Method of obtaining information about entities in a liquid sample

Abstract

Methods of obtaining information about entities in a liquid sample are disclosed. An observation volume is defined by a pair of confinement surfaces of a reservoir containing a sample. The confinement surfaces define a plurality of traps. Information about residence times of target entities in the traps is obtained and used to determine information about the target entities. A ratio of an average separation between the confinement surfaces outside of the traps and a maximum dimension of each target entity is equal to or less than 50.

Description

[0001] METHOD OF OBTAINING INFORMATION ABOUT ENTITIES IN A LIQUID SAMPLE

[0002] The present disclosure relates to methods and apparatus for obtaining information about entities in a liquid, such as molecules in solution, particularly by observing diffusion dynamics of the entities.

[0003] There is a demand for high resolution characterisation of biomolecular properties in the solution phase under native conditions. A range of molecular analysis techniques are available that can characterise molecular species and resolve the states present in a heterogeneous mixture. These methods focus on the measurement of various molecular properties such as mass, charge, hydrodynamic radius or size, and 3d-conformation, and the interrogation mechanisms involved generally feature the use of applied fields e.g., light, electrical fields, temperature gradients and flow, as well as the combination of external fields with micro- or nanoscale transport principles. Surface immobilization, as well as strong external electrical and optical fields can however perturb the integrity and function of biomolecules, whose 3D conformation often arises from the concerted effect of weak intramolecular forces.

[0004] It is an object of the present disclosure to provide alternative or improved ways of obtaining information about entities such as molecules in a liquid sample.

[0005] According to an aspect of the invention, there is provided a method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: a ratio of the average separation between the confinement surfaces outside of the traps in the observation volume and a maximum dimension of each target entity is equal to or less than 50.

[0006] The method has been found to allow high-precision characterisation of entities to be achieved with no or only weak perturbations being applied to the entities of interest. The method can be performed using relatively simple and inexpensive equipment, such as unmodified wide-field optical microscopes. Arranging for the ratio of the average separation between confinement surfaces and a maximum dimension of each target entity to be equal to or less than 50 amplifies sensitivity to size and / or shape. Rapid, high- throughput molecular characterisations are demonstrated in a broad spectrum of bioanalytical application areas.

[0007] Optionally, the confinement surfaces are substantially parallel to each other outside of the traps in the observation volume, such that the separation between the confinement surfaces is substantially uniform outside of the traps in the observation volume. This configuration facilitates manufacture and reliability, as well as minimising deviations in the environment around different traps.

[0008] Optionally, an average separation between the confinement surfaces in the traps is at least 2 times larger than the average separation between the confinement surfaces outside of the traps in the observation volume. This configuration has been found to provide sufficiently different behaviour in the traps compared to outside of the traps.

[0009] Optionally, the separation between the confinement surfaces in the traps is substantially the same for at least a majority of the traps. Making the traps as similar as possible facilitates interpretation of observations of the target entities and / or analysis of measured residence times.

[0010] Optionally, the number of traps in the observation volume is at least 50. Providing a large number of traps allows accurate results to be obtained through averaging. High throughput can be simultaneously achieved by observing a large number of traps simultaneously. Optionally substantially all of the traps in the observation volume are observed simultaneously and / or at least 50 are observed simultaneously.

[0011] Optionally, the liquid sample and / or the confinement surfaces in the observation volume are configured such that the residence times of the target entities are predominantly determined by non-electrostatic effects. Suppressing electrostatic effects allows properties that depend on other factors, such as shape and / or size, to be measuring with greater sensitivity.

[0012] Optionally, the sample is additionally provided in a further observation volume, the further observation volume being defined by a pair of further confinement surfaces of the reservoir containing the sample, wherein the pair of further confinement surfaces face into the further observation volume from opposite sides of the further observation volume, and wherein the further confinement surfaces define a plurality of further traps in which a separation between the further confinement surfaces is larger than an average separation between the further confinement surfaces outside of the further traps in the further observation volume. Providing multiple, separately observable observation volumes (e.g., the observation volume and one or more of the further observation volumes) allows multiplexed measurements to be made. Observation of target entities in different observation volumes allows the target entities to be simultaneously probed differently in the same sample, for example to obtain information about different characteristics of the target entities and / or to vary relative sensitivities of the measurements to different characteristics.

[0013] Optionally, the liquid sample, the confinement surfaces in the observation volume, and the further confinement surfaces in the further observation volume, are configured such that the residence times of the target entities in the traps of the observation volume are influenced by electrostatic effects to a different extent than the residence times of the target entities in the further traps of the further observation volume. Optionally, the residence times of the target entities in the traps of the observation volume are predominantly determined by non-electrostatic effects; and / or the residence times of the target entities in the further traps of the further observation volume are predominantly determined by electrostatic effects. This configuration allows electrostatic and non-electrostatic properties of the target entities to be probed simultaneously, thereby allowing a richer spectrum of information to be obtained about target entities without compromising throughput.

[0014] Optionally, the determining of information about the target entities comprises determining information about binding of the first target entities to the second target entities to form third target entities, optionally the determining of information comprising determining variations in an amount of the third target entity in the sample as a function of time. Thus, the method may be used to measure on- and off-rates of binding where two species (i.e., first and second target entities) are mixed and the amount of bound or unbound complex is observed to increase / decrease with the passage of time. This is an important measurement modality in bioanalytics.

[0015] Optionally, the method comprises driving flow of the sample through the observation volume. Driving flow of the sample increases throughput by increasing a rate at which target entities encounter the traps.

[0016] According to an alternative aspect of the invention, there is provided a method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the number of traps in the observation volume is at least 50.

[0017] According to an alternative aspect of the invention, there is provided a method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample contains first target entities and second target entities and the determining of information about the target entities comprises determining information about binding of the first target entities to the second target entities to form third target entities, optionally the determining of information comprising determining variations in an amount of the third target entity in the sample as a function of time.

