WO2024150012A1

WO2024150012A1 - Detecting and analysing analytes

Info

Publication number: WO2024150012A1
Application number: PCT/GB2024/050074
Authority: WO
Inventors: James Alan BROZIK; Andrew James Thompson; Michael Joe MARTINEZ; Evan Lewis TAYLOR
Original assignee: SMI Drug Discovery Limited
Priority date: 2023-01-12
Filing date: 2024-01-12
Publication date: 2024-07-18
Also published as: GB202300442D0

Abstract

Methods for detecting and/or analysing analytes in a sample are described. The methods are useful for detecting and/or analysing single molecules. Devices and kits for detecting and/or analysing a sample are also described, and the use of the device are, also described. Particularly, the present invention uses a super resolution optical microscope, having a resolution of 100 nm or less.

Description

DETECTING AND ANALYSING ANALYTES

FIELD OF THE INVENTION

The present invention relates to a method for detecting and/or analysing analytes in a sample, and to devices and kits for doing the same. Particularly, the present invention relates to the use of a super resolution optical microscope.

BACKGROUND OF THE INVENTION

Optical microscopy is a microscopy technique that uses light to produce an image. The use of optical microscopy to observe biological molecules and their interactions is well known in the art. Conventional optical microscopes have a resolution of around 0.2 pm. In cell biology and pathology, this resolution is ideal for studying larger subcellular organelles. For example, a lysosome stands at 0.2 - 0.3 pm in diameter, while larger organelles such as a nucleus stand between 5 - 20 pm in diameter. However, to study smaller organelles, such as ribosomes, macromolecular assemblies, and macromolecules, electron microscopy is generally used.

The use of optical microscopy to observe biological processes in particular has undergone huge advances in the last decade, and optical microscopy is now used to image single molecules. There have been significant advances in resolution including high resolution, often called, super resolution microscopy with resolutions of 100nm or less. High resolution microscopy with resolution of 15 to 20nm, or even lower, such as 1.5nm, is desirable, particularly for drug discovery. Such high resolution is particularly effective for single molecule imaging, not only for detection of a molecule but also to understand the interactions between molecules, such as between a protein and its small molecule ligand, substrate, or inhibitor.

An optical microscope for single molecule imaging is disclosed in international patent application with publication No. WO 2020/245579 with the same applicant as the present patent application. Broadly, the optical microscope of international patent application with publication No. WO 2020/245579 comprises, in a single housing, a first optical microscope in the form of a confocal microscope, and a second optical microscope in the form of a total internal reflection fluorescence microscope. Significantly, to provide this very high resolution, the second optical microscope is used to correct drift from the first optical microscope. Single molecules can range in size from on the order of picometres to nanometres. Cisplatin, a common chemotherapy agent, has a diameter of around 50 - 150 nm. Thus to observe a molecule and determine its binding interactions requires higher resolution than a conventional optical microscope. Indeed, understanding how drugs bind to their receptors is the most fundamental purpose of pharmacological research that enables the effective development of novel therapeutics. Monitoring kinetic properties is critical, but often based on limited macroscopic measurements. Currently, deterministic measurements are made using methods that yield ensemble averages (e.g. radioligand binding, surface plasmon resonance (SPR), X-ray crystallography). Techniques such as single-channel patch clamp go some way towards monitoring the properties of single molecules, but are composite measurements of both ligand binding and a functional response, and can only be used for receptors that are functionally coupled to ion channels.

Current pharmacokinetic models for drug metabolism are also based entirely on macroscopic kinetics experiments and crude deterministic kinetic models, devoid of the actual stochastic molecular detail of the enzymes, receptors or other samples; resulting in numerous promising drugs never reaching clinical trials. There is a need to provide a tool for researchers, healthcare institutions, governmental drug regulatory bodies, and the pharmaceutical industry to monitor the underlying molecular scale properties such as drug-protein, antibodyepitope and protein-protein interactions by providing the raw data for realistic stochastic kinetic modelling. The incorporation of these concepts and measurements could result in greater drug safety and an increase in life-saving pharmaceuticals reaching clinical trials.

Furthermore, current techniques to detect an analyte in a sample comprise mass spectrometry, PCR, ELISA, and gel electrophoresis. In particular, to detect for biological material from an infectious agent, and thus diagnose a subject with a particular disease, PCR or ELISA are generally used, with PCR being the gold standard diagnostic technique. In the case of nucleic acid detection, such methods almost always require an initial nucleic acid amplification step. PCR can also be used for determining whether the subject is infected with a variant of the infectious agent, using amplification primers which are specific for the mutated gene. There are a multitude of drawbacks to the aforementioned techniques, including the amount of time and cost needed to run the experiments, and accuracy of result.

There is therefore a need to provide a tool for medical practitioners, researchers and the like to detect for analytes in a sample. In particular, there is a need to provide a technique to quickly and accurately determine the presence or absence of a particular analyte in a sample. Such techniques are critical for fast and reliable diagnosis of a particular disease, and whether a sample comprises a variant of an infectious agent. This is critical in preventing the spread of disease, and providing the correct treatment to the subject, and in doing so limit the spread of drug resistance among infectious agents.

SUMMARY OF THE INVENTION

The Applicant has appreciated that the use of optical microscopy, at a resolution of 100 nm or less, in the imaging of analytes provides a new and innovative means to observe and detect analytes in a sample. Methods using optical microscopes having a resolution of 100 nm or less allow for single molecule imaging, wherein the capture and detection or observation of single molecules is enabled. The Applicant has appreciated that this technology has application in many areas of analytics, such as disease diagnosis, determination of microbial variant, and drug discovery.

In particular, the applicant has recognised that single molecule microscopy can be used to image single analytes, such as a drug, and how they interact with other molecules. Such a technique can enable detailed pharmacokinetic measurements to be obtained with high precision. Such high-resolution imaging provides stochastic determinations at the molecular level, providing critical detail on the configuration and kinetics of molecules on their interaction with other molecules. This is crucial in determining how a particular disease initiates and progresses. In the realm of drug discovery, this information is key for determining how a molecule can be targeted to prevent, mitigate, and resolve a disease.

The Applicant has also appreciated the application of single molecule microscopy in analytical detection. Providing means to image single molecules with nanometer resolution opens the door for novel methods of diagnosis, wherein imaging of a particular analyte provides for detection of the analyte in absolute terms. No amplification of the analyte by replication is required due to the super resolution of the imaging enabling single molecules to be imaged at a resolution below the wavelength of light. The Applicant has also appreciated that such analytical determination has application outside of detection of biological molecules. For instance, single molecule imaging has potential to detect for environmental contaminants, explosives, medical drugs, recreational drugs, and other particulates.

The Applicant has further appreciated that optical microscopes having a resolution of 100 nm or less can image fragments of a larger molecule with high precision. As such, the Applicant has recognised that molecules can be fragmented to increase the number of analytes that can be imaged, thus essentially amplifying the analyte. Confidence in the detection of the parent analyte is therefore increased without amplification of the analyte by replication.

According to the invention, there is provided a method comprising observing an analyte using a microscope, wherein the analyte is captured on an analytical surface by an immobilised capture probe. Optionally, the microscope is an optical microscope with a resolution of less than 100 nm.

An example of an optical microscope having a resolution of 100 nm or less is disclosed in international patent application No. PCT/GB2020/051336, with publication No. WO 2020/245579. The content of PCT/GB2020/051336 is hereby incorporated by reference.

According to the invention, there is provided a method for detecting and/or analysing an analyte in a sample, comprising observing an analytical surface that has been contacted by the sample, using an optical microscope with a resolution of 100 nm or less, wherein the analytical surface comprises a capture probe immobilised to a solid phase, the capture probe for specifically, or selectively, capturing the analyte, such that if the analyte is present in the sample, the analyte is captured by the capture probe and can be observed.

In other words, if analyte is present in the sample, it would be captured by the capture probe and could be detected and observed. If there is no analyte in the sample, no analyte may be detected or observed. If the analyte is captured by the capture probe, the interaction of the analyte and the capture probe could be analysed. The specificity or selectivity of the capture probe for the analyte means that if there are multiple analytes, such as multiple biomolecules, in a sample, a specific analyte of interest can be captured.

The resolution of the microscope may thus allow the imaging and analysis of a single molecules. Preferably, the microscope has an optical resolution of 50 nm or less, 20 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, or 1.5 nm or less.

The method may comprise the step of contacting a sample with the analytical surface. So, if the sample comprises the analyte, the analyte would contact the analytical surface and could be captured.

Detecting and/or or analysing the analyte may comprise observing the analyte, either at a fixed time point, over a series of time points, or continuously over a period of time. The analyte may comprise a nucleic acid (e.g. DNA or RNA), peptide, polypeptide, protein, peptide nucleic acid, lipid, carbohydrate (e.g. sugar). The analyte may, for example, be a receptor, ligand, antibody or antibody fragment (e.g. Fab, scFV or dAb), antigen, whole cell (either fixed or live) or organelle.

Optionally, the capture probe specifically binds to the analyte directly. Alternatively, the capture probe may bind to the analyte indirectly. For example, the capture probe may specifically bind to a linker moiety (or complementary molecule) which is coupled to the analyte. The binding to a linker moiety may be particularly advantageous if the analyte is already known, but the analytical surface may comprise a capture probe which is not able to specifically bind the analyte. This may allow the binding of any analyte of interest to an analytical surface which includes standard capture probes. The linker moiety may comprise a nucleic acid (e.g. DNA or RNA), peptide, polypeptide, protein, or peptide nucleic acid.

