forming a nanopore aperture through the opposed membrane of SiNx, in the region of the cavity to form a continuous channel; or

[0031] (ii-b) Forming a nanopore aperture through one of the membranes of SiNx;

[0032] (iii-b) Removing a portion of the other SiNx membrane and a portion of the silica substrate in the region of the nanopore aperture to form a cavity in the silica substrate in the form of a continuous channel; wherein steps (ii-a) and (iii-a) or (ii-b) and (iii-b) are followed by steps (iv) and (v):

[0033] (iv) Forming a layer of SiC>2 on exposed surfaces, including the surface of the nanopore aperture formed in step (iii-a) or (ii-b) and the substrate surfaces;

[0034] (v) Modifying the layer formed in (iv) by chemical or physical means.

[0035] In accordance with a second aspect of the invention there is provided a chip comprising a coated nanopore in a silicon nitride membrane on a silica substrate, wherein the chip is obtainable by the method according to the first aspect of the invention.

[0036] In accordance with a third aspect of the invention there is provided use of a chip according to the second aspect of the invention in nanomedicine, development and characterisation of vehicles for vaccine or drug delivery, and / or gene therapy.

[0037] In accordance with a fourth aspect of the invention there is provided a coated chip comprising a nanopore in a silicon nitride membrane on a silica substrate; wherein the chip comprises a cavity formed within the silica substrate and connected to the nanopore to form a continuous channel, wherein the nanopore comprises an aperture connecting the exterior of the chip to the cavity; wherein the chip comprises multiple layers; wherein the multiple layers comprise a central silica substrate coated with a first SiNxlayer above, and a second SiNxlayer below the central silica substrate respectively; wherein the surfaces of the chip, including the inner surfaces defining the nanopore aperture is covered with a layer of SiC>2, which is coated with a layer of further material.

[0038] The invention provides a method for producing reproducible solid state nanopores of defined size and shape in a silicon nitride (SiNx) membrane on a silica substrate. This approach can significantly speed up the pore fabrication process and enhance the accuracy, throughput, and reliability of the pores when in use, for instance in Tunable Resistive Pulse Sensing (TRPS) measurements. The methods disclosed herein ensure the highest reproducibility of the pores, enabling standardised measurements in bio-chemical settings - for instance in nanomedicine, gene-therapy and for the measurement of extracellular vesicles (EVs). Additionally, the methods disclosed herein make pore fabrication more cost-effective, which is essential for using TRPS in clinical studies. In addition, compared with the prior art TPU pores discussed above, the glass-on-glass pores of the present invention can be made much smaller, thus allowing for the detection of smaller biomolecules. The addition of a top coating further enables the properties of the nanopores to be “tuned” to their desired end purpose.

[0039] In this invention the chip is formed by a multi-step process involving deposition of SiC>2 followed by further coatings on top of the SiC>2 to optimise the nanopore surface to the specific application at hand. Final layer coatings may be chosen for instance to reduce blockages, increase wetting and / or reduce non-specific binding. In other embodiments it may be possible to reduce the charge of the pore surface to enable highly accurate zeta potential measurements. The nanopore size and geometry may also be tailored to the final use of the chip, for instance in resistive pulse sensing applications, specifically for the detection of AAVs or in gene therapy.

[0040] Brief Description of the Figures

[0041] Figure 1 shows a nanopore chip and aperture, made using semiconductor manufacturing methods, with the region of the free-standing silicon nitride membrane (1) and the nanopore aperture (2) detailed;

[0042] Figure 2 shows a chip cross section after using Atomic Layer Deposition (ALD) to introduce a thin coating of silicon dioxide to the chip in Figure 1 ;

[0043] Figure 3 illustrates a chip produced following the process according to the invention wherein a subsequent hydrophilic coating has been applied to the chip shown in Figure 2. Labels show the cavity in the silica substrate (1); the silica substrate layer (2), the upper, or front (3), and lower, or back (4), layers of silicon SiNx; the layer of silicon dioxide deposited by ALD (5); and the layer of polymer coating (6);

[0044] Figure 4 shows a section of the nanopore chip with dimensions indicating diameter (d) and thickness (t);

[0045] Figures 5 and 6 show current traces (including an example of a single blockade from each) recorded in Example 1 ;

[0046] Figure 7 shows a current trace (including an example of a single blockade from that trace) recorded in Example 2; Figure 8 shows a current trace (including an example of a single blockade from that trace) recorded in Example 3;

[0047] Figure 9 shows high-resolution carbon XPS scans of wafers 1 (left), 2 (middle), and 3 (right) demonstrating the presence of PEG functional groups in Example 4 (solid bars - untreated, unshaded bars - PEGylated); and

[0048] Figure 10 shows a sequence of current traces over a 1 hr 50min run time with a single PEG- coated nanopore, with insets expanding some of those traces to show the current baseline and superimposed blockades.

[0049] Detailed Description of the Invention

[0050] The present invention provides a method as described in the first aspect above. All methods of the invention comprise steps (i), (iv) and (v). The ordering of the individual features making up steps (ii) and (iii) may differ, and therefore steps (ii) and (iii) of the method may proceed in the order (ii-a) followed by (iii-a), or (ii-b) followed by (iii-b).