[0018] According to an alternative aspect of the invention, there is provided a method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample is additionally provided in a further observation volume, the further observation volume being defined by a pair of further confinement surfaces of the reservoir containing the sample, wherein the pair of further confinement surfaces face into the further observation volume from opposite sides of the further observation volume, and wherein the further confinement surfaces define a plurality of further traps in which a separation between the further confinement surfaces is larger than an average separation between the further confinement surfaces outside of the further traps in the further observation volume; and the liquid sample, the confinement surfaces in the observation volume, and the further confinement surfaces in the further observation volume, are configured such that the residence times of the target entities in the traps of the observation volume are influenced by electrostatic effects to a different extent than the residence times of the target entities in the further traps of the further observation volume.

[0019] According to an alternative aspect of the invention, there is provided a method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample contains a plurality of monomers and the target entities are multimers of the monomers.

[0020] Embodiments of the disclosure will be further described by way of example only with reference to the accompanying drawings.

[0021] Figure l is a schematic side sectional view of a measurement apparatus configured to perform a method of obtaining information about entities in a liquid sample.

[0022] Figure 2 is a schematic side sectional view of an example trap in an observation volume.

[0023] Figure 3 is a schematic side sectional view of an alternative example trap.

[0024] Figure 4 is a perspective view illustrating illumination of an example observation volume 4 comprising an array of traps.

[0025] Figure 5 depicts: (left) an image obtained by an optical measurement device of a portion of a sample in an observation volume 4; and (right) a portion of the image corresponding to a single one of the traps shown at a sequence of different times corresponding to different respective captured frames.

[0026] Figure 6 is a graph depicting probability density distributions, P(At), of durations At of escape events for target entities of different sizes.

[0027] Figure 7 depicts: (top) example observation volume geometries adjacent to traps, the observation geometries differing by having adjacent confinement surfaces separated by different amounts; and (bottom) a graph depicting simulated and measured variations of residence / escape times tescof different sized target entities for the observation volume geometries shown in Figure 7 (top).

[0028] Figure 8 is a graph depicting application of the method to inferring protein masses.

[0029] Figure 9 is a graph depicting application of the method to distinguishing single basepair differences in double stranded DNA molecules.

[0030] Figures 10-12 illustrate the method being used to detect differences of 18 Da between chemically different derivatives (maleimide and NHS) of the same fluorescent organic dye molecule. Figure 13 illustrates use of the method to detect differences in molecular shape amongst DNA molecules of identical mass and charge.

[0031] Figure 14 schematically depicts a portion of a reservoir containing an observation volume (defined by a pair of confinement surfaces) and a further observation volume (defined by a pair of further confinement surfaces).

[0032] Figure 15 shows three graphs demonstrating application of methods of the present disclosure in the context of studies on insulin. The rightmost panel shows spectra of escape times for different amounts of unlabelled insulin compared to labelled insulin. The middle panel shows curves demonstrating different off-rates for disassociation of hexameric insulin for different background media. The leftmost panel shows curves of bound insulin fraction against amount of unlabelled insulin to derive molecular affinity for two different types of insulin (Novorapid - leftmost curve; Apidra - rightmost curve).

[0033] The present disclosure provides a method of obtaining information about entities in a liquid sample. Figure l is a schematic side sectional view of a measurement apparatus 2 configured to perform the method.

[0034] A sample is provided in an observation volume 4. The observation volume 4 is defined by a pair of confinement surfaces 11, 12 of a reservoir 6. The reservoir 6 contains the sample. Typically, the reservoir 6 defines a volume that is larger than the observation volume 4. The observation volume 4 is only a portion of the volume defined by the reservoir 6. Surfaces other than the two confinement surfaces 11, 12 may therefore face into the reservoir 6 and contribute to containing the sample in the reservoir 6. The other surfaces may, for example, face into the reservoir 6 in a portion of the reservoir 6 outside of the observation volume 4. As described below with reference to Figure 14, the reservoir 6 may comprise one or more further observation volumes 104 that are fluidically interconnected. The sample may or may not be stationary in the reservoir 6. In some configurations, a flow of the sample is driven through the observation volume 4. The observation volume 4 represents a portion of the reservoir 6 in which the sample may be observed (e.g., by obtaining image data or video data by performing measurements on the sample and / or by performing visual inspection (manual observations), such as by looking through a microscope at the sample).

[0035] The confinement surfaces 11, 12 face into the observation volume 4 from opposite sides of the observation volume 4. In the example of Figure 1, an uppermost confinement surface 11 faces substantially downwards into the observation volume 4 and a lowermost confinement surface 12 faces substantially upwards into the observation volume 4.

[0036] The confinement surfaces 11, 12 define a plurality of traps 14. As described in further detail below, each trap 14 is configured to increase a probability of an entity in the sample being observed in the trap 14 in comparison to a region outside of the trap 14. This effect may be referred to as a trapping effect of the trap 14. As described in further detail below, information about residence times of target entities in the traps 14 may be obtained, for example by measurements to obtain image and / or video data or by visual inspection. Information about the target entities may be determined from the obtained information about residence times of target entities in the traps 14.

[0037] Examples of a trap 14 are depicted schematically in Figures 2 and 3. For each example, a corresponding observation volume 4 may comprise any number of the traps 14, such as 50 or more, optionally 100 or more. The traps 14 may all be the same or some of the traps 14 may be different from each other. Thus, a profile of each trap 14 may be the same for at least a subset of the traps 14 and / or the profile of each trap 14 may be different for at least a subset of the traps 14. These possibilities are applicable for all shapes of traps 14, not just for the examples of Figures 2 and 3.