The term “specifically bind” is used herein to describe the interaction of molecules with one another via attractive intermolecular forces sufficient for the molecules to bind. Such forces include ionic, covalent, and/or intermolecular bonds, such as hydrogen bonds, dipole-dipole bonds, and van der Waals forces. The molecules have at least partial complementarity, that is, there is sufficient chemical complementarity between chemical groups of the molecules to allow the molecules to bind. For example, specific binding may occur between receptors and complementary ligands (e.g. signalling molecules), antibodies and complementary antigens, or between nucleic acids with complementary base pairs. Specific binding may occur between drugs and receptors, or between nucleic acids and amino acids.

Optionally, the methods may be carried out on a biological sample obtained from a subject, which may contain the analyte. The subject may be a human or animal. The biological sample may be, for example, any biological sample that could contain biological material of an infectious agent to allow for detection of the agent. The biological material could be nucleic acid, peptide, polypeptide or protein. The biological sample could be a blood, plasma, urine, stool, serum, sputum, cell, oral, nasal, vaginal, or any other bodily tissue sample. The biological sample may comprise biomarkers indicative of whether or not the subject has a disease or condition.

The presence of an analyte in the sample may be known prior to carrying out the method. For example, rather than using a sample from a subject, the sample may have been formulated to include a molecules, such as candidate drug molecules. An immobilised capture probe means that the capture probe is retained at a particular location on the solid phase. Consequently, when an analyte is captured by the capture probe, the analyte would also be immobilised to the solid phase. This may allow the analyte to be readily observed on the analytical surface using the microscope. The analytical surface may thus also be referred to as an imaging surface.

The solid phase forming part of the analytical surface must be of a suitable optical purity, or optical quality, for use with a high-resolution microscope. Preferably, the solid phase is an inorganic material, more preferably glass (such as soda lime glass, borosilicate glass or quartz glass). Suitable requirements may be as set forth in ISO 8037. Other transparent materials may be suitable. For instance, the solid phase may comprise an organic material, such as a polymer, e.g. a plastics material..

Immobilisation of the capture probe to the solid phase may be achieved by covalent coupling (for example, coupling via groups consisting or comprising silane, azide, acetylene, maleamide, carboxylic acid, primary amines, succinimidyl esters or epoxides). Preferably, the capture probe is covalently attached to a glass solid phase.

The solid phase may thus be a surface of an analytical device, to which the sample is applied. For example, the analytical surface may be a surface of a microscope slide, an internal surface of a multi-well plate (such as a 6, 12, 24, 48, 96, 384 or 1535 well plate), an internal surface of a microfluidic device or an internal surface of a sample container. Further examples of analytical devices are provided below.

Optionally, the capture probe comprises a biomolecule. Optionally, the biomolecule comprises a polynucleic acid (e.g. DNA or RNA), an amino acid, peptide, polypeptide, protein. The biomolecule may comprise an oligonucleotide, a peptide nucleic acid, an aptamer or an antibody. Advantageously, biomolecular capture probes may be able to specifically bind to biological analytes present in a sample which have at least partial complementarity to the capture probe. For instance, if the capture probe comprises one or more nucleotides, it may specifically bind to an analyte comprising one or more nucleotides of at least partial nucleotide base complementarity. Further advantageously, the use of biological capture probes may allow the binding kinetics of the capture probe and associated analyte to be analysed, or it may allow the binding kinetics to be manipulated, by reaction conditions for instance, such that the binding of analyte to the capture probe can be controlled. The analytical surface may comprise a plurality of immobilised capture probes.

For example, the analytical surface may comprise a plurality of immobilised capture probes, each of which is for specifically, or selectively, capturing the same analyte. This may increase the chances a particular analyte being captured, or may assist in the quantification of a particular analyte in the sample.

Preferably, the method of the invention is for detecting and/or analysing a plurality of different, or distinct, analytes in the sample. So, the analytical surface may comprise a plurality of immobilised capture probes, each capture probe for specifically, or selectively, capturing a different, or distinct, analyte. Consequently, if any of the plurality of analytes is present in the sample, the, or each, analyte can be captured by its corresponding capture probe. Advantageously, this may allow detection of multiple analytes from a single sample on a single analytical surface. For example, this may be advantageous, for diagnosis of multiple diseases simultaneously, or allow multiple biomarkers indicative of a particular disease, to be identified. The plurality of distinct analytes may be detected simultaneously.

Preferably, the analytical surface comprises a plurality of capture zones (also referred to as binding regions). The capture zones may be referred to as microzones because they are microscopic. Each capture zone may be discernible by high-resolution microscopy.

Each capture zone may be specific for a particular analyte. So, if a particular analyte is present in a sample, it may bind to a particular capture zone. Each capture zone may thus comprise a capture probe for specifically capturing a different, or distinct, analyte. Spatially distributed or spatially distinct capture zones may allow for easy distinction between captured analytes because each distinct analyte may be detected and identified by its capture location alone. Thus, the use of single molecule microscopy allows for the detection of each analyte of interest by targeting each analyte to a distinct capture zone.

Each capture zone may comprise a plurality of the same capture probe. For example, there may be a first capture zone comprising multiple first capture probes, each of which is for specifically capturing a first analyte. There may be a second capture zone separated from the first capture zone, which comprises multiple second capture probes, each of which is for specifically capturing a second analyte.

Each capture zone may comprise a mixture of different capture probes. For example, there may be a first capture zone comprising a first and a second capture probe, the first capture probe being for specifically capturing a first analyte and the second capture probe for specifically capturing a second analyte. There may be a second capture zone spatially distinct from the first capture zone, which comprises a third and a fourth capture probe, the third capture probe for specifically capturing a third analyte and the fourth capture probe being for specifically capturing a fourth analyte.

There may be at least two, at least three, at least five, at least ten, at least twenty or at least fifty capture zones, at least one hundred, or at least five hundred capture zones.

Each capture zone may comprise at least two, at least five, at least ten, at least twenty, at least one hundred, at least one thousand or at least five thousand capture probes.

The use of single molecule imaging may allow individual molecules to be detected, observed and counted. Methods of the invention may involve observing and/or counting individual molecules. So, the analyte, or plurality of distinct analytes, may be quantified at each capture zone. Advantageously, this may allow detection and quantification of analytes in absolute terms, negating the requirement for amplification by replication of the analyte. Quantification of each analyte may increase the confidence in a detection result. Detecting more than one specific analyte from a sample may increase the confidence of the detection. Detecting and/or quantifying multiple distinct analytes from a particular infectious agent, for example, may increase the confidence of a diagnosis.

Furthermore, the advantage single molecule microscopy offers is the ability to quantify the presence of analytes with extremely high levels of accuracy. The presence of single molecules can be assessed simply by visualising them, and accurate quantification can be achieved by simply counting those that are visible. If specific molecular types can first be captured within a specific location, the identity of the single molecules can also be ascertained simply by their presence in that location. With the use of several different locations that are each specific for different molecular types it is then possible to detect and quantify the number of each different molecular type that is visible.

Optionally, each analyte is a fragment of a larger analyte. So, each fragment may be specifically captured by a capture probe of the analytical surface. Advantageously, splitting a larger molecule into a number of fragments, and detecting each fragment may allow the amplification of signal, as each fragment is detected with single molecule resolution. Optionally, fragmenting the larger molecule may allow for detection of smaller fragments that have different emission intensities. In this instance, there may be a greater quantity of signals detected, but the emission intensity may be less than if the parent molecule was unfragmented.

Fragmentation allows for the detection of each individual fragment of the parent (unfragmented) analyte, as each fragment may be captured by a capture probe specific for the fragment in a capture zone which is specific for that fragment. Spatially distributed or spatially distinct capture zones may allow for easy distinction between captured fragments because each distinct fragment may be detected and identified by its capture location alone. This provides more detailed information about the identity of the analytes in the sample.

This signal amplification may be demonstrated by comparing the signal intensity where a single (unfragmented) analyte is detected, with the signal intensities where multiple, different fragments of the analyte are detected, wherein each different fragment binds to a separate capture zone on the analytical surface. Amplification may also be demonstrated by counting the number of labelled fragments relative to the labelled parent, unfragmented analyte.

Different fragments may also bind to complementary capture probes in the same capture zone. Amplification may be demonstrated by counting the number of labelled fragments relative to the labelled parent, unfragmented analyte.

In summary, if a parent molecule is fragmented to produce several smaller child fragments, and each of these child fragments can be detected in different locations, the total number of counted molecules across all locations may be increased relative to counting of the parent molecule alone. In this way it is possible to count an increased number of individual child fragments and an amplification relative to the parent signal may be achieved. Through appropriate chemistry it is also possible to specifically detect multiple different molecules in the same location. In this instance, amplification may be achieved by detecting and counting an increased number of child fragments that are present in the same location.

The analyte may be fragmented by cleaving or breaking at specific positions of the analyte. Advantageously, this may allow for the detection of known, specific fragments. Alternatively, the analyte may be fragmented at non-specific positions. This may allow for the detection of non-specific fragments.