[0051] This method can alternatively be described as follows:

[0052] A method of forming a coated nanopore in a silicon nitride membrane on a silica substrate, the method comprising either

[0053] (i-a) Depositing layers of SiNxon opposing surfaces of a sheet of a silica substrate to form opposed SiNxmembranes;

[0054] (ii-a) Removing a portion of one of the SiNxmembranes and a portion of the silica substrate to form a cavity in the silica substrate which is open on one side and bounded by the opposed membrane of SiNxon the other;

[0055] (iii-a) Forming a nanopore aperture through the opposed membrane of SiNx, in the region of the cavity to form a continuous channel;

[0056] (iv-a) Forming a layer of SiO2 on exposed surfaces, including the surface of the nanopore aperture formed in step (iii-a) and the substrate surfaces;

[0057] (v-a) Modifying the layer formed in (iv-a) by chemical or physical means; or

[0058] (i-b) Depositing layers of SiNxon opposing surfaces of a sheet of a silica substrate to form opposed SiNxmembranes;

[0059] (ii-b) Forming a nanopore aperture through one of the membranes of SiNx;

[0060] (iii-b) Removing a portion of the other SiNxmembrane and a portion of the silica substrate in the region of the nanopore aperture to form a cavity in the silica substrate in the form of a continuous channel; (iv-b) Forming a layer of SiC>2 on exposed surfaces, including the surface of the nanopore aperture formed in step (iii)-b and the substrate surfaces;

[0061] (v-b) Modifying the layer formed in (iv)-b by chemical or physical means.

[0062] In an alternative, the present invention provides a method of forming a coated nanopore in a silicon nitride membrane on a silica substrate, comprising

[0063] (i) Depositing layers of SiNxon opposing surfaces of a sheet of a silica substrate to form opposed SiNxmembranes;

[0064] (ii) Removing a portion of one of the SiNxmembranes and a portion of the silica substrate to form a cavity in the silica substrate which is open on one side and bounded by the opposed membrane of SiNxon the other;

[0065] (iii) Forming a nanopore aperture through the opposed membrane of Si Nx, in the region of the cavity to form a continuous channel;

[0066] (iv) Forming a layer of SiC>2 on exposed surfaces, including the surface of the nanopore aperture formed in step (iii) and the substrate surfaces;

[0067] (v) Modifying the layer formed in (iv) by chemical or physical means.

[0068] The first aspect of the invention provides a five step process for the production of one or more nanopores in a silicon nitride membrane on a silica substrate. “Silica” may also be referred to as “glass” or as silicon dioxide, an oxide of silicon with the chemical formula SiC>2. In the invention, a central layer of silica is fused on both of its planar surfaces to silicon nitride, otherwise referred to as SiNx. Silicon nitride is a chemical compound of the elements silicon and nitrogen. SisN4 (trisilicon tetranitride) is the typical form of silicon nitride. In thin films there might be deviations from this stoichiometry, and the resulting SiNxprovides a lower stress thin film than SisN^ Stoichiometry may also depend on the presence of traces of contaminants stemming from the deposition precursors, for instance Cl' ions.

[0069] A nanopore is a channel or aperture passing through the silicon nitride membrane. Its dimensions are on the nanoscale, i.e. typically in the range 1 to 1000nm. In this invention, the substrate that the membrane is bound to is silica, rather than silicon (which is typically used for most nanofabrication processes), to minimise the noise in electrical sensing zone applications. The silica substrate gives strength and body to the resultant nanopore chip while keeping a very low capacitance.

[0070] The ultimate layered ensemble, with silicon nitride membranes on the silica substrate, is generally provided in the form of a “chip”, which is generally small (for instance, 5mmx5mm cross section with a thickness around 250pm) and has the nanopore channel running through the centre. On an industrial scale, many chips may be produced from a larger piece of silica substrate known as a wafer, which is then divided into the constituent chips for use. The chip may alternatively be referred to as a “device” in this disclosure.

[0071] The fabrication process comprises the following five steps:

[0072] Step 1 : The first step of the invention comprises depositing layers of SiNxon opposing surfaces of a sheet of silica (SiO2) to form opposed silicon nitride membranes. The sheet of silica may have a thickness in the range 200 to 550 pm or 200 to 500pm and is typically around 250 pm. If the sheet of silica is viewed as a plane then a layer of SiNxis deposited above and below the plane of the silica.

[0073] Silicon nitride may be deposited onto the substrate using chemical vapor deposition (CVD). One of these SiNxlayers becomes the membrane through which the nanopore aperture is formed. The thickness of the layers is equal to or greater than the ultimate desired thickness of the membrane. The initial thickness can be up to 300nm, for instance it may be in the range 20 to 300nm, with thicker layers formed initially as they are inherently stronger than thinner ones. The silicon nitride can be thinned down later in the manufacturing process, typically to the range of 30 - 50nm, which allows for aperture diameters of 80 - 100nm at a thickness I diameter ratio of 0.5 (i.e. d = 2t where d and t are exemplified in Figure 4), all dimensions which are particularly beneficial for AAV measurements.