[0038] In each trap 14, a separation between the confinement surfaces 11, 12 is larger than an average separation between the confinement surfaces 11, 12 outside of the traps 14 (but still in the observation volume 4). The confinement surfaces 11, 12 may, for example, be substantially parallel to each other outside of the traps 14, such that the separation between the confinement surfaces 11, 12 is substantially uniform outside of the traps 14. For example, the confinement surfaces 11, 12 may be substantially planar outside of the traps 14. In the examples of Figures 2 and 3, the confinement surfaces 11, 12 are substantially planar and parallel to each other outside of the traps 14. The confinement surfaces 11, 12 may be described as parallel plates or parallel slits or parallel plate slits in these (and other) examples. A gap between the confinement surfaces 11, 12 may be referred to as a slit. In the examples of Figures 2 and 3, the confinement surfaces 11, 12 are separated from each other by a distance outside of the traps 14. The traps 14 may each be defined by an indentation in one or both of the confinement surfaces 11, 12. Figure 2 depicts an example in which a trap 14 is defined by an indentation in only one of the confinement surfaces (confinement surface 11). Figure 3 depicts an example in which a trap 14 is formed by a combination of mutually facing indentations in both of the confinement surfaces 11, 12. A separation between the confinement surfaces 11, 12 is h2in each trap 14. In the example of Figure 2, this is achieved by providing a single indentation having a depth of h2— h . In the example of Figure 3, this is achieved by providing two indentations having a combined depth of h2— h . In the particular example of Figure 3, each indentation has the same depth of (h2— h-^ / 2 but the indentations could also have different depths.

[0039] As exemplified in Figures 2 and 3, the separation between the confinement surfaces 11, 12 may be arranged to be uniform within each trap 14. The traps 14 may, for example, be defined by a cylindrical indentation in one or both confinement surfaces 11, 12. In the example shown in Figure 2, a trap 14 is defined by a cylindrical indentation with diameter d and length h2— h . In the example of Figure 3, two cylindrical indentations with diameter d are provided, each having a length (h2— h1) / 2. In other implementations, the separation between the confinement surfaces 11, 12 may be non-uniform.

[0040] The separation between the confinement surfaces 11, 12 in the traps 14 may be substantially the same for at least a majority of the traps 14. Traps in which the separation between the confinement surfaces is the same may have the same or a similar trapping effect, which may facilitate analysis of observations of the behaviour of entities of interest in the sample. For example, at least a majority of the traps 14 may be formed by substantially identical indentations in one or both of the confinement surfaces 11, 12.

[0041] A sample having target entities of interest is provided in the reservoir 6 and information about residence times of the target entities in the traps 14 is obtained (e.g., by measuring the residence times). As described in further detail below, the method of the present disclosure amplifies a measured response by arranging for the following condition to be satisfied: a ratio of an average separation between the confinement surfaces 11, 12 outside of the traps in the observation volume and a maximum dimension of each target entity is equal to or less than 50, optionally equal to or less than 30, optionally equal to or less than 10, optionally equal to or less than 5. The maximum dimension of the target entity is defined as the diameter of the smallest notional bounding sphere that can contain the target entity. When the above condition is satisfied target entities not located in traps 14 take up a relatively large proportion of a gap between the confinement surfaces 11, 12. The separation between the confinement surfaces 11, 12 is larger in the traps 14. In some implementations, an average separation between the confinement surfaces in the traps 14 is at least 2 times larger, optionally at least 3 times larger, optionally at least 5 times larger, optionally at least 10 times larger, than the average separation between the confinement surfaces 11, 12 outside of the traps 14.

[0042] An optical measurement device 20 may be used to measure the residence times of target entities in the traps 14. The optical measurement device 20 may be configured to perform optical measurements through one or both of the confinement surfaces 11, 12. The optical measurement device 20 may be configured to obtain image data and / or video data. The optical measurement device 20 may be configured to operate in a reflection mode or a transmission mode. In the example of Figure 1, the optical measurement device 20 is configured to perform optical measurements through the confinement surface 12. The optical measurement device 20 may, for example, be configured to perform wide-field optical microscopy (e.g., by comprising a wide-field optical microscope). In some embodiments, the target entities are configured to fluoresce. The target entities may, for example, be tagged with fluorescent markers. In such embodiments, the optical measurement device 20 may be configured to excite the fluorescence to make entities of interest more detectable. The optical measurement device 20 may comprise an illumination unit configured to direct light towards the observation volume 4 and a detection unit configured to detect light emitted from the observation volume 4.

[0043] Information about the residence times may be obtained by visually inspecting the observation volume through one of the confinement surfaces. The observation volume may be observed, for example, by a user looking through an optical microscope or observing an output on a display of an optical microscope. Alternatively, the information about the residence times may be obtained by visually inspecting image data or video data obtained by performing optical measurements through one or both of the confinement surfaces.

[0044] Obtaining information about residence times may comprise measuring residence times to obtain numerical values representing the residence times and / or a distribution of residence times. Alternatively or additionally, information about residence times may be obtained by indirect measurements or observations. For example, the obtaining of information about residence times may comprise determining a proportion of the traps that contain a target entity in an image, in a plurality of images, and / or during a predetermined observation time. A sample containing target entities having characteristics (e.g., size and / or shape) that favour longer residence times will be expected to have such target entities present in a higher proportion of the traps 14 than would be -the case without such target entities. A single frame image or a single visual observation may be sufficient in this case to determine whether or not a large proportion of the traps 14 contain target entities and therefore whether or not the sample contains target entities having the characteristics that favour longer residence times. The determination of information about the target entities from the obtained information about residence times may thus comprise determining a measure of a number or concentration of target entities in the sample that have a characteristic that promotes trapping of the target entities in the traps. This type of application may be referred to as “molecular counting".

[0045] The optical measurement device 20 and the observation volume 4 may be configured such that the optical measurements simultaneously observe at least 50 of the traps 14, optionally at least 100 of the traps 14, optionally at least 200 of the traps 14, optionally at least 500 of the traps 14. Observing a large number of traps 14 simultaneously allows accurate results to be obtained with high throughput. Observing a large number of traps 14 improves accuracy by averaging. Observing a large number of traps 14 simultaneously provides high throughput. Throughput may be further increased by driving flow of the sample through the observation volume 4. Driving a flow of the sample through the observation volume 4 may increase the rate at which target entities encounter the traps 14. The flow may be driven using a pumping system and / or by the application of an electric field (e.g., via electro-osmosis).