Optionally, the method comprises fragmenting the analyte to form analyte fragments. The analyte may be fragmented enzymatically, for example using a peptidase or a nuclease. A suitable nuclease may be a site-specific nuclease. The analyte may be fragmented by exposure to divalent cations, pH treatment or heat treatment. The sample may be exposed to a means, or conditions, for fragmenting the analyte. For example, an enzyme may be contacted with the sample. So, if the analyte is present in the sample, the analyte would be fragmented.

The method may involve the detection of multiple analytes, each of which has been fragmented. So, for example, there may be a first set of analyte fragments from a first analyte and a second set of analyte fragments from a second analyte. The first set of analyte fragments may be captured in a first capture zone and a second set of analyte fragments may be captured in a second capture zone.

To assist in detecting and/or analysing the analyte, the analyte may be coupled to a detection moiety. The detection moiety may be covalently bound, or fused, to the analyte. This may be particularly applicable when the analyte is already known. A sample may be prepared with the analyte of interest coupled to the detection moiety. Suitable coupling or cross-linking reagents are known in the art.

Alternatively, a detection probe may comprise the detection moiety, and the detection probe may be specific, or selective, for the analyte. Consequently, only analytes of interest may be detectable.

The detection probe may specifically bind the analyte directly, so it may be least partially complementary to the analyte. The interaction between the analyte and the detection probe may thus comprise ionic and/or intermolecular bonds, such as hydrogen bonds, dipole-dipole bonds, and van der Waals forces. Advantageously, this may allow for the capture and detection of the analyte wherein its presence in the sample is unknown. This may also allow for the analyte to be recovered from the sample in its original form. If the detection probe binds the analyte and the capture probe also binds to the analyte, the detection probe may bind to a different part of the analyte to the detection probe, so that the analyte can bind both simultaneously.

Optionally, the detection probe comprises a biomolecule. The biomolecule may comprise a nucleic acid, an amino acid, peptide, polypeptide or peptide nucleic acid.

The detection moiety may be a fluorescent label or any other type of label that can be detected visually, such as a phosphorescent label or a label that can be detected using fluorescence resonance energy transfer. The detection probe may be formed by the coupling the detection moiety to a biomolecule.

For example, the detection moiety may be covalently coupled to the biomolecule.

Optionally, the method for detecting an analyte in a sample may comprise coupling the detection moiety to the analyte. Coupling the analyte to the detection moiety may involve binding the analyte to the detection probe.

The method may involve contacting the detection moiety with the sample, so the detection moiety can couple with any analyte which may be present in the sample. In this way, the detection moiety may couple with the analyte prior to the analyte being captured by the capture probe. The sample may be contacted simultaneously with the detection moiety and the capture probe.

The method may involve contacting the detection moiety with the analytical surface, so the detection moiety can be coupled to any captured analyte. So, the detection moiety may couple with the analyte after the analyte has been captured by the capture probe.

The detection moieties, or detection probes, may be selected such that different analytes may be identified based on a specific visual signal. For example, different analytes may be identified by different coloured signals.

For example, a first analyte may specifically bind a first detection probe, the first detection probe comprising a first detection moiety. A second analyte may specifically bind a second detection probe comprising a second detection moiety. The first and second detection moieties may generate distinguishable signals.

The invention may involve formation of a complex including the analyte, the capture probe and the detection moiety. In other words, the capture probe may be bound to the analyte and the analyte may, at the same time, be coupled to the detection moiety.

Optionally, at least one reference element is used for positional drift correction of an image taken by the optical microscope. Reference elements are described and discussed in WO 2020/245579, and are described in more detail below.

Optionally, the at least one reference element comprises a polystyrene bead, nanodiamond, fluorophore, or a protein, polyethyleneimine, antibody, or lipid-coupled fluorophore. The analytical surface may comprise an immobilised reference element. For example, the reference element may be immobilised to the solid phase of the analytical surface. The immobilised reference element may be immobilised by binding to biological materials. Optionally, the reference element is immobilised by peptide coupling. Optionally, the reference element is immobilised by covalent coupling, including coupling via groups chosen from silane, azide, acetylene, maleamide, carboxylic acid, primary amines, succinimidyl esters, epoxides. Optionally, the reference element may be formed by etching of the analytical surface.

Optionally, the analyte comprises biological material from one or more infectious agents. Optionally, the infectious agent is a virus, bacterium, fungus, or parasite.

Optionally, methods of the invention may comprise determining whether the sample comprises one or more infectious agents, and/or may allow detection of different strains of infectious agents. The method may comprise capturing for a particular nucleotide sequence, and/or amino acid sequence, of the one or more infectious agents, in the sample.

Optionally, the biological molecule comprises wild-type nucleotide sequence, or wild-type amino acid sequence. The term “wild-type nucleotide sequence” is used herein to mean a nucleotide sequence of the infectious agent that is in its non-mutated, or recognised reference, form. Similarly, the term “wild-type amino acid sequence” is used herein the mean the amino acid sequence encoded by the wild-type nucleotide sequence.

In particular, methods of the invention may comprise determining whether the sample comprises one or more infectious agents having wild-type nucleotide sequence and/or wildtype amino acid sequence, wherein the method comprises capturing and detecting for wildtype nucleotide sequence, and/or wild-type amino acid sequence, in the sample.

Optionally, the biological molecule comprises mutant-type nucleic acid sequence, or mutanttype amino acid sequence.

In particular, the methods of the invention may comprise determining whether the sample comprises one or more genomic variants of the one or more infectious agents, wherein the method comprises capturing and detecting for mutant-type nucleotide sequence, and/or mutant-type amino acid sequence, in the sample. Advantageously, such methods may be used to diagnose a subject with a particular disease, or multiple diseases. As such, a suitable treatment regimen can be administered to the subject to mitigate and resolve the disease or relieve their symptoms.

Furthermore, such methods can be used to determine the genomic and phenomic status of an infectious agent with regard to a particular nucleic acid or amino acid sequence. As such, suitable treatment can be administered to the subject, which is suitable for the particular strain of infectious agent infecting the subject. A potential application may be to determine resistance of microbes to antimicrobial agents. Determining whether a nucleotide sequence, or encoded amino acid sequence, is mutated may allow the determination of whether the strain of infectious agent infecting the sample is resistant to a particular antimicrobial agent. This may limit the development and/or spread of antimicrobial resistance because the antimicrobial agent will be administered only to those subjects likely to be effectively treated by the antimicrobial agent, and not to those subjects infected with resistant strains.

Methods of the invention may be for identifying a biomarker profile of a subject. For example, it may allow identification of a biomarker profile indicative of a particular disease state.

Optionally, the analyte is selected from one or more of: environmental contaminants, explosives, medical drugs, recreational drugs, particulates, or a biological or disease marker.

The method may comprise comparing signals from molecules specifically captured by capture probes, and signals from molecules that have not been specifically captured. Signals from molecules that have not been specifically captured may thus provide a background signal. Signals from molecules that have not been specifically captured may be from molecules on the analytical surface, but not captured by a capture probe, after contact with the sample

According to the invention, there is provided an analytical device for use, or adapted for use, with an optical microscope having a resolution of 100 nm or less, comprising an analytical surface, the analytical surface comprising a capture probe immobilised to a solid phase, and the capture probe for specifically, or selectively, capturing an analyte.

The analytical surface may be as already described above. For example, there may be a plurality of capture probes, each capture probe being specific, or selective, for an analyte or analyte fragment. Preferably, the analytical surface comprises a plurality of distinct capture zones, each capture zone capturing a particular analyte. The device may comprise an analyte wherein the analyte is captured by the, or each, capture probe. The analyte may be as already described above. For example, there may be a plurality of distinct analytes or analyte fragments.

The device may comprise a detection moiety coupled to the, or each, analyte. The detection moiety may be as already described herein. For example, there may be multiple detection moieties, each bound to an analyte.

The device may be in the form of a sample enclosure, comprising a cover, wherein the cover is spaced from the analytical surface to form a space in which to locate the sample, or through which the sample can flow. Optionally, the cover may be transparent to light.

The device may comprise a well, comprising one or more capture probes. The device may comprise a plurality of wells, each well comprising one or more capture probes. Each well may comprise a distinct capture probe. So, each well may be a distinct capture zone, as described above. Each well may comprise a plurality of capture zones, each comprise one or more distinct capture probes. There may be at least 1 , 2, 8 12, 20 or 50 wells.

The device may comprise an inlet channel through which the sample can enter the, or each well, and an outlet channel through which the sample can exit the, or each, well.

The device may comprise an immobilised reference element.

The device may comprise the structure as illustrated in Figures 8 and 9.

The device may be in combination with a microscope, preferably a microscope having a resolution of 100 nm or less. For example, the device may be positioned on a viewing platform of the microscope.

According to the invention there is provided a use of the analytical device with a microscope having a resolution of 100 nm or less.

According to the invention, there is provided a method comprising contacting a sample, or analyte, with the analytical device. The method may comprise coupling a detection moiety to the analyte. This may involve contacting a detection moiety with the sample, or contacting a detection moiety with the device.

The method may comprise observing the device following contact with the sample or analyte, using an optical microscope, preferably using an optical microscope with a resolution of 100 nm or less.

According to the invention there is also provided a kit comprising: the analytical device; and i) a detection moiety for coupling to an analyte; and/or ii) a separate linker moiety for coupling to an analyte, the linker moiety specific, or selective, for a capture probe of the device.

The kit may comprise reagents for coupling the detection moiety to the analyte, and/or comprise reagents for coupling the linker moiety to the analyte. Such reagents may be crosslinking agents.