[0074] Step 2: Step 2 comprises removing a portion of the silica glass and a portion of the lower SiNxmembrane formed in step (1), to form a cavity. A portion of the layer of SiNxopposing the layer for which the nanopore is intended is first removed. Masking, photolithography, and reactive ion etching may be used to open up a hole in the opposing SiNxlayer, which then allows free access to an agent which is able to remove the silica.

[0075] Silica may be removed by isotropic etching with 49% HF, which is a suitable reagent for this process. The etching is halted when the HF has fully penetrated the silica and leaves a freestanding membrane of SiNxon the membrane opposing the etching point, at the apex of the cavity formed through silica substrate. The cavity generally takes a dome-like shape. The HF may also remove the SiNxaround the injection point, although this reaction occurs at a slower rate.

[0076] Step 3: The third step of the method according to the first aspect of the invention comprises forming a nanopore channel through the free-standing SiNxmembrane, which is much narrower than the cavity formed in the substrate from the HF etching mentioned above. Steps 2 and 3 may be performed in any order. Therefore, in one embodiment, the cavity is formed first, followed by the formation of the nanopore channel through the freestanding SiNx membrane. The method comprises steps (ii-a) and (iii-a), and step (iii-a) comprises forming a nanopore aperture through the remaining layer of SiNxwhich forms a free-standing membrane over the cavity formed in step (ii-a).

[0077] In another embodiment, the nanopore channel is formed first, and the cavity is formed afterwards. The method comprises steps (ii-b) and (iii-b), and step (ii-b) comprises forming the nanopore aperture through the layer of SiNxwhich forms a free-standing membrane over the cavity after it is formed in step (iii-b).

[0078] The fabrication of the nanopore channel can be done using various methods, such as focussed ion beam (FIB), or a combination of E-beam lithography and reactive ion etching (RIE), or laser ablation. These preferred methods for forming the pores involving lithographic fabrication result in reproducible pores of defined size and shape.

[0079] Step 4: A silicon oxide layer on top of SiNxis created to render the nanopores hydrophilic and amenable to further coating processes as set out in step 5. Generally in the fourth step a layer of SiC>2 is deposited on all exposed surfaces of the chip; this includes the inner surfaces of the cavity and nanopore channel formed in the second and third steps, as well as any exposed surfaces of the silica substrate and SiNxmembranes. As an alternative to deposition the layer of silicon oxide can be formed by oxidation of the SiNxmembrane formed previously.

[0080] The silica layer thus produced is generally substantially uniform in thickness.

[0081] Uniform silica layers can be formed in a controlled way on top of the SiNxpore using methods such as Atomic Layer Deposition (ALD). Alternatively, oxygen plasma, UV / ozone or wet acid and base based methods can be used to create a layer of SiC>2 on SiNx.

[0082] The silica (SiC>2) material introduced in the fourth step may be the same material as that forming the glass in the silica substrate. However, it may have a different crystalline structure, as, for instance, it originates from the ALD deposition process.

[0083] The layer of silicon oxide formed in step 4 may have a thickness in the range 1 to 30nm, and this is typically the case when using Atomic Layer Deposition (ALD). ALD is a monolayer by monolayer deposition process. The ultimate layer is alternatively 1 to 30 or 1 to 10nm thick. The thickness of a SiC>2 monolayer is generally between 0.25-0.5 nm and hence at least 20 ALD cycles are needed to create a 10 nm layer. Regular chemical vapour deposition can be used to create thicker layers but is generally less controlled. Oxidation of SiNxvia oxygen plasma and ozone typically creates a SiO2 layer that is less than 10 nm in thickness.

[0084] The SiO2 layer formed in step 4 is an intermediate coating for the further modifications set out in step 5.

[0085] Step 5: In the fifth step of the invention the layer formed in the fourth step is modified by chemical or physical means. This may involve applying a further coating of a material, preferably a hydrophilic material. In this step, the SiO2 top layer formed in the previous step can be modified with other coatings, such as organosilanes or PEG silanes, to suit the application at hand. Zwitterionic coatings, e.g. based on amino acids such as sulfobetaine (SB) and carboxybetaine (CB), may also provide useful surface coatings. In one embodiment, the silica surface formed in step 4 may be silanised using an organosilane coating, and then further modified to provide a zwitterionic surface for less biofouling.

[0086] Accordingly, the coating of this step may comprise a material that reduces the effect of fouling or any other nonspecific binding between a sample and the chip surface.

[0087] Alternatively, the coating of this step may comprise a material that modifies the charge of the chip surface when wetted with electrolyte.

[0088] The thickness of the layer formed in step 5 is generally in the range 1-30nm, 1-20nm or 1- 15nm, e.g. 1-10nm or 1.5nm. For most applications a monolayer is desired, and hence the coating will typically be only several nm thick. In the case of PEG silane, for instance, the thickness will strongly depend on the number of PEG units.