[0046] As will be explained below, the residence times of entities in traps depends on properties of the entities such as size and / or shape. The method exploits this relationship to determine information about the entities. Referring again to Figure 1, the method may, for example, use a data processing system 30 to determine information about the target entities from the information obtained about the residence times, for example by analysing measurements of the residence times. The data processing system 30 may receive an output from the optical measurement device 20. The data processing system 30 may, for example, receive a time series of frames of image data from the optical measurement device 20. In an embodiment, the data processing system 20 is configured to analyse the frames to detect frames in which a target entity is present in one or more locations corresponding to traps 14. The data processing system 20 may thereby measure residence times of target entities in the traps 14. The data processing system 20 may analyse the measured residence times to determine information about the target entities. The information about the target entities is thus determined based on the measured residence times. The determined information may comprise information about the size of the target entities, the shape of the target entities, or the size and the shape of the target entities. The method may be configured to detect changes in size and / or shape of the target entities as a function of time. The determined information may alternatively or additional include other (i.e., other than size and shape) physical / chemical information about the target entities, information about a binding affinity relevant to the target entity, and / or information about concentrations or amounts of the target entity in a sample. The data processing system 20 may comprise any suitable combination of computer hardware, including for example CPUs, RAM, SSDs, motherboards, network connections, firmware, software, and / or other elements known in the art that allow the data processing system 20 to perform the required computing operations. The data processing system 20 may be referred to as a computer or computing system. The required computing operations may be defined by one or more computer programs. The one or more computer programs may be provided in the form of media, optionally non-transitory media, storing computer readable instructions. When the computer readable instructions are read by the computer, the computer performs the required method steps. The data processing apparatus (or computer or computer system) may consist of a self-contained unit, such as a general-purpose desktop computer, laptop, tablet, or mobile telephone. Alternatively, the computer may consist of a distributed computing system having plural different computers connected to each other via a network such as the internet or an intranet.

[0047] The method may be performed with the sample substantially unperturbed. For example, the sample may be substantially still (i.e., with substantially no net flow within the sample) during the measurements of the residence times. Alternatively, as depicted schematically in Figure 1, a flow control system may be provided to drive flow of the sample through the observation volume 4 during the measurements of the residence times. In the example shown, the flow control system comprises an input unit 3 driving a flow of the sample into the observation volume 4 and an output unit 5 receiving a flow of sample out of the observation volume 4. Either or both of the input unit 3 and the output unit 5 may, for example, comprise any suitable combination of pumps, conduits, flow controllers, valves, seals, etc. required to provide the required flow. The input unit 3 and the output unit 5 may be fluidically connected outside of the observation volume 4 to provide a closed circuit in which the sample can flow, with individual portions of the sample flowing repeatedly through the observation volume 4. This may be appropriate where the sample is relatively scarce and / or expensive. Alternatively, the flow control system may be configured to drive flow of sample in a one-way flow through the observation volume 4 (e.g., by configuring the output unit 5 to operate as a sink or disposal system), with each portion of the sample only being driven through the observation volume 4 once before being discarded. As mentioned above, driving a flow of the sample may increase throughput. Alternatively or additionally, the sample may be subjected to other external perturbations such as pressure drops, magnetic fields, changes in temperature, temperature gradients, changes in sample composition (e.g., pH, salt, osmolytes etc.), changes in illumination conditions, etc.

[0048] As mentioned in the introductory part of the description, there is a demand for performing high resolution measurements of entities in a liquid sample but existing techniques for achieving this can involve application of undesirable perturbations to entities being measured. Embodiments described herein allow high-precision characterisation of entities to be achieved with no or only weak perturbations being applied to the entities of interest. The methods described demonstrate that observing entities (e.g., molecules) in solution diffusing within the confines of a geometrically modulated nanostructured landscape (e.g., comprising the confinement surfaces 11, 12 and traps 14 mentioned above) using a simple wide-field optical microscope (e.g., an example of the optical measurement device 20 described above) can provide a means to precisely characterise a molecular species (an example of a target entity) and / or distinguish molecular states in solution. In example configurations described herein, translational and configurational entropic effects that arise in a geometrically modulated nanoscale environment are tailored to achieve significant amplification of the measured response to molecular size and shape in solution. This is achieved by arranging for a ratio of the average separation between confinement surfaces 11, 12 and a maximum dimension of each target entity to be equal to or less than 50 as mentioned above. The power and versatility of methods of the present disclosure, allowing rapid, high-throughput molecular characterisation, are demonstrated below in a broad spectrum of bioanalytical application areas.

[0049] In an example implementation for demonstration purposes, a sample was provided by introducing fluorescent molecules at a concentration of 1-10 nM in PBS buffer into a series of parallel plate slits of height (see discussion below concerning Figures 4 and 5 for more details about the parallel plate slits). Inward facing surfaces of the parallel plate slits provide confinement surfaces 11, 12 as discussed above. The height defines the separation between the confinement surfaces 11, 12. The parallel plate slits carry arrays of periodic cylindrical indentations of total height h2. The cylindrical indentations are examples of the traps 14 discussed above. The indentations that provide the traps 14 may be referred to as "pocket" nanostructures. The traps 14 act as thermodynamic traps for single molecules in solution. The molecules in this study carry on average a single ATTO532 fluorescent label.