The kit may further comprise a microscope, preferably a microscope having a resolution of 100 nm or less.

According to the invention there is also provided a kit for detecting and/or analysing an analyte in a sample comprising a microscope having a resolution of 100 nm or less, and the analytical device.

An example of a microscope that can achieve a resolution of 100 nm or less is a confocal microscope. An alternative example of an optical microscope having a resolution of 100 nm or less, according to the invention, or for use according to the invention, is described in more detail below.

High resolution microscopy with a spatial resolution of 100 nm or less, preferably 20 nm or less (such as 15 to 20 nm) is desirable.

The Applicant has also appreciated that an optical microscope and, in particular a super resolution microscope, comprising two different optical microscopes that each operate simultaneously using a different mode to view a sample, provide enhanced resolution images of a sample. The optical microscope may be a multi-modal or bi-modal optical microscope. Molecular-scale resolution may allow single molecules to be observed. High resolution microscopy with a spatial resolution of 100 nm or less, preferably 20 nm or less (such as 15 to 20 nm) is desirable.

An example of an optical microscope of the invention, or for use according to the invention, may be as described in the following numbered paragraphs.

1 . An optical microscope comprising: a first optical microscope; and a second optical microscope with a different mode of operation to the first optical microscope; wherein the optical microscope is configured such that the first optical microscope and the second optical microscope simultaneously view a sample.

2. An optical microscope according to numbered paragraph 1 , wherein the first optical microscope uses a first light source and the second optical microscope uses a second light source; and the first light source is different to the second light source.

3. An optical microscope according to numbered paragraph 1 or numbered paragraph 2, further comprising an objective lens, wherein the first light source and the second light source pass through the objective lens.

4. An optical microscope according to numbered paragraph 3, wherein the objective lens has a numerical aperture of at least 1 .37.

5. An optical microscope according to numbered paragraph 4, wherein the objective lens has a numerical aperture of at least 1 .45.

6. An optical microscope according to any of numbered paragraphs 3 to 5, wherein the optical microscope is configured such that a sample for imaging is located below the objective lens.

7. An optical microscope according to any preceding numbered paragraph, wherein the optical microscope is a high-resolution optical microscope or a super resolution optical microscope.

8. An optical microscope according to any preceding numbered paragraph, wherein the first optical microscope is a confocal microscope.

9. An optical microscope according to any preceding numbered paragraph, wherein the second optical microscope is a total internal reflection fluorescence microscope.

10. An optical microscope according to any preceding numbered paragraph, wherein the optical microscope is housed in a single housing.

11. An optical microscope according to numbered paragraph 10, wherein the single housing is in one and only one piece. 12 . An optical microscope according to any preceding numbered paragraph, wherein the optical microscope comprises a dichroic mirror to reflect light from the first optical microscope on to the sample and to allow light from the sample to pass through to a detector.

13. An optical microscope according to any preceding numbered paragraph, wherein the optical microscope comprises a dichroic mirror to reflect light from the second optical microscope on to the sample and to allow light from the sample to pass through to a detector.

14. An optical microscope according to any preceding numbered paragraph, wherein the optical microscope comprises a dichroic mirror to route light from the sample from the first optical microscope and from the second optical microscope to respective detectors.

15. An optical microscope according to any preceding numbered paragraph, wherein an optical route from the sample to a detector comprises a tube lens.

16. An optical microscope according to any preceding numbered paragraph, wherein the optical microscope comprises a position sensor configured to receive light reflected from the sample from a laser of the second optical microscope.

17. An optical microscope according to numbered paragraph 16, wherein the position sensor is in communication connection with a computer.

18. An optical microscope according to numbered paragraph 17, wherein the computer is configured to provide signals to a sample stage to move the sample stage dependent on the received light reflected from the laser of the second optical microscope.

19. An optical microscope according to numbered paragraph 18, wherein the computer is configured to provide signals to a sample stage to move the sample stage vertically dependent on the received light reflected from the laser of the second microscope.

20. An optical microscope according to any preceding numbered paragraph, wherein a computer of the optical microscope is configured to capture and store a plurality of images of a sample over time.

21. An optical microscope according to numbered paragraph 20, wherein the computer processes the plurality of images to provide an output image.

22. An optical microscope according to any preceding numbered paragraph, wherein the second optical microscope is used to correct drift from the first optical microscope.

23. An optical microscope according to any of numbered paragraphs 20 to 22, wherein the second optical microscope is used to correct drift from the first optical microscope and/or the sample in the X,Y plane or horizontal plane based on the stored plurality of images of the sample over time.

24. An optical microscope according to numbered paragraph 22 or numbered paragraph 23, wherein the second optical microscope is used to correct drift from the first optical microscope and/or sample using at least one reference element located relative to the sample.

25. An optical microscope according to numbered paragraph 24, wherein the at least one reference element has a diffraction limited intensity distribution of emitted light.

26. An optical microscope according to any preceding numbered paragraph, wherein the first optical microscope is used to correct drift from the first optical microscope and/or the sample in the Z direction or vertical direction.

27. An optical microscope according to any of numbered paragraphs 24 to 26, further comprising a beam splitter and at least two detectors configured to detect light from the sample split by the beam splitter from the first optical microscope.

28. An optical microscope according to numbered paragraph 27, wherein the detectors are at a calibrated focal plane within an axial confocal volume of the first optical microscope.

29. An optical microscope according to any of numbered paragraphs 27 and 28, wherein the detectors comprise avalanche photo diodes, such as single photon counting avalanche photo diodes.

For example, the optical microscope of the invention may comprise a first optical microscope; and a second optical microscope with a different mode of operation to the first optical microscope; wherein the first optical microscope is a confocal microscope and the second optical microscope is a total internal reflection fluorescence microscope, wherein the optical microscope is configured such that the first optical microscope and the second optical microscope simultaneously view a sample and wherein the second optical microscope is used to correct drift from the first optical microscope.

The optical microscope may provides a direct optical microscopy method of monitoring analyte binding that provides single molecule imaging (SMI). This may enable observations of phenomena such as ligand-receptor interactions and single-protein tracking. In these approaches, the interactions of molecules such as purified receptors, enzymes or other biological samples with their ligands, substrates or other interacting molecules, are monitored by time-lapse optical microscopy. This may allow observations of single receptors bound with one or more of their ligands, state changes in protein membrane interactions and augmented protein-protein interactions. The resultant moving images of these events can be analysed to determine key components of the interactions, such as the number, position, and rate constants of individual molecules or proteins. As described, the optical microscope of the invention has application in detection and/or analysis of an analyte in a sample. The analyte may be a biological molecule, the detection of which confirms a particular health or disease state. The use of single molecule microscopy also allows variants of an infectious agent to be detected for, to determine an appropriate treatment regimen for the subject. The analyte may be a non-biological molecule, such as an environmental contaminant, explosive, medical drug, recreational drug, or other such particulates. Use of single molecule microscopy for detection of an analyte provides absolute quantification of the analyte, which negates the need for amplification of the analyte, or the signal associated therein. The application of single molecule microscopy in detection of analytes therefore provides a simple, rapid, and accurate method for detection of the analyte, allowing for fast diagnosis and prevention of spread of disease.

The microscope of the invention may automatically correct for positional movements to enable the observation of single molecule interactions at high spatial resolution (such as 10Onm or less, for example, 15 to 20nm or even as low as 1 ,5nm). The positional movement correction, in the Z direction, may be in real time. That is to say, as an image is sampled, positional movement or drift in the Z direction may be corrected as the sample is imaged, such as by moving the sample stage. In contrast, the positional movement correction, in the X and Y directions (horizontal plane), may be carried out after the event. In other words, captured images of a sample may be corrected for drift after they have been captured.

The microscope of the invention may be an automated super-resolution kinetic microscope that utilises simultaneous fast confocal imaging, laser scanning, and total internal reflection fluorescence (TIRF) microscopy techniques to measure the stochastic binding kinetics of interacting molecules with nano-scale spatial resolution. Lasers may be accessed by computer, and a fast acousto-optic laser scanner (or galvanometer) may be used to scan samples and identify regions of interest via automated positioning. Nanometer-scale drift correction of confocal images in the X-direction and Y-direction may be accomplished with embedded fiducial markers excited in TIRF mode by a laser (TIRF laser wavelength can be in the visible or near infrared depending on the fiducial marker chosen; fiducial markers used are explained further below). X-direction and Y-direction frame-by-frame super resolution triangulation and correction may be accomplished by post-processing without the need of imaging stage feedback (imaging stage feedback limits the spatial resolution). In other words, the X,Y plane drift correction may be carried out after imaging a sample. The drift correction based on a plurality of images may not result in movement of the sample stage. It may result results in computer processing after the image capturing event. In other words, computer processing of already captured and stored images. Z-direction correction is accomplished with a position sensor coupled to the Z-focus on the instrument and is achieved in real time to keep the sample in the focus continuously. Automated temperature cycles may be used for sample preparation and in situ conjugation chemistry. Software algorithms are used that take into account movements and point-spread reconstruction.