[0089] A major advantage of following method steps one to four is that the surface of the nanopores thereby produced can be easily modified. Wet chemistry and chemical deposition methods are available to modify the SiO2 surface of the nanopores with a wide range of materials to provide tunable properties such as hydrophobicity, hydrophilicity, high surface charge, no surface charge, and antifouling, inter alia. Custom made coatings can be tuned to the nanopores to enable reproducible, highly accurate TRPS measurements. Low or no surface charge is particularly beneficial for zeta potential measurements, while hydrophilicity greatly aides fast wetting of the nanopore when electrolyte is first applied to it.

[0090] The method of the invention may further comprise one or more steps of depositing a layer of photoresist to aid the other steps of the process. The second aspect of the invention provides a chip comprising a coated nanopore in a silicon nitride membrane on a silica substrate, wherein the coated nanopore is obtainable (or obtained) by the method according to the first aspect of the invention. These nanopores are reproducible pores with optimised aspect ratio for concentration, size and surface charge measurements. Such a nanopore is illustrated in Figure 3. The cavity (1) through the fused silica substrate (2) is shown along with the upper and lower surface coatings of SiNx(3,4). The SiNxlayers are further coated with a layer of SiC>2 (5), typically deposited by ALD. A final polymer coating (6) is then applied to the SiC>2 layer (5).

[0091] The third aspect of the invention provides use of the chip described above in nanomedicine, development and characterisation of vehicles for vaccine delivery, and / or gene therapy. More detail on these uses is given below.

[0092] The fourth aspect of the invention provides a coated chip comprising a nanopore in a silicon nitride membrane on a silica substrate. Features of this coated chip may apply to the first aspect of the invention also. The chip comprises a cavity formed within the silica substrate and connected to the nanopore to form a continuous channel, wherein the nanopore comprises an aperture connecting the exterior of the chip to the cavity. Typically the cavity is generally hemispherical. By generally hemispherical is meant that the cavity is substantially hemispherical. It may take a dome-like shape forming approximately half a sphere in the silica substrate material. By “continuous channel" is meant that the nanopore and hemispherical cavity are linked and in fluid communication with each other, when the chip is submerged in a fluid or fluid is delivered to both sides of the chip.

[0093] The chip comprises multiple layers which include a central glass layer coated with a first SiNx layer above, and a second SiNxlayer below the central glass layer respectively. The surface of the chip, including the surface of the aperture is covered with a layer of SiC>2, which is further coated with a layer of further material. By central is meant that the glass layer is present in the interior of the chip and is sandwiched by layers on both of its surfaces. The layers above and below the central glass layer need not be of the same thickness.

[0094] In the fourth aspect, the nanopore comprises a thickness / diameter ratio in the range 0.25 to 0.8; and wherein the nanopore comprises a diameter in the range 10 to 500nm, preferably 50 to 200nm. In one embodiment, the thickness / diameter ratio is in the range 0.4 to 0.6; and diameter is in the range 10 to 300nm. Preferably the diameter is in the range 10 to 200nm or 50 to 300nm and most preferably 50 to 200nm, 70 to 150nm, 70 to 120nm or 80 to 100nm. The chip may be produced using the method described above in the first aspect of the invention.

[0095] The nanopore is formed in a support material (silica) with a cavity on one side, which leads to a SiNxmembrane which has a smaller aperture. The diameter of the nanopore is in the range 10 to 500nm or 10 to 300nm, preferably 10 to 200nm, even more preferably 50 to 200nm or 70 to 150nm, as detailed above, whereas the largest diameter of the generally hemispherical cavity in the substrate is much larger than this, and may be for instance in the range 100-1000pm, for instance around 500pm.

[0096] The nanopores made in the invention using standard lithographic methods on a wafer scale have a very high reproducibility regarding dimensions and geometry.

[0097] The pores are generally cylindrical in shape, with the length of the pores being determined by the thickness of deposited material. The pore size can be accurately controlled using the above-mentioned fabrication methods. In this way the aspect ratio of the nanopore can be exactly tuned to achieve maximal sensitivity of the nanopore sensor and ensure accurate measurements of the analyte particle characteristics, concentration and surface charge / zeta potential. A crucial advantage of the device is that the particle speed through the pore can be accurately controlled by balancing pressure-driven flow and electrokinetic transport mechanisms, all of which can be calculated precisely with the knowledge of the pore geometry.

[0098] The nanopore may be defined as having a diameter and a thickness as illustrated in Figure 4, which illustrates a nanopore having a cylindrical shape. The diameter is the dimension parallel to the direction of the glass plane and is the distance across the channel from one edge to the next. The thickness is the length of the nanopore from the top to the bottom of the channel in the membrane, in the direction perpendicular to the plane of glass in the silica substrate. In the method according to the first aspect of the invention, the pore preferably comprises an aspect ratio of thickness to diameter from 0.25 to 0.8, preferably 0.4 to 0.6. More preferably this aspect ratio is in the range 0.45 to 0.55.

[0099] The nanopore may comprise a shape other than cylindrical, such as conical. The diameter for these measurements is then taken as the smallest diameter in the direction parallel to the plane of silica substrate.