[0050] As depicted schematically in Figures 4 and 5, the diffusive dynamics of molecules in these arrays of traps 14 were imaged using a wide-field microscope acting as optical measurement device 20. In the examples shown, the observation volume is formed by a plurality of parallel plate slits separated by support structures 15. The support structures 15 provide mechanical support to the confinement surfaces 11, 12 that define the traps 14. The support structures 15 may therefore allow the total volume of the observation volume to be made larger. The slits and support structures 15 run generally from the top left towards the bottom right in Figure 4 and from top to bottom in Figure 5. In other configurations, the support structures may take a different form. In some configurations the support structures may be omitted. By imaging the diffusive dynamics it is possible extract average residence times of target entities in the traps 14. As depicted in the lower right portion of Figure 5, for example, the measuring of residence times may comprise detecting for how many frames 16 an entity is detected to be present in a trap 14. The presence of an entity in a trap 14 in a given frame 16 may be determined for example by detecting whether or not an average detected intensity in a portion of an image corresponding to the trap 14 is above or below a predetermined threshold. If the intensity is above the threshold an entity is present (e.g., causing the average intensity to be higher due to fluorescence). When the intensity is below the threshold an entity is not present (e.g., due to a lack of detected fluorescence). In the example depicted, an entity is detected to be present during the second and third frames 16 and then escapes, which corresponds to an “escape event” having a duration of At. Escape events of shorter duration are also observed in the fifth and ninth frames 16.

[0051] In an embodiment, the method comprises obtaining a probability density distribution of durations At of escape events. Average residence (escape) times, tesc, of entities in the traps 14 can be extracted from exponential fits to the probability density distributions. Figure 6 shows examples of such probability density distributions / ’(At) (with P(At) on the vertical axis and At on the horizontal axis) for four different entities: insulin (mass = 6.44kDa; rH= 1.16nm; illustrated schematically as molecule 41 in Figure 6), carbonic Anhydrase (mass = 29.65kDa; rH= 2.58nm; illustrated schematically as molecule 42 in Figure 6), Transferrin (mass = 76.05kDa; rH= 3.69; illustrated schematically as molecule 43 in Figure 6), and Stm-I protein (disordered) (mass = 46kDa; rH= 5.99nm; not illustrated in Figure 6). Curves 51-54 in Figure 6 correspond respectively to insulin, carbonic Anhydrase, Transferrin, and Stm-I protein (disordered). The calculated average residence times tescof 9.65ms, 17.19ms, 25.18ms, and 53.70ms scale as expected with the sizes rHof the respective target entities.

[0052] An expected average residence time of molecules (or other entities) in a trap 14, which is referred to below as the escape time tesc, may be written as follows: where W = AFe(+ Fentis the free energy difference between molecular states in the pocket (trap) and the slit (outside of the trap), T is the absolute temperature and kBis

[0053] L2Boltzmann's constant. Furthermore, troc — denotes a residence time of the molecule in a pocket region (trap) of radius L, in the absence of any geometric modulation of the slit (i.e., — = 1). Here D =kBTis the diffusion coefficient of an object with Stokes' radius, rH, in h±’ 6nrp'HJa fluid of viscosity J ,

[0054] In some implementations, the liquid sample and / or the confinement surfaces 11, 12 are configured such that the residence times of the target entities are predominantly determined by non-electrostatic effects. This may be achieved, for example, by arranging for the liquid sample to comprise a salt concentration that is high enough to suppress electrostatic effects. The salt concentration may, for example, be at least 50mM, optionally at least lOOmM. Operating at such high salt concentrations ensures that the electrostatic contribution is negligible, which implies that AFe(« 0 and the trap is purely entropic. A similar effect may be achieved, alternatively or additionally, by configuring the confinement surfaces 11, 12 to have an electrostatics-suppressing coating, such as a polymer such as polyethylene glycol (PEG). When the electrostatic is suitably suppressed, such that AFe(« 0, the following holds: where the translational entropy contribution may be estimated as AStrans= kBln(-^), and Sconfigwhich enters the picture for non-spherical objects will be discussed in detail later.

[0055] For a small hard sphere, when rH« h , we have Sconfig= 0. Substitution of VF = T Stransin Eq. (1) shows that the average escape time is enhanced by a factor h2 / h1compared to the free-diffusion value.

[0056] This enhancement effect is demonstrated in Figure 7, in which Brownian Dynamics simulations of molecular diffusion in the observation volume 4 are used to extract simulated escape times (symbols), which are fit to theoretical functions (lines). This is performed for three different configurations of the observation volume 4: configuration 1, configuration 2 and configuration 3. In configuration 1, the confinement surfaces 11, 12 are separated by = lOnm and the depth of each trap 14 is h2= 300nm, as depicted schematically by image 61 in Figure 7. In configuration 2, the confinement surfaces 11, 12 are separated by = 25nm and the depth of each trap 14 is h2= 300nm, as depicted schematically by image 62 in Figure 7. In configuration 3, the confinement surfaces 11, 12 are separated by h = 70nm and the depth of each trap 14 is h2= 300nm, as depicted schematically by image 63 in Figure 7. Curves corresponding respectively to configurations 1-3 are labelled 71-73 in Figure 7.

[0057] For comparison, experimentally measured residence times tescin devices of slit height h = 21nm were converted to molecular hydrodynamic radii (rHor R) using the theoretical function relating tescand rescto provide curve 74 in Figure 7. The inferred values are found to agree well with independent experimental measurements and theoretical values.

[0058] The progressive (non-linear) increase in steepness and curvature of curves 73, 72, 74 and 71 demonstrates the power of the enhancement effect on residence times tescthat is achieved by increasing the confinement between the confinement surfaces 11, 12 outside of the traps 14 relative to the confinement within the traps 14, i.e., by increasing h2 / h1. The enhancement effect results from entropic effects and may therefore be referred to as an entropic enhancement.

[0059] The entropic enhancement of residence times in the traps 14 is beneficial to the method since it makes residence times long enough to be detectable with high accuracy and / or for a wider range of target entities of interest, thereby fostering high throughput direct imaging of the escape process on entities (e.g., molecules) in parallel, using both low incident optical power densities, and a large-area camera-based detector. High-throughput, highly parallel observation of the escape process provides the ability to attain high precision measurements of the hydrodynamic radius in a short measurement time (in exemplified in Figure 7).