The microscope of the invention may incorporate automated temperature cycling that enables conjugation chemistry, followed by visualisation by automated optical microscopy. A confocal and TIRF arrangement may enable the simultaneous monitoring of both target elements (e.g. analytes) and embedded fluorescent reference elements independently of one another. The reference elements may be excited by a laser in TIRF mode and the fluorescence is directed towards an electron multiplying charge coupled device (EMCCD) camera, and a super-resolution constellation map may be generated for every frame in the data collection. A second laser or lasers may be configured for fast scanning confocal laser excitation and the fluorescence from the sample is directed to a single photon counting avalanche photo-diode (APD). Examples include a combination of simultaneous superresolution confocal imaging, super-resolution wide-field imaging, photon-by-photon spatial- tagging, and the time-tagging of collected photons. Software algorithms use the data gathered from this instrument to reconstruct super-resolution images.

Optical measurements may be made via an automated optical system that comprises filters, mirrors, laser scanners, lenses and lens combinations to focus and position laser light onto a sample via an objective lens that sits directly above the sample (as illustrated in Figures 6 and 7 and described in detail below). The imaging stage may provide an environment that is both thermally and vibrationally stable, and contains integrated channels that enable the microfluidic application of buffers and test compounds to the sample. Automated confocal laser scanning of the sample may account for any positional movements within the sample and may identifies regions of interest that are interrogated at higher spatial and temporal resolution via an automated computational process. Subsequent frame-to-frame drift correction and imaging processing may provides a means to interrogate the kinetic properties of single molecules over very long period of time (more than 24 hours if needed) leading to an unprecedented level of statistical confidence.

The microscope of the invention may comprises an enclosure containing an integral imaging stage, microfluidics, laser optics that enable simultaneous laser scanning confocal and total internal reflection fluorescence (TIRF) microscopy, and an imaging system. The flow chamber may be mounted on an imaging stage manufactured from, for example, invar steel, a nickel-iron alloy that is noted for its low coefficient of thermal expansion and low heat conductivity. The flow chamber may be immobilised on the imaging stage with clamps that incorporate microfluidic channels that directly couple with the etched glass channels in the flow chamber and enable test solutions to pass across the contained samples. The imaging stage may incorporate a temperature-controlled device or Peltier device that allows automated temperature cycling for sample preparation and for imaging at user-defined temperatures that may be controlled by a computer with milli-Kelvin precision.

The microscope may provide a means of automated optical microscopy that scans for reference and target elements, and focusses and captures images for analysis. A system for accounting for changes in the positions of reference and target elements within the sample may enable automation of image processing, and super-resolution analysis of the kinetic properties that define the interaction of single molecules. An advantage may be that correction in the X-plane and Y-plane or horizontal plane is performed after measurements have been made. As such, positional drift may be continuously monitored and a permanent record of the correction is made. This may increase accuracy as images may be compared as a continuous sequence of events before and after the point of measurement and any sudden and unexpected changes in the behaviour of the reference and target elements may be later identified and re-assessed for accuracy. As such, the microscope may simultaneously monitors both the experimental outcome and the corrective changes that were necessary to achieve it. Drift correction in the Z-plane may also be provided, and may be performed in real time to the keep the sample continually in focus. This may be accomplished with sub-diffraction limited resolution by continuously monitoring the back reflection from the TIRF laser on a position sensor and adjusting focus on the microscope objective with nanometer precision.

Sample stabilisation may be achieved through vibrational damping and the use of invar or invar steel, which displays low levels of expansion or contraction with temperature changes. Image stability may also be assisted by the use of fluid-based cooling systems to reduce vibration within the instrument and a benchtop active-air vibration isolation system with PID (proportional integral derivative) control.

Reference elements or fiducial markers, may be attached to biological materials and solid surfaces, for example, it may be attached to the solid phase. These reference elements may be polystyrene beads, fluorescent molecules (for example, Alexa 532 and ATTO-700), antibody coupled fluorophores, lipid-coupled fluorophores, proteins that do not bleach or blink, nanodiamonds, or polyethyleneimines coupled with fluorescent probe molecules. Reference elements attached to biological materials may be, for example, elements attached to free thiol groups by coupling with MTS-fluorophore reagents, photo-crosslinking compounds, antibody labels, or fluorophore elaborated ligands. Solid surfaces (for example, glass slides, cover slips) may be modified, for example, by covalent coupling (for example, coupling via groups consisting or comprising of silane, azide, acetylene, maleamide, carboxylic acid, primary amines), peptide coupling of an amino terminated silane surface, or antibody labelling of protein coated slides. The reference element may be part of the sample or added in addition to the sample and may be placed within, or outside, the field of view of the sensor.

The position of the reference element may be calculated from the images captured on an image sensor, such as an EMCCD camera. These reference elements may have a diffraction limited intensity distribution (described by an Airy function and approximated by a 2D- Gaussian function) that may be accessible under a microscope and may be used to very accurately locate its position in two dimensions by computing the center of the point spread function of the emitted light. The intensity of the point emitter may be visible when illuminated by a source of excitation such as a laser, LED illuminator, filament lamps, halogen lamps or flash lamps, and may be, for example fluorescent emission. The selection of the appropriate excitation may be determined by the physico-chemical and spectral properties of the reference and target elements. One, two, or more excitation sources may be used to enable the excitation of one or more reference elements and target elements.

The positions in the X- and Y-plane may be determined by fitting the intensities of the point spread functions to an Airy, 2D-Gaussion, or centroid. In which case, the peak of the fit gives the super-resolution position.

Resolution below the diffraction limit in the Z-plane may be accomplished with the addition of a 50:50 beam splitter and a second APD. In which case, the focal planes of both APDs may be set at unique calibrated focal planes within the axial confocal volume of the instrument and the Z-localization of point emitters may be determined by comparison of the point spread in each plane. Refinement of the Z-plane may be further refined by Z-piezo objective scanning with nanometer positional accuracy.

The image sensor may be a device that detects light signals, such as the photon streams emitted by fluorescently labelled reference elements or target elements. Such sensors may include EMCCD cameras, CCD cameras, CMOS cameras or APDs (coupled with a laser scanner) which are used for both image acquisition of the reference element and target element. Image capture of both elements enables them to be viewed, taken or saved from the sensor and the position of each element may be known with nanometer precision in two dimensions that are commonly referred to as the X- and Y-planes. Z-sections of thicker samples, such as cells, are constrained by the diffraction limit in the Z-plane. Frame-to-frame drift correction may be performed in the X-plane and Y-plane. Comparing and correcting sequences of images over extended periods of time using positional information from the reference element may be gathered both before and after the image being corrected at a specific time point. This provides a method by which the target element may be accurately rendered without distortion from movement.

BRIEF DESCRIPION OF THE DRAWINGS

Figure 1 is a schematic diagram showing the capture of an analyte to the analytical surface according to an embodiment of the invention;

Figure 2 is a schematic diagram showing the capture of fragments of a larger analyte according to an embodiment of the invention;

Figure 3 is a schematic diagram showing the capture of fragments of a larger analyte, and detection of genomic variants, according to embodiments of the invention;

Figure 4 is a schematic diagram showing the capture of different antibody isotypes to the analytical surface according to an embodiment of the invention; and

Figure 5 is a schematic diagram showing the diagnosis of two disease states according to the capture of disease markers.

Figure 6 shows representative parallel experiments to demonstrate the signal arising from fluorescently labelled analytes binding to regions with specificity. Each well has two binding regions (capture zones); one region to bind Analyte 1 and a second region to bind Analyte 2. In Figure 6A, the top panel contains Analyte 1 only, the middle panel contains Analyte 2 only, and the bottom panel contains both Analytes 1 + 2 at the same concentrations as the top and middle panels. Figure 6B is a histogram of the integrated intensities at each of the locations (Region 1 & Region 2). Figure 6C shows a schematic representation of the experiments shown in Figure 6A and 6B. Figure 7 illustrates amplification of signal intensity by fragmentation. In Figure 7A, amplification is demonstrated by comparing signal intensities where a single fragment is bound to a binding region (Fragment 1 OR Fragment 2), compared to the signal intensity where both are simultaneously bound to a binding region (Fragment 1 AND Fragment 2). Figure 7B shows a comparison of the integrated intensity for the addition of separate signals from Fragment 1 and Fragment 2 alone (left-hand column), compared to the signal when Fragment 1 and Fragment 2 are combined (right-hand column). For each experiment, mean ± sem, n = 5. Figure 7C shows a schematic representation of the experiments shown in Figure 7A and 7B.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Targeting of analytes to specific regions on a surface

For many applications it can be helpful to distribute different analytes or molecules to specific regions on a surface (Figure 1 and Figure 6). For single molecule approaches this is particularly helpful as it can enable the capture, imaging, and analysis of different molecules in different capture zones of an imaged surface.

Capture and imaging can be achieved using molecular probes that are target specific. Capture of an analyte is achieved using capture probes specific for either the analyte (shown in Figure 2) or a complementary molecule (detection moiety)) coupled to the analyte (secondary molecule in Figure 1 ). In either case, the analyte is captured to specific locations on the analytical surface via the capture probes. This targeting of analytes allows for their analysis at the site of capture.