[0100] Typically, in the first aspect of the invention, the nanopore comprises a diameter in the range 10nm to 500nm or 10 to 300nm, preferably in the range 10 to 200nm or 50 to 300nm and most preferably 50 to 200nm, 70 to 150nm, 70 to 120nm or 80 to 100nm. The nanopores thus allow for particle detection as small as 1 nm. Current electrical sensing zone methods for nano / microparticle detection have a lower limit of approximately 40-50 nm, mainly due to the pore geometry and electronics used in various devices. The new hydrophilic SiNxpores of this invention bridge the gap between molecular detection (e.g. Oxford Nanopores: DNA detection in biological nanopores) and the current lower limit of 40-50 nm for nanoparticle detection, using electrical sensing zone methods. In theory, the upper limit is determined by the diameter of the nanopore. However, to minimise the chances of pore blockages, particles larger than half the size of the pore should be filtered out from the analyte prior to the measurement using typical filtration devices.

[0101] Nanopores as described in this invention have particular use for the detection of particles in the range 10-50 nm, and accordingly in one preferred embodiment, d is in the range 70- 120nm. However the method according to the first aspect of the invention can be used can used for smaller or larger pores according to the intended use, with the diameter and thickness of the pores adapted accordingly (e.g. larger thickness due to larger layers).

[0102] The invention may find use in many different applications. A preferred use is in particle detection, for instance using resistive pulse sensing (RPS), including Tunable Resistive Pulse Sensing (TRPS). Typically, in use, the device will be placed between two reservoirs that are filled with electrolyte solution. An electrical potential difference is applied between electrodes in these two reservoirs which leads to a steady state ionic baseline current through the nanopore. When an insulating nanoparticle occludes a portion of the volume of the nanopore, it transiently reduces the ionic current and produces a “resistive pulse” that provides information about the physical properties of the molecule.

[0103] A further aspect of the invention provides use of the nanopores described above in biomedical applications, for instance in nanomedicine, biomolecule sensing applications or gene therapy. The nanopores may be used to measure various properties of a biological sample including concentrations, loading, charge, and surface charge.

[0104] A biological sample to be analysed may contain, for instance, AAVs, small lipid nanoparticles (LNPs) or extracellular vesicles (EVs). Extracellular vesicles (EVs) are small lipid membrane enclosed bodies of size greater than 30 nm in diameter. EVs can be classified into several different subtypes, based on biogenesis, such as exosomes (100 nm) and apoptotic bodies (>100 nm). As mentioned in the background to the invention, physicochemical properties of EVs are critical quality attributes that must be measured in a standardised way to assure quality, potency, stability, and batch to batch consistency of EV samples. There is currently a lack of standardised methods for the characterisation of those quality attributes. The present invention addresses this problem by providing nanopores which are reproducible and able to accurately measure the concentrations of these particles.

[0105] The present invention may be useful in nanomedicine formulations. They may have use in therapeutic and diagnostic applications in the pharmaceutical industry. Such formulations are generally complex and may comprise for instance a variety of carrier and nanomaterials, such as liposomes, lipid nanoparticles, extracellular vesicles, polymeric particles, micelles, metal colloids, and dendrimers. The present invention can be used to accurately assess the properties, such as size, surface charge and hydrophobicity. Thus the present invention provides use of a chip as described above in nanomedicine, development and characterisation of vehicles for drug or vaccine delivery, and / or gene therapy.

[0106] Hence, the present invention provides accurate and high-resolution nanoparticle characterisation which can assist with fine tuning of nano-bio interactions

[0016] , and also during FDA regulatory submission, review, and acceptance.

[0107] In one embodiment, the invention is used to detect or analyse recombinant adeno-associated viruses (rAAVs). As mentioned in the background to the invention, due to their small size, the measurement of these particles with currently available electrical sensing zone methods is not possible. The present invention can be used to assess three potential critical quality attributes of rAAVs: capsid titer, content ratio, and aggregate content. Using a chip according to the invention together with the TRPS method can provide a more accurate method for these measurements than light-scattering-based methods.

[0108] In another embodiment, the invention is used for monitoring and / or characterisation of lipid nanoparticles (LNPs).

[0109] References

[0110] 1. Fraikin, J.L., et al., A high-throughput label-free nanoparticle analyser. Nature Nanotechnology, 2011. 6(5): p. 308-313.

[0111] 2. Spectradyne, US009945802.

[0112] 3. Resun, CN109580718A.

[0113] 4. Pollard, M., R. Maugi, and M. Platt, Multi-resistive pulse sensor microfluidic device. Analyst, 2022. 147(7): p. 1417-1424. 5. Figura-Analytics, WO2021079153A1.

[0114] 6. Waugh, M., et al., Solid-state nanopore fabrication by automated controlled breakdown. Nature Protocols, 2020. 15(1): p. 122-143.

[0115] 7. Zhang, B., et al., Bench-top method for fabricating glass-sealed nanodisk electrodes, glass nanopore electrodes, and glass nanopore membranes of controlled size. Analytical Chemistry, 2007. 79(13): p. 4778-4787.

[0116] 8. de Vreede, L.J., et al., Wafer-scale fabrication of fused silica chips for low-noise recording of resistive pulses through nanopores. Nanotechnology, 2019. 30(26).