[0060] The statistically dominated measurement imprecision on tescdepends on N the number of escape events observed as 1 / A / TV. In an example implementation, N = 104is achieved in approximately 1 minute of measurement time using a molecular concentration of about 0.1-10 nM in solution. An ~1% measurement imprecision on tescis therefore expected, and by implication also on rH. Given the approximate scaling of molecular 1 weight with R for globular molecules, ~1% imprecision on tesctranslates to about 3% measurement imprecision in mass. For small molecules (mol. wt. ~1 kDa), or elongate (e.g., rod shaped) molecules such as double stranded DNA (dsDNA), where rHscales approximately linearly with mass, < 1% measurement imprecision on rHimplies the ability to detect single carbon atom differences between molecular species, and single basepair differences in dsDNA. This is demonstrated in Figures 8-12 discussed below. Increasing the field of view and / or improving the array design (e.g., by optimising the sizes and / or separations of traps 14 etc.) would allow the same level of precision to be achieved more quickly, for example in a few seconds worth of measurement time, or much greater precision may be achieved in the same time, for example in a minute-long measurement window.

[0061] Figures 8-12 demonstrate application of the method to a variety of molecules over three orders of magnitude in molecular weight. Inferences of protein mass are performed (Figure 8), single basepair differences in double stranded DNA molecules are distinguished (Figure 9), and differences of 18 Da are clearly detected between chemically different derivatives (maleimide and NHS) of the same fluorescent organic dye molecule (Atto 542) (Figures 10-12).

[0062] The next section considers alteration of the height of the slit and examines the impact thereof on the molecular escape process. For larger entities (e.g., modelled as spheres), or shallower slits, such that the slit height, approaches the diameter of a sphere, 2R (given by the regime 2R < « h2) a non-linear enhancement of escape times with increasing object size R is expected (as illustrated by curves 72 and 71 in Figure 7). This non-linearity arises from the fact that R, which is the same as rHfor spheres, appears both in tr, the prefactor kinetic term that governs diffusive transport of the molecule in the potential well, as well as in the exponential entropic part that arises from nanoscale confinement (see Eq. 1). More specifically, the ratio of the effective number of translational states accessible to the molecule in the pocket (trap) vs. the slit (outside of the trap) may be expressed more accurately rather than h2 / h1. Since 2R « h2, Eq. (3) now reads:

[0063] Therefore when 2R is a substantial fraction of the slit height the escape time acquires a multiplicative size factor = 1 1 — The experimental readout now increases strongly with the diameter of the object (target entity) which offers the prospect of greatly enhancing disparities in measured escape timescales for molecules of similar diameter. Brownian Dynamics simulations of the escape process show good agreement with the simple arguments and theoretical expressions outlined above. The simulated escape time data are captured remarkably well by the simple functional forms based on Eq. (4). It is found that a coefficient of approximately 0.2 that accounts for various aspects of the image-based detection process, and inclusion of an offset, t0« 5 ms, ensures that an 7? ( 1 — — — ) + toe quantitatively matches the simulated escape times. We then use this functional form to convert measured tescvalues to measurements of object size, R.

[0064] The discussion thus far has focussed on the sensitivity of the method to the size of a spherical or globular molecule. However, as Eq. (2) indicates, the depth of the entropic potential well can consist of more than just a translational entropy. As in electrostatic trapping, it is expected that configurational entropy will play a role in the escape process. Configurational entropy may include both orientational states, e.g., for a rigid anisometric object, as well as conformational states for flexible polymers. Spatial confinement in the slit region imposes a further constraint on the number of orientational or conformational states accessible to the molecule compared to the "unconfined" pocket-state. Thus, extreme confinement - which may be described as the regime where approaches at least one geometric dimension of the object - can generate an additional contribution to W. We lump entropic contributions from orientational and conformational states together into a "configurational entropy" term which can report on the geometry and / or 3D conformation of the object. For example, consider a hypothetical case of a rod-shaped object of length Z, with the same hydrodynamic radius and translational entropy difference in the slit / pocket geometry (i.e., the geometry of the observation volume 4) as an equivalent sphere, but whose rotational states in the slit are limited by the constraint that the slit height (separation between the confinement surfaces 11, 12) approaches I. If the number of configurational states accessible to the molecule in the pocket (trap 14) state and slit (out of trap 14) state are given by c2and c respectively, then the factor — > 1 enters Eq. (1) in ci a multiplicative fashion, similar to arguments used for the translational factor h2 / h1above. The following may thus be expected: which implies an even greater enhancement of escape times on account of the configurational properties of the molecule. Thus, by constraining the number of accessible states: translational and configurational - of the molecule in the slit, the entropic term can introduce a significant size and shape dependence of our readout that goes well beyond that provided by the purely diffusive prefactor alone. This enhancement factor is given by the expression2- (yy 1 — . Since the system can be designed such that y- ~ 10-100 or greater, the method enables strong shape-based discrimination of molecules that may be otherwise identical (in hydrodynamic radius, mass and / or charge). The approach may be referred to as escape time size and shape spectrometry (ETsy).

[0065] Thus, methods of the present disclosure may comprise determining information about the size and / or shape of target entities based on information about residence times (obtained by measurements or observations). The determining of the information may comprise detecting changes in size and / or shape of the target entities. The determining of the information may comprise detecting the size and / or shape of the target entities. Alternatively or additionally, the method may comprise distinguishing between two or more different types of target entity based (e.g., predominantly) on differences in shape between the two or more different types of target entity. The different types of target entity may be substantially identical in respect of one or more of the following: mass, charge, hydrodynamic radius (optionally all three). The different types of target entity may include one or more of the following: DNA molecules or portions of DNA molecules; and protein molecules or portions of protein molecules. The method is particularly sensitive to non-spherical objects and may therefore be particularly effectively applied in cases where the target entity is non-spherical. For example, the method may be particularly effectively applied in cases where an aspect ratio of the target entity is at least 2, optionally at least 5, optionally at least 10. The method may, however, be applied to a wide range of target entities. The target entities may for example comprise one or more of the following: an elongate molecule (including DNA or a portion of DNA as mentioned above); RNA (or a portion of RNA); a lipid nanoparticle; a virus; a biological molecule (additionally or alternatively to those mentioned above); a colloidal particle; a nanoparticle; a quantum dot; and a polymer.