The analytical surface comprises a plurality of distinct capture zones (Figures 1 -5), wherein each capture zone comprises a distinct capture probe for capturing a specific analyte. Figure 1 shows capture zones for capturing a specific analytes, the analytes being coupled to a complementary molecule which specifically binds to the capture probe. In this way, a plurality of distinct analytes can be targeted to regions of the analytical surface from a single sample, wherein each distinct analyte binds to a capture probe in a capture zone. The spatial distribution of the capture zones allows each analyte to be distinguished and independently analysed. One means to know the location of analytes is to print capture molecules directly onto a surface at different locations (not shown). Capture molecules are immobilised at specific locations on the analytical surface, and are each specific for binding to one or more analyte. Binding of analytes to the capture molecules disposed at specific locations on the analytical surface allows for imaging and analysis of the analytes, using a detection label either intrinsic to the target molecule or using a detection probe. An alternative method is to print and covalently couple adaptor molecules onto a surface. An adaptor can be an oligonucleotide or protein that has a unique signature that binds to a complementary molecule (a detection moiety) wherein the complementary molecule has at least partial complementarity to the adaptor molecule, via highly specific molecular interactions (Figure 1 ). Examples of such adaptor molecules could include DNA oligonucleotides, amino acids, aptamers or antibodies.

If different adaptor molecules are used at different locations, complementary molecules that are specific to the adaptor molecule will target those specific locations. If the complementary molecule is fused to a secondary molecule (e.g. an analyte), that secondary molecule will be similarly targeted to a specific location. As an illustrative example, the complementary molecule could be an antibody, aptamer or any other molecule that can specifically interact with other molecules. The use of adaptors therefore provides a means to specifically target a secondary molecule to a region of a surface. This may be particularly important for single molecule imaging, as each individual captured molecule can be analysed separately. This could potentially highlight changes in the strength of an interaction which is indicative of a change in the analyte properties. This may enable rare events to be measured that would be lost in the noise of traditional ensemble approaches.

The complementary molecule and/or secondary molecule comprise a fluorescent label such that the capture of the secondary molecule to a region of the analytical surface can be detected by the optical microscope.

The adaptor molecules are covalently coupled to the surface, and are specific for a particular complementary molecule. A secondary molecule of interest (an analyte) is coupled to a suitable complementary molecule (detection moiety), and is therefore targeted to a specific region.

This approach allows researchers to modify and then target their own modified secondary molecules to specific locations on a surface. In one example, it is possible to conjugate protein 1 with complementary strand T, protein 2 with complementary strand 2’ and protein 3 with complementary strand 3’ (Figure 1 - slide 6). Upon adding a mix of these to the surface, each of the distinct complementary molecules will specifically bind only to one of the three adaptors that are located in each of the three discrete regions.

Figure 6 demonstrates the capture of two different fluorescently labelled analytes to two different binding regions (also referred to as capture zones) on an analytical surface (also referred to as an analytical device). The figure shows two binding regions (Region 1 and Region 2) printed on the surface of single wells in a 384 well microtiter plate. The capture probes used were:

REGION 1 printed with: TTTTTTTTTTTTTTTTTTTT (SEQ ID NO:1 )

REGION 2 printed with: GGGGGGGGGGGGGGG (SEQ ID NO:2)

The analytes shown in Figure 6 are fluorescently labelled nucleotide complements of the two capture probe sequences:

1 . AAAAAAAAAAAAAAAAAAAA (20mer) (SEQ ID NO:3)

2. CCCCCCCCCCCCCCC (15mer) (SEQ ID NO:4)

The images were captured using an optical microscope with resolution of 100 nm or less. Such an optical microscope is described herein, and in WO 2020/245579.

The signal was analysed by measuring the integrated intensity (e.g. total fluorescence within each capture zone). The individual molecules in each binding region may also have been counted.

Example 2

Detection of an analyte in a sample

Knowledge of the precise localisation of capture probes on the analytical surface can provide researchers with a means to specifically determine whether an analyte is present in a sample by its binding to capture probes alone. An analyte bound to a capture probe is detected using labelled detection probes (Figure 2 - slide 5 and 6), bound to the analyte.

The analytical surface comprises a plurality of distinct capture zones, wherein each capture zone comprises capture probes specific for a particular analyte. In this way, a plurality of distinct analytes can be detected from a single sample, wherein each distinct analyte binds to a capture probe in a distinct capture zone. The spatial distribution of the capture zones allows each analyte to be distinguished. The distribution of captured molecules across these distinct capture zones can provide information about the nature of the analyte (e.g. influenza A viruses are divided into subtypes based on their 18 different hemagglutinin subtypes and 11 neuraminidase subtypes. Capture on any of these proteins would be indicative of influenza A infection, with specific subtypes being distinguished by signals in specific capture zones that target each of the individual protein types).

Thus, the capture of said analytes to particular regions indicates their presence in the sample. Similarly, the use of multiple capture zones allows researchers to specifically determine the presence of multiple different analytes and use their locations to discriminate between them.

Example 3

Amplification of detection signal obtained from an analyte by fragmentation

Measurement of biological samples is often performed by amplification of a specific signal. For example, the detection of small quantities of DNA can be achieved by the amplification of specific target regions of the DNA using polymerase chain reaction (PCR), to generate sufficient quantities that they can be visualised. For methods such as enzyme-linked immunosorbent assay (ELISA), protein signals can be amplified by linking specific target probes to enzymes that catalyse a reaction that results in the accumulation and measurement of a coloured product. For detection of other materials, a range of approaches can be used, including the amplification of signals generated by light scattering, light emission, light absorbance, electrical changes, temperature changes and many others.

The use of single molecule microscopy enables individual molecules to be directly observed at a resolution below the wavelength of light. The ability of single molecule microscopy to image fragments of analytes provides a new opportunity for amplifying signals. In particular, the use of fragmentation of molecules into smaller parts that can each be detected. Fragmentation changes a measured signal from that of a single molecule to one characterised by multiple smaller disconnected parts which have their own signal. In one example, one molecular signal is fragmented into six individual parts that can be detected as separate signals (Figure 2 - slides 1 , 2, and 3).

The approach is particularly suitable and desired when combined with a method that utilises the capture of the different fragment types to specific well-defined locations of an analytical surface (capture zones). Here, capture probes are immobilised to capture zones on an analytical surface, and are specific for individual fragments of an analyte. After the fragments have bound to the capture probes, detection probes specific for the fragment bind to the fragments, allowing for their detection. If detection of a target can be performed by its localisation on a surface, the fragmentation of a target molecule into smaller parts can enable the specific and independent detection of each separate part, and in doing so, provide an enhanced signal over that of the unfragmented target molecule alone (Figure 2 - slides 4, 5, and 6). In particular, the capture of distinct fragments of the parent molecule to spatially distinct regions of the analytical surface provides spatial distribution of the detection signals. This allows for detection of the fragments as individual units, and enables absolute quantification of each separate fragment. This in turn provides means to quantify the specific fragments in the sample (Figure 2 - slide 6). An unfragmented molecule will essentially be detected as one signal, due to spatial constraint of the detection signals (Figure 2 - slide 5; Figure 3 - slide 1 ). Fragmentation therefore allows for detection and quantification of individual fragments, rather than whole molecules.

The use of single-molecule microscopy enables each of the fragments to be assessed as individual units that can be designed to have more, less, or equal signal intensity to the parent molecule. Fragmentation of a larger molecule increases the number of molecules that can be detected, but the signal intensity may be lower, higher, or the same as the signal intensity of the unfragmented parent molecule. This will depend on the emission intensity of each fragment.

In one instance a microbial genome may be present at very low concentrations in a sample. For example, if there are 10 microbial genomes in 1000 ml, it is likely that when taking a 1 ml sample, many of the samples will not contain a genome. If the genome is fragmented into more parts, for example, 100 or more, there is a greater probability that each sample will contain a fragment of the original genome. This is highly relevant when probing for sequences of nucleic acid which are unique to a particular infectious agent, as each separate sequence of nucleic acid represents a unit that can be detected. Fragmenting the genome into such individual sequences allows for increased likelihood of a sample comprising a sequence of nucleic acid for detection, and also allows for detection of individual sequences of nucleic acid at distinct regions of the analytical surface.

Nucleotide-containing molecules such as DNA and RNA are amenable to such fragmentation, through physical processes such as shearing, through chemical methods such as the use of divalent cations at elevated temperatures, or the use of enzymes. Both DNA and RNA can be specifically cleaved using a class of enzymes called nucleases, both of which are well described in the literature (Sulej, A. A et al, 2012, Sequence-specific cleavage of the RNA strand in DNA-RNA hybrids by the fusion of ribonuclease H with a zinc finger. Nucleic Acids Research 40, 1 1563; Zellmann, F. et al, 2019, Site-Specific Cleavage of RNAs Derived from the PIM1 30-UTR by a Metal-Free Artificial Ribonuclease. Molecules 24, 807; Pray, L. A. 2008, Restriction enzymes are one of the most important tools in the recombinant DNA technology toolbox. But how were these enzymes discovered? And what makes them so useful? Nature Education 1 , 38). Digestion of DNA and RNA by nucleases represents one method by which fragmentation-based amplification of a single molecule signal can be achieved with high specificity that is governed by the inherent properties of the nucleases.

For methods such as PCR, an ensemble average of a population of genomes is measured, with a lower threshold that must be exceeded for signal detection. To achieve this minimum signal, the genome must be amplified to reach sufficient quantities for detection. With PCR, a specific region of the genome is usually amplified by careful design of oligonucleotide primers that enclose the region of interest. Amplification therefore demonstrates the presence or absence of that region when its replication reaches sufficient levels for the signal to be detected.

With single molecule imaging, the same regional identity can be achieved by detecting for sequences of nucleic acid of the infectious agent using fluorescent detection molecules specific for the nucleic acid. With single molecule imaging, distinct sequences of nucleic acid can be detected as individual signals, meaning that breaking a single genome into many parts increases the signal as effectively as amplifying the signal by replication (Figure 3).