[0117] 9. Sowerby, S.J., M.F. Broom, and G.B. Petersen, Dynamically resizable nanometre-scale apertures for molecular sensing. Sensors and Actuators B: Chemical, 2007. 123: p. 325-330.

[0118] 10. Vogel, R., et al., Quantitative sizing of nano / microparticles with a tunable elastomeric pore sensor. Analytical chemistry, 2011. 83: p. 3499-3506.

[0119] 11. Balan, A., et al., Suspended Solid-state Membranes on Glass Chips with Sub 1-pF Capacitance for Biomolecule Sensing Applications. Scientific Reports, 2015. 5.

[0120] 12. Chien, C.C., et al., Single-Stranded DNA Translocation Recordings through Solid-State Nanopores on Glass Chips at 10 MHz Measurement Bandwidth. Acs Nano, 2019. 13(9): p. 10545-10554.

[0121] 13. McNeil, S.E., Nanoparticle therapeutics: a personal perspective. Wiley Interdisciplinary Reviews-Nanomedicine and Nanobiotechnology, 2009. 1(3): p. 264-271

[0122] 14. Nel, A.E., et al., Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater, 2009. 8(7): p. 543-557.

[0123] 15. Wilhelm, S., et al., Analysis of nanoparticle delivery to tumours. Nature Reviews Materials, 2016. 1(5).

[0124] 16. Lynch, I., et al., The bio-nano-interface in predicting nanoparticle fate and behaviour in living organisms: towards grouping and categorising nanomaterials and ensuring nanosafety by design. BioNanoMaterials, 2013. 14(3-4): p. 195-216.

[0125] 17. Gimpel, A.L., et al., Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Molecular Therapy-Methods & Clinical Development, 2021. 20: p. 740-754.

[0126] 18. Lee, MH., Kumar, A., Park, KB. et al. A Low-Noise Solid-State Nanopore Platform Based on a Highly Insulating Substrate. Sci Rep 4, 7448 (2014).

[0127] 19. Eggenberger, O. M., Ying, C., & Mayer, M. (2019). Surface coatings for solid-state nanopores. Nanoscale, 11(42), 19636-19657.

[0128] EXAMPLES

[0129] The invention will now be illustrated by the following Examples. EXAMPLE 1 : Hydrophilic Atomic layer deposition (ALD)-coated silicon nitride-on-glass nanopores, 100nm diameter

[0130] Fabrication process

[0131] Nanopores are fabricated to the form illustrated in Figures 1 and 2. The fabrication method starts by creating layers of 100nm silicon nitride (SiN) on both sides of a 250|jm thick silica substrate. For each chip to be etched on the wafer, an aperture is created in the lower face of the SiN by photolithography followed by Reactive Ion Etching (RIE). This aperture allows 49% HF to be administered for isotropic bulk etching through the silica substrate and the lower silicon nitride, which is terminated to give a hemispherical cavity exposing a free-standing SiN membrane (a). An electron-beam lithography (EBL) and further RIE step is run to etch 100nm diameter nanopore apertures (b) in the free-standing membrane, before the wafer is diced into 5mm x 5mm chips with a dicing saw. Finally, all surfaces of the nanopores are fully coated in a thin silicon dioxide coating (Figure 2) by Atomic Layer Deposition (ALD). In this Example the nanopore was cleaned of all residues with 10% Piranha solution but not modified with any further coating.

[0132] Measurement process

[0133] Measurements were made using the Tunable Resistive Pulse Sensing (TRPS) technique with components modified from Izon Science’s Exoid instrument. One nanopore chip is held in a Fluid Cell, which encases the chip and has liquid reservoirs, containing Phosphate-Buffered Saline (PBS) electrolyte, on either side of the chip but whose contents are only in contact though the narrow channel of the nanopore aperture. A pair of electrodes, each in contact with one reservoir, allow a potential difference to be applied through the solution and continuously measure the current flowing through it at a sampling rate of 64ksps.

[0134] Carboxylated polystyrene particles (Bangs Laboratories), with a mean diameter of 24nm and at a concentration of 5 x 1011particles / ml are delivered to one of the liquid reservoirs and a pressure differential of ~1 atmosphere is applied, manually with a syringe pump, between the reservoirs. T ranslocation events of particles flowing through the aperture, under hydrodynamic and electric forces, present as deviations in the baseline current and demonstrate the use of the nanopore in detecting and characterising particles in this size range. An example of a recorded current trace, which shows blockade events due to translocations of these particles, is supplied (Figure 5). An identical pore is used to make like measurements on a population of unmodified polystyrene particles (Thermofisher Scientific) with a mean diameter of 41 nm a concentration of 5 x 1011particles / ml. An example current trace is presented in Figure 6.

[0135] EXAMPLE 2: 9-12 PEG-trimethoxysilane-coated silicon nitride-on-glass nanopore, 200nm diameter

[0136] Fabrication process

[0137] The fabrication process for the nanopore aperture in this Example was as for Example 1. In this Example, the nanopore diameter is etched to 200nm and the surfaces are not coated with silicon dioxide by ALD; instead, the silicon nitride surface is oxidised to a thin monolayer of SiC>2 through exposure to UV-generated ozone. The whole nanopore was then coated with a further monolayer of 9-12 PEG-trimethoxysilane by soaking in a dilute mixture of the silanes in distilled water.