[0066] In an embodiment, the target entity comprises a complex of an optical marker bound to a predetermined binding target of interest. The liquid sample may contain a plurality of optical markers that are optically distinguishable from each other (e.g., of different colour) and configured to bind to a corresponding plurality of different binding targets. This allows different binding targets of interest to be identified in the same sample.

[0067] In an embodiment, the determining of information about the target entities comprises determining information about binding of the first target entities to the second target entities to form third target entities (which may be referred to as a complex). Optionally the determining of information comprising determining variations in an amount of the third target entity in the sample as a function of time. This approach may be used to measure on- and off-rates of binding where two species (i.e., first and second target entities) are mixed and the amount of bound or unbound complex is observed to increase / decrease with the passage of time. This is an important measurement modality in bioanalytics. The bound fraction (where the first and second target entities are bound together to form the third target entity) is distinguished from the unbound fraction (where the first and second target entities are unbound) by virtue of escape times as in a usual affinity measurement. In measurements of this type, the rates at which the fractions increase / decrease with time may be determined, for example by observation of the sample in real time (with the clock on reaction time starting at the point of the mixing of the two species). In one class of implementation, the first target entities and the second target entities are monomers, optionally identical monomers (different instances of the same monomer), as discussed in further detail below. Alternatively or additionally, the first target entities and the second target entities may have different compositions relative to each other.

[0068] In an embodiment, the method of the disclosure is applied to the case where the sample comprises a plurality of monomers and the target entities are multimers (which may be referred to as oligomers) of the monomers. A multimer (or oligomer) is a molecule made up of multiple monomers (smaller molecules) that are held together by weak bonds. The process of forming a multimer (or oligomer) may be referred to as multimerization (or oligomerization). In such embodiments, the determining of information may comprise determining information about the size, shape and / or abundance of each of one or more multimer orders of the multimers. A multimer order is the number of monomers in the multimer. For example, a dimer is a multimer of order 2, consisting of two monomers. A Km er is a multimer of order K (where K is an integer), consisting of K monomers. The method may in principle be applied to any monomer and / or multimer by selecting dimensions of the confinement surfaces and traps to provide appropriate sensitivity. The method has been found to be particularly effective in situations where the monomers comprise proteins, such a monomeric insulin. In an embodiment, an optical marker is attached to each of a minority of the monomers in the sample to form labelled monomers. Optionally, a ratio of the labelled monomers to unlabelled monomers in the sample is less than 1: 103, optionally less than 1: 104.

[0069] The oligomerization of insulin represents an archetypal medically and pharmaceutically relevant protein oligomerization problem. Insulin is physiologically active in the monomeric state but at high concentrations of ca. 100 / zM readily forms oligomers including dimers and multi-hexamers that are pharmacologically relevant, with the characterisation problem thus spanning nearly two orders in molecular weight. Monomeric insulin has a molecular weight of ~6 kDa, which is currently at the lower end of the sensitivity of most techniques based on scattered light. Higher energy X-ray scattering has been used to characterize insulin in suspension but the method is infrastructure intensive. In general although nanopore-based characterization approaches and light-scattering techniques are label-free they do not cope well with high concentrations of material, typically 0.5 mM in pharmacological preparations, or with a complex background, which precludes characterization of processes and states in complex media such as serum. On the other hand, separation and sieving methods such as Analytical ultracentrifugation (AUC) and size-exclusion chromatography (SEC) can in general deal well with a high-level of background material but these methods are slow and require large amounts of sample. Furthermore SEC entails large dilution factors and matrix-species interactions both of which may interfere with oligomerization equilibria altering the delicate balance between multimeric states. Although the analysis of insulin oligomeric state using AUC is free of these limitations the method lacks resolution. The method of the present disclosure has been found to provide beneficial performance in comparison with these prior art alternatives.

[0070] The inventors have demonstrated use of the method to characterize the interactions and oligomeric state of pharmacological insulin preparations both in buffer solution as well as in simulated serum. In this practical application, the method comprises introducing a small amount of singly fluorescently labelled insulin (i.e., with insulin monomer units labelled with an optical marker). The labelled insulin acts as a “spectator” species in a bath of unlabelled molecules of any identity. Weak interactions characteristic of protein oligomerization imply that the higher order molecular complexes that form are relatively unstable, characterized by high off-rates. Thus rapid exchange and equilibration ensures that a small concentration of labelled spectator is incorporated into the existing palette of higher order states that may now be detected by virtue of the fluorescing spectator species. Because spectator molecules are in relative low abundance (e.g., at 1 : 104dilution) with reference to the unlabelled protein whose interactions are probed it may be assumed that the vast majority of intermolecular interactions remain unaffected by covalent labelling of the probe species. Running the equilibrated suspension through a landscape of entropic traps and recording the escape time spectrum, for example in a 1 -minute measurement window, has been found to yield a read-out of shape and size of the various multimeric states present in solution. Note that only individual complexes that incorporate a labelled spectator molecule are visible in the measurement with the remaining complexes passing through the field of view undetected.