Application of this approach can be useful when low quantities of microbial genomes are present in a patient sample. By fragmenting the microbial genomes, a larger number of signals can be detected. It is possible to simultaneously capture and detect several fragments from different regions of the genome and consequently increase confidence in the result.

Such a method can also be used to identify genome variants if regions contain capture probes which are specific for the mutant nucleic acid sequence associated with a variant (Figure 3 continued).

Amplification of signal can also be seen where different fragments bind to one binding region. Figure 7 shows one binding region (Region 1 +2) printed on the surface of single wells in a 384 well microtiter plate. The capture probes, printed on the same binding region, are as follows:

REGION 1 +2 printed with: TTTTTTTTTTTTTTTTTTTT (SEQ ID NO:1 ) and GGGGGGGGGGGGGGG (SEQ ID NO:2)

The analyte added to the well in each experiment shown is one of the following fluorescently labelled nucleotide complements of the two capture probe sequences:

1 . AAAAAAAAAAAAAAAAAAAA (SEQ ID NO:3)

2. CCCCCCCCCCCCCCC (SEQ ID NO:4)

3. 1 + 2 mixed

An alternative approach is to create a single oligonucleotide with a specific sequence recognised by a restriction enzyme (in the example: below Gsal which digests CCCAG^AC) to enzymatically produce two fragments that will similarly bind to the binding region. E.g

CCCCCCCCCCCCCCCAGCAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:5)

After digestion becomes:

CCCCCCCCCCCCCCCAG (SEQ ID NO:6)

CAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:7)

The signal was analysed by measuring the integrated intensity (e.g. total fluorescence within the capture zone). The individual molecules in each binding region may also be counted.

Example 4

Targeting molecules to regions of an analytical surface

The distribution of specific adaptor molecules at known capture zones allows the targeting of molecules of interest (e.g. an analyte) to those same specific regions if the latter molecules are conjugated to a molecule that is specific for the adaptor. By utilising adaptor molecules and their respective complementary molecules, one can change the molecule of interest according to assay preference, and use the same adaptor and complementary molecules to target the molecule to the surface. Examples of adaptors include, but are not limited to, antibodies, oligonucleotides, aptamers, or protein nucleic acids. Alternatively, capture molecules are immobilised to the analytical surface which bind to the molecule of interest directly and capture it to the surface.

Such an approach is particularly useful in the single molecule context where, for example, specific Ig subtypes are being identified from serum or blood samples (Figure 4). In such an instance, different isotype specific antibodies (e.g. IgA, IgD, Ig E, IgG, IgM, or subtypes such as lgG1 , lgG2, lgG3, lgG4, lgA1 , lgA2) can be targeted to different, specific locations on a surface of an analytical chip through interaction with specific adaptors or capture molecules. In another instance a secondary detection antibody can be used to detect a specific antigen, meaning that in this instance the fluorescent signal demonstrates the presence of the antigen, and its location in the capture zones (which contains isotype-specific capture antibodies) determines the specific isotypes of the antibody that is raised against that antigen. In such a way, it is possible to apply a single biological sample to the analytical surface and simultaneously detect and quantify all the constituent parts of the analyte. The power of single molecule methods is that absolute quantification can be achieved simply by counting the number of signals within each capture zone, wherein each capture zone is specific for each distinct analyte.

For example, in the above embodiment, the quantity of different antibody isotypes can be simultaneously determined from the application of a single patient sample to the chip surface (Figure 4). The locations and counts of the resultant signals in each capture zone will represent specificity and abundance, respectively.

Example 5

Diagnosis of disease state

The invention can also be used to diagnose a particular state of an individual. In this instance, the explicit arrangement of disease-specific capture molecules can be used to provide a diagnosis for one or more diseases using the same sample. For example, as shown in Figure 5, if capture zone 1 (region 1 ) is has capture probes, either adaptors or capture molecules, specific for disease 1 and capture zone 2 (region 2) has capture probes, again either adaptors or capture molecules, specific for disease 2, it is possible to determine which of these two diseases (singly, both or neither) the patient has acquired. Here, the target molecules for disease 1 and 2 which bind to the capture probes are biological markers for the disease, such as a nucleic acid sequence, amino acid sequence, peptide, or protein. In an instance where a patient is infected with only a single disease, a positive signal will only be seen in the region that contains capture molecules that correspond to that particular disease. The positive signal derives from a detection probe which binds specifically to a biological molecule from the particular infectious agent.

Diagnosis by this method provides increased statistical confidence in the result if there is more than one test for a single disease type as the probability of the separate tests can be multiplied. In this instance, there are multiple capture zones specific for one infectious agent. Each capture zone will bind specifically to a distinct biological molecule from the infectious agent. In some cases, the biological molecule will comprise nucleic acid, and the capture zones will be specific for distinct genes of the infectious agent which enable diagnosis. As an example, to diagnose SARS-CoV-2, one capture zone may be specific for the RNA- dependent RNA polymerase gene (RdRPgene), or the spike protein gene (Sgene), and one capture zone may be specific for the nucleocapsid protein gene (A/ gene). Detection probes specific for such genes are employed to allow detection of the bound analyte.

Knowledge of both positive and negative results can also be helpful in a clinical setting where there is a potential of comorbidity, or where there are different diseases with similar symptoms.

The number of regions can be 2 or more, with no upper limit to the number. In such an instance, tens, hundreds or more regions can be used to increase confidence and to allow the diagnosis of multiple diseases, disease variants, or disease progression

Example 6

Detection of an analyte

In another example, the molecule being detected is not limited to detecting biological molecules. Single molecule imaging is agnostic to the type of molecule being detected. It is therefore similarly possible to detect molecules such as environmental contaminants, explosives, medical drugs, drugs of abuse, particulates, and many other molecules. Capture probes specific for such a molecule can be employed on the analytical surface to capture the target molecule. With a suitable fluorescent detection molecule or using the inherent fluorescence of the molecule being detected, it is possible to capture, and directly detect and quantify signals that are specific to the single molecules being assayed. of the invention are defined in the following numbered clauses.

1 . A method for detecting and/or analysing an analyte in a sample, comprising observing an analytical surface that has been contacted by the sample, using an optical microscope with a resolution of 100 nm or less, wherein the analytical surface comprises a capture probe immobilised to a solid phase, the capture probe for specifically capturing the analyte, such that if the analyte is present in the sample, the analyte is captured by the capture probe and can be observed.

2. A method according to clause 1 , comprising contacting the sample with the analytical surface.

3. The method according to clause 1 or clause 2, wherein the capture probe specifically binds to the analyte or the capture probe specifically binds to a linker moiety which is coupled to the analyte.

4. The method according to clause 3, comprising coupling the linker moiety to the analyte.

5. The method according to any preceding clause, wherein the capture probe comprises a biomolecule, optionally wherein the biomolecule comprises a nucleic acid, an amino acid, peptide, polypeptide, protein or peptide nucleic acid.

6. The method according to any preceding clause, wherein the analytical surface comprises a plurality of immobilised capture probes, and each capture probe is for specifically capturing the same analyte.

7. The method according to any preceding clause, for detecting and/or analysing a plurality of different analytes in the sample.

8. The method according to clause 7, wherein the analytical surface comprises a plurality of immobilised capture probes, and each capture probe is for specifically capturing a different analyte. 9. The method according to clause 8, wherein the analytical surface comprises a plurality of distinct capture zones, each capture zone comprising a capture probe for specifically capturing a different analyte.

10. The method according to clause 9, wherein each capture zone comprises a plurality of the same capture probe for specifically capturing the same analyte.

11 . The method according to any of clauses 7 to 10, wherein each different analyte is a fragment of a larger analyte.

12. The method according to clause 11 , comprising treating the sample to fragment any of the larger analyte present in the sample, to form each analyte fragment.

13. The method according to clause 12, comprising contacting an enzyme with the sample, optionally wherein the enzyme is a peptidase or a nuclease.

14. The method according to any preceding clause, wherein a detection moiety is coupled to the, or each, analyte for assisting the detection and/or analysis of the or each analyte.

15. The method according to clause 14, wherein the detection moiety is covalently bound to the, or each, analyte.

16. The method according to clause 15, wherein a detection probe comprises the detection moiety and the detection probe is specific for the, or each, analyte, optionally the detection probe specifically binds the, or each, analyte.

17. The method according to clause 16, wherein the detection probe comprises a biomolecule, optionally wherein the biomolecule comprises a nucleic acid, an amino acid, peptide, polypeptide, protein or peptide nucleic acid.

18. The method according to any of clauses 14 to 17, wherein the detection moiety is a fluorescent label.

19. The method according to any of clauses 14 to 18, comprising contacting the detection moiety: i) with the sample, to couple the detection moiety to any of the, or each, analyte present in the sample; or ii) with the analytical surface, to couple the detection moiety with any of the, or each, analyte which may have been captured. 20. The method according to any preceding clause, wherein the solid phase is glass and/or the or each capture probe is covalently immobilised to the solid phase.

21 . The method according to any preceding clause, wherein the optical microscope comprises: a first optical microscope; and a second optical microscope with a different mode of operation to the first optical microscope; wherein the first optical microscope is a confocal microscope and the second optical microscope is a total internal reflection fluorescence microscope, wherein the optical microscope is configured such that the first optical microscope and the second optical microscope simultaneously view a sample and wherein the second optical microscope is used to correct drift from the first optical microscope.