[0138] Measurement process

[0139] Measurements were made with an identical configuration of nanopore, fluid cell, and measurement electronics as in Example 1. In this Example, unmodified 150nm-diameter polystyrene particles were run at a sampling frequency of 256ksps. An example current trace showing blockades due to translocations of these particles is presented in Figure 7.

[0140] EXAMPLE 3: Detection of Adeno-associated Viruses with hydrophilic silica-coated silicon nitride-on-glass nanopores, 100nm diameter

[0141] Fabrication process

[0142] The fabrication process for the nanopore aperture in this Example was as for Example 1 , to the same nominal specifications including aperture size and silica coating. No polymer coating is applied in this Example.

[0143] Measurement process

[0144] Measurements were made with an identical configuration of nanopore, fluid cell, and measurement electronics as in Example 1. A sample of adeno-associated viruses of an unknown concentration was delivered to one chamber of the fluid cell. At a sampling rate of 64ksps and a voltage differential of 100mV, with no external pressure applied, recordings were made of blockades due to translocations of AAVs through the nanopore aperture. An example current trace is presented in Figure 8. EXAMPLE 4: longevity of a 9-12 PEG-trimethoxysilane-coated silicon nitride-on- silicon nanopore

[0145] This Example shows the effect of a polyethylene glycol (PEG) coating on nanopore performance. PEG has been chosen for its antifouling effect and this seen in extreme pore longevity in a Resistive Pulse Sensing application. The attachment of the PEG coating is by a silane functional group and is demonstrated by both XPS and Resistive Pulse Sensing measurements with polystyrene standards.

[0146] While the chips used in this Example use silicon instead of silica as a substrate, the outer SiO2 surface coating on the SiNx membrane is identical in both cases. This example demonstrates the binding of PEG silane to the SiO2 surface, the effectiveness of the SiO2as a platform for functionalisation, and the beneficial effect of this particular modifier.

[0147] Fabrication process

[0148] The chips used in this Example are fabricated by Goeppert LLC. They are comprised of a 250|jm silicon substrate, with 1 pm thick SiO2 deposited on both sides, for capacitive noise reduction, and 100 nm thick SiNx layers of which one forms a free-standing membrane and hosts a 200 nm-diameter aperture. In this example, the chip cavity is etched with KOH, to remove the silicon, and a buffered oxide etch (BOE) to remove the underlying SiO2 to expose the membrane. In other respects, the fabrication process is the same as Example 1.

[0149] Prior to coating with PEG silane, chips were cleaned in dilute piranha solution for 2 hrs. PEG silane (3-[methoxy(polyethyleneoxy)9-12]propyltrimethoxysilane, Gelest Inc.) was dissolved in a 95% ethanol water mixture (w / w) at a concentration of 50 mg / ml. Chips were immersed for 2-3 hrs in the PEG silane solution with the back-etched side facing downwards, and then thoroughly rinsed (at least 3 times) in MilliQ water.

[0150] Measurement process

[0151] X-ray photoelectron spectroscopy (XPS), also known as ESCA (Electron Spectroscopy for Chemical Analysis), was used to monitor the presence of PEG molecules on the chip surface. This is a non-destructive technique which provides chemical analysis of the outermost 5 - 10 nm of any vacuum-compatible solid. The sample is illuminated with monochromatic X-rays which have sufficient photon energy to cause the photoemission of the core level electrons whose binding energies are characteristic of the elements present. By analyzing the kinetic energy of these emitted electrons, the elemental composition and chemical state of the sample's surface can be determined, as the binding energy of each electron is unique to its element and chemical environment. The position and intensity of the binding energy peaks provide both chemical (e.g. oxidation state) and quantitative (> ~0.1 atom %) information for all elements except hydrogen. A Kratos AXIS Supra Plus XPS system was used for the elemental analysis of the chips.

[0152] Untreated chips and PEG silane-coated chips from three different source wafers were analysed with XPS to demonstrate the attachment of the PEG coating layer. The results are supplied in Figure 9.

[0153] XPS results can be assessed in terms of absolute amounts, which equals the areas of binding energy signals, or relative amounts, where signal areas are first weighted and then compared with each other in percentage distributions. In terms of high-resolution scans of the C-content, the distribution of C-O, C=O and C-C bonds is within 2% of the corresponding absolute results, and hence only relative % results are presented here.

[0154] For high-resolution XPS carbon scans, the C-0 content (absolute and relative) is highest for PEGylated chips. This applies for all three tested wafers. This result demonstrates that this PEG silane, which hosts a high density of C-0 bonds, has been attached to the chip surface either by physisorption or covalent bonding.