22. The method according to any preceding clause, wherein the analyte comprises a nucleic acid, a peptide, polypeptide, protein, peptide nucleic acid, lipid or carbohydrate.

23. The method according to any preceding clause, wherein the analyte comprises a receptor, ligand, antibody, antibody fragment, or antigen.

24. The method according to any preceding clause, wherein the analyte comprises biological material from one or more infectious agents.

25. The method according to clause 24, wherein the biological molecule comprises wildtype nucleotide sequence, and/or wild-type amino acid sequence.

26. The method according to clause 25, wherein the method is for determining whether a sample comprises one or more infectious agent having wild-type nucleotide sequence and/or wild-type amino acid sequence, wherein the method comprises capturing and detecting for wildtype nucleotide sequence, and/or wild-type amino acid sequence, in the sample.

27. The method according to clause 26, wherein the biological molecule comprises mutanttype nucleotide sequence, or mutant-type amino acid sequence.

28. The method according to clause 27, wherein the method is for determining whether a sample comprises one or more genomic variant of the infectious agent, wherein the method comprises capturing and detecting for mutant-type nucleotide sequence, and/or mutant-type amino acid sequence, in the sample. 29. An analytical device for use, or adapted for use, with an optical microscope having a resolution of 100 nm or less, comprising an analytical surface, the analytical surface comprising a capture probe immobilised to a solid phase, the capture probe for specifically capturing an analyte.

30. The device according to clause 29, wherein the capture probe specifically binds directly to the analyte or specifically binds to a linker moiety which is coupled to the analyte.

31 . The device according to clause 29 or 30, wherein the capture probe comprises a biomolecule, optionally wherein the capture probe comprises a nucleic acid, an amino acid, peptide, polypeptide, protein or peptide nucleic acid.

32. The device according to any of clauses 29 to 31 , comprising a plurality of immobilised capture probes for capturing the same analyte.

33. The device according to any of clauses 29 to 32, comprising a plurality of immobilised capture probes, and each capture probe is for specifically capturing a different analyte.

34. The device according to clause 33, wherein there are a plurality of capture zones on the analytical surface, each capture zone comprising a capture probe for specifically capturing a different analyte.

35. The device according to clause 34, wherein each capture zone comprises a plurality of the same capture probe for specifically capturing the same analyte.

36. The device according to any of clauses 32 to 34, wherein each different analyte is a fragment of a larger analyte.

37. The device according to any of clauses 29 to 36, further comprising an immobilised reference element.

38. The device according to clause 37, wherein the reference element comprises a polystyrene bead, nanodiamond, fluorophore, or a protein, polyethyleneimine, antibody, or lipid- coupled fluorophore.

39. The device according to clause 37 or 38, wherein the solid phase is glass and/or the or each capture probe is covalently immobilised to the solid phase. 40. The device according to any of clauses 29 to 39, comprising an analyte captured by the, or each, capture probe.

41 . The device according to clause 40, comprising a detection moiety coupled to the analyte.

42. The device according to any of clauses 29 to 41 , in combination with an optical microscope, optionally the microscope having a resolution of 100 nm or less.

43. The device according to any of clauses 29 to 42, in the form of a sample enclosure, comprising a cover, wherein the cover is spaced from the analytical surface, forming a space in which to locate a sample, or through which a sample can flow.

44. The device according to clause 43, wherein the analytical surface comprises a well, or a plurality of wells, in which the or each capture probe is located.

45. The device according to clause 44, comprising a plurality of wells, each well defining a capture zone as described in clause 33 or clause 34.

46. The device according to clause 44 or clause 44, comprising an inlet channel through which the sample can enter the, or each well, and an outlet channel through which the sample can exit the, or each, well.

47. A method comprising contacting a sample with the device defined in any of clauses 29 to 46.

48. A method according to clause 47, comprising contacting a detection moiety with the sample, or contacting the detection moiety with the device, to couple the detection moiety to an analyte.

49. The method according to clause 48, wherein a detection probe comprises the detection moiety and the detection probe is specific for the analyte, optionally wherein the detection probe specifically binds the analyte..

50. The method according to clause 48 or clause 49, wherein the detection moiety is a fluorescent label. 51 . The method according to any of clauses 47 to 50, comprising observing the device using an optical microscope with a resolution of 100 nm or less.

52. The method according to clause 51 , wherein the optical microscope comprises: a first optical microscope; and a second optical microscope with a different mode of operation to the first optical microscope; wherein the first optical microscope is a confocal microscope and the second optical microscope is a total internal reflection fluorescence microscope, wherein the optical microscope is configured such that the first optical microscope and the second optical microscope simultaneously view a sample and wherein the second optical microscope is used to correct drift from the first optical microscope.

52. A kit, comprising: a device as defined in any of clauses 29 to 46; and i) a detection moiety for coupling to an analyte; and/or ii) a separate linker moiety for coupling to an analyte, the linker moiety specific for a capture probe of the device.

53. The kit according to clause 52, comprising one or more reagent for coupling the detection moiety to the analyte, and/or comprising one or more reagent for coupling the linker moiety to the analyte.

54. The kit according to clause 53, further comprising a microscope having a resolution of 100 nm or less.

55. A kit, comprising a microscope having a resolution of 100 nm or less, and a device as defined in any of clauses 29 to 46.

56. The kit according to clause 54 or clause 55, wherein the microscope comprises: a first optical microscope; and a second optical microscope with a different mode of operation to the first optical microscope; wherein the first optical microscope is a confocal microscope and the second optical microscope is a total internal reflection fluorescence microscope, wherein the optical microscope is configured such that the first optical microscope and the second optical microscope simultaneously view a sample and wherein the second optical microscope is used to correct drift from the first optical microscope. 57. Use of an analytical device as defined in any of clauses 29 to 46, with an optical microscope having a resolution of 100 nm or less.

Claims

2. A method according to claim 1 , comprising contacting the sample with the analytical surface.

3. The method according to claim 1 or claim 2, wherein the capture probe specifically binds to the analyte or the capture probe specifically binds to a linker moiety which is coupled to the analyte.

4. The method according to claim 3, comprising coupling the linker moiety to the analyte.

5. The method according to any preceding claim, wherein the capture probe comprises a biomolecule, optionally wherein the biomolecule comprises a nucleic acid, an amino acid, peptide, polypeptide, protein or peptide nucleic acid.

6. The method according to any preceding claim, for detecting and/or analysing a plurality of different analytes in the sample.

7. The method according to claim 6, wherein the analytical surface comprises a plurality of immobilised capture probes, and each capture probe is for specifically capturing a different analyte.

8. The method according to claim 7, wherein the analytical surface comprises a plurality of distinct capture zones, each capture zone comprising a capture probe for specifically capturing a different analyte.

9. The method according to any of claims 6 to 8, wherein each different analyte is a fragment of a larger analyte.

10. The method according to claim 9, comprising treating the sample to fragment any of the larger analyte present in the sample, to form each analyte fragment.

11 . The method according to claim 10, comprising contacting an enzyme with the sample, optionally wherein the enzyme is a peptidase or a nuclease.

12. The method according to any preceding claim, wherein a detection moiety is coupled to the, or each, analyte for assisting the detection and/or analysis of the or each analyte.

13. The method according to claim 12, wherein the detection moiety is covalently bound to the, or each, analyte.

14. The method according to claim 13, wherein a detection probe comprises the detection moiety and the detection probe is specific for the, or each, analyte, optionally the detection probe specifically binds the, or each, analyte.

15. The method according to any of claims 12 to 14, wherein the detection moiety is a fluorescent label.

16. The method according to any of claims 12 to 15, comprising contacting the detection moiety: i) with the sample, to couple the detection moiety to any of the, or each, analyte present in the sample; or ii) with the analytical surface, to couple the detection moiety with any of the, or each, analyte which may have been captured.

17. The method according to any preceding claim, wherein the solid phase is glass and/or the or each capture probe is covalently immobilised to the solid phase.

18. The method according to any preceding claim, wherein the analyte comprises a nucleic acid, a peptide, polypeptide, protein, peptide nucleic acid, lipid or carbohydrate, optionally wherein the analyte comprises a receptor, ligand, antibody, antibody fragment, or antigen.

19. Use of an analytical device with a microscope having a resolution of 100 nm or less, the analytical device comprising an analytical surface, the analytical surface comprising a capture probe immobilised to a solid phase, the capture probe for specifically capturing an analyte.

20. The use according to claim 19, wherein the analytical surface comprises a plurality of immobilised capture probes, and each capture probe is for specifically capturing a different analyte.

21 . The use according to claim 20, wherein the analytical surface comprises a plurality of capture zones, each capture zone comprising a capture probe for specifically capturing a different analyte.

22. The use according to claim 20 or claim 21 , wherein each different analyte is a fragment of a larger analyte.

23. The use according to any of claims 18 to 22, wherein the solid phase is glass and/or the or each capture probe is covalently immobilised to the solid phase.

24. The use according to any of claims 18 to 23, wherein the device is a glass slide, a multiwell plate or a microfluidic device.

25. A kit comprising: i) an analytical device, and ii) a microscope with a resolution of 100 nm or less; and/or wherein the analytical device is for use, or adapted for use, with an optical microscope with a resolution of 100 nm or less, and the analytical device is as defined in any of claims 19 to