[0155] Resistive Pulse Sensing measurements are made with identical equipment and samples to Example 1. A series of current recordings through a PEGylated chip are supplied in Figure 10 and exemplify the antifouling effect of this coating. Runs are made with 100nm diameter carboxylated polystyrene particles (Bangs Laboratories) and 100nm diameter unmodified polystyrene particles (Sigma Aldrich) and show clear translocations without detrimental blocking over almost two hours of continuous run time. The hydrostatic pressure applied across the nanopore is controlled between 0 - 2000Pa and translocation rates are seen to change accordingly, with a typical rates at 500Pa of 300particles / min for the carboxylated particles and 180particles / min for the neutral particles.

Claims

1. CLAIMS1. A method of forming a coated nanopore in a silicon nitride membrane on a silica substrate, comprising(i) Depositing layers of SiNxon opposing surfaces of a sheet of a silica substrate to form opposed SiNxmembranes; followed by steps (ii-a) and (iii-a), or (ii-b) and (iii-b):(ii-a) Removing a portion of one of the SiNxmembranes and a portion of the silica substrate to form a cavity in the silica substrate which is open on one side and bounded by the opposed membrane of SiNxon the other;(iii-a) Forming a nanopore aperture through the opposed membrane of Si Nx, in the region of the cavity to form a continuous channel; or(ii-b) Forming a nanopore aperture through one of the membranes of SiNx;(iii-b) Removing a portion of the other SiNx membrane and a portion of the silica substrate in the region of the nanopore aperture to form a cavity in the silica substrate in the form of a continuous channel; wherein steps (ii-a) and (iii-a) or (ii-b) and (iii-b) are followed by steps (iv) and (v):(iv) Forming a layer of SiC>2 on exposed surfaces, including the surface of the nanopore aperture formed in step (iii) and the substrate surfaces;(v) Modifying the layer formed in (iv) by chemical or physical means.

2. Method according to claim 1 comprising steps (ii-a) and (iii-a), and step (iii-a) comprises forming a nanopore aperture through the remaining layer of SiNxwhich forms a free-standing membrane over the cavity formed in step (ii).

3. Method according to claim 1 comprising steps (ii-b) and (iii-b).

4. Method according to any preceding claim wherein the nanopore aperture comprises a diameter and a thickness and the nanopore comprises a thickness / diameter ratio in the range 0.25 to 0.8, preferably in the range 0.4 to 0.6.

5. Method according to any preceding claim wherein the nanopore comprises a diameter in the range 10 to 500nm, preferably 10 to 300nm, more preferably 10 to 200nm, even more preferably 50 to 200nm or 70 to 150nm.

6. Method according to any preceding claim wherein in step (v) a coating is applied to the layer formed in step (iv).

7. Method according to claim 6 wherein the coating comprises a hydrophilic material.

8. Method according to claim 6 or 7 wherein the coating comprises a material that reduces the effect of fouling or other nonspecific binding between a sample and the chip surface.

9. Method according to any one of claims 6 to 8 wherein the coating comprises a material that modifies the charge of the chip surface when wetted with electrolyte.

10. Method according to any one of claims 6 to 9 wherein the coating in step (v) comprises a material selected from organosilanes and PEG silanes.11 . Method according to any preceding claim wherein the SiC>2 is deposited in step (iv) by atomic layer deposition (ALD).

12. Method according to any preceding claim wherein the nanopore in step (iii-a) or (ii-b) is formed by electron beam lithography and masked Reactive Ion Etching (RIE), or by Focused Ion Beam milling.

13. Method according to any preceding claim wherein the sheet of silica substrate comprises a thickness in the range 200-550pm, preferably 200-500pm.

14. Method according to any preceding claim wherein the layers of SiNxformed in step (i) have, independently, a thickness in the range 20 to 300nm.

15. Method according to any preceding claim wherein the layer of SiC>2 formed in step (iv) has a thickness in the range 1 to 30nm, preferably 1 to 15nm.

16. A chip comprising a coated nanopore in a silicon nitride membrane on a silica substrate, wherein the coated nanopore is obtainable by the method according to any preceding claim.

17. A coated chip comprising a nanopore in a silicon nitride membrane on a silica substrate; wherein the chip comprises a cavity formed within the silica substrate and connected to the nanopore to form a continuous channel, wherein the nanopore comprises an aperture connecting the exterior of the chip to the cavity; wherein the chip comprises multiple layers; wherein the multiple layers comprise a central silica substrate coated with a first SiNxlayer above, and a second SiNxlayer below the central silica substrate respectively; wherein the surfaces of the chip, including the inner surface defining the nanopore aperture are covered with a layer of SiC>2, which is coated with a layer of further material.

18. A coated chip according to claim 17 wherein the nanopore comprises a thickness / diameter ratio in the range 0.25 to 0.8, preferably in the range 0.4 to 0.6; and wherein the nanopore comprises a diameter in the range 10 to 500m, preferably 10 to 300nm, most preferably 50 to 200nm.

19. Use of a chip according to any one of claims 16 to 18 in nanomedicine, development and characterisation of vehicles for drug or vaccine delivery, and / or gene therapy.

20. Use of a chip according to any one of claims 16 to 18 for particle detection using resistive pulse sensing.

21. Use according to claim 19 or 20 for monitoring and / or characterisation of adeno- associated viruses (AAVs).

22. Use according to claim 19 or 20 for monitoring and / or characterisation of lipid nanoparticles (LNPs).

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