[0071] The method may also be used to perform kinetics measurements, for example by measuring the dissolution of multimers upon dilution into serum, closely mimicking the situation immediately post-injection into the body. The inventors found that the rate of dissolution of multimers into the physiologically active monomeric state is different for different preparations and correlates well with measured overall Kds inferred for the multimerization process and off-rates. Embodiments of the method of the present disclosure that use fluorescence detection, where a label (optical marker) serves the important function of conferring (bio)chemical specificity on the readout, which is highly valuable in applications focused on detecting species of interest in a complex mixture. The labelled species, which may be present at trace concentrations, can act as a reporter of molecular or complex-state in the presence of high background concentrations (mM) of unlabelled, interacting matter of any mass, identity and compositional complexity. The method of the present disclosure is well suited to detecting and measuring the physical properties of multi-subunit macromolecular complexes that form on account of weak interactions. These complexes are challenging to detect by other techniques that not only contend with restrictions on molecular weight, concentration, solution composition, but also often rely on the application of strong external fields that can perturb the integrity of fragile molecular assemblies. Rapid solution-phase direct sensing offered by methods of the present disclosure will also benefit investigations of long-standing problems such as pathological protein aggregation.

[0072] Figure 15 demonstrates application of the method in the context of studies on insulin.

[0073] The panel on the right in Figure 15 shows a series of "spectra" of measured escape times in a sample containing a fixed amount of labelled insulin acting as the "spectator species", and increasing amounts of unlabelled insulin (the top graph represents no additional unlabelled insulin, the middle graph represents adding l[iM unlabelled insulin, and the bottom graph represents adding 600 / J.M unlabelled insulin). The shift of the peaks to the right (higher escape times, tesc) from the top graph to the bottom graph shows the increasing size of the molecular complexes that form in solution (vertical lines indicate expected values for the monomer, dimer, hexamer etc.) as the amount of unlabelled insulin increases, and demonstrates the ability of the method to sensitively detect sizes of different target entities.

[0074] The middle panel in Figure 15 shows the ability to measure off-rates, i.e., the rate at which these complexes dissociate when the sample is diluted (this is an indication of how fast hexameric insulin present in a pharma product would turn into its bioavailable / bioactive monomeric form in the body). Three curves are shown, corresponding to different background media: saline, simulated serum, and human serum. The method is sensitive enough to detect clear differences in behaviour for the three different background media.

[0075] The left panel in Figure 15 is a different measure of molecular affinity inferred from the data in the rightmost panel. By assessing the amount of hexameric form as a function of insulin concentration we fit a curve and extract an affinity constant or Kdvalue for the formation of hexamers for two different types of insulin (Novorapid - leftmost curve; Apidra - rightmost curve). Values in the range of Kd> 10 micromolar are indicative of weak interactions. All these measures shown in Figure 15 are of great interest in biomolecular characterization in general and there are few / no techniques out there that will achieve all of this together, especially on small molecules, and with the speed, precision and negligible sample requirement of the method of the present disclosure.

[0076] An advantage of the ETsy approach in comparison to prior art alternatives is that (i) it can function at thermodynamic and mechanical equilibrium, in the absence of applied fields and in native solution conditions, and (ii) the experimental readout, which arises from direct observation of thermal diffusion in a geometrically modulated landscape, is highly amenable to computation and / or simulation and comparisons with theoretical models of a given molecular structure. Because of the sensitivity of the read-out to shape, and the feasibility of relating the readout to 3D structural models the method provides a route to screening conformational states and performing coarse-grained structural biology interpretations in native solution. Demonstration of this functionality in the context of DNA molecules of identical mass and charge is discussed below with reference to Figure 13.

[0077] Figure 13 depicts detection of differences in molecular shape amongst DNA molecules of identical mass and charge using the method of the present disclosure. For large values of (> 70 nm), all three DNA nanostructures (molecular structures and coarse-grained shapes illustrated on bottom row) have identical tescvalues. Subtracting the tescvalue corresponding to the “tile” structure from the measured tescfor the other two DNA nanostructures places all three species at At = 0 (top row). Progressively decreasing the height of the slit (separation between confinement surfaces 11, 12), places orientational or configurational constraints on the target entity (molecule). This results in large shape or 3d conformation-dependent differences in escape time, as reflected in progressively increasing Atescvalues between the three DNA nanostructures considered (middle and bottom row), as indicated by the increasing separation between the peak 81 corresponding to the tile structure, the peak 82 corresponding to the bundle structure, and the peak 83 corresponding to the rod structure.

[0078] Figure 13 thus demonstrates the ability of the method to leverage the extreme confinement regime to enhance disparities in tescbetween molecules that are effectively identical in mass and charge but differ in 3D conformation. To elucidate this principle we focus on DNA nanostructures that carry almost exactly the same number of basepairs but differ significantly in 3D geometry, progressively increasing in compaction from an extended rod state of length I to a more condensed square-tile state. Interestingly, these DNA nanostructures are experimentally indistinguishable from each other in free solution diffusion coefficient measurements and are also expected to be rather similar in their calculated hydrodynamic friction factors. Holding h2= 300 nm constant and decreasing h- from the regime where > I we note the ability to significantly alter measured tescvalues as we enter the regime where h -> I. The progressively increasing disparity in tescnoted as -> I can be exploited to construct single molecule conformation spectra in a mixture of different molecular. Note that every measurement in such a molecular conformation spectrum arises from a separate molecule. We currently record about N = 200-300 hops on average from each individual molecule before it either photobleaches or diffuses out of the field of view. This yields -3-5% measurement imprecision per molecule at present which can be improved by optimisation.

[0079] The ETsy method can also be used to measure binding affinities over several orders of magnitude in affinity constant. A further use of ETsy involves inferring the structure of molecules, or molecular complexes in solution. A computational platform that accurately simulates ETsy escape times and applies these results in conjunction with molecular structural modelling is very likely to be able to shed light on the most plausible coarsegrained structure or family of molecular-level structures of the molecule or complex under study. Such measurements may therefore furnish an additional orthogonal dimension of solution phase structural information that may inform high resolution structural methods, molecular structural databases and / or Al approaches to structural modelling. Finally, the highly parallel nature of the imaging process is amenable to low copy number detection of molecules of interest in a test sample, opening doors to applications in diagnostics.

[0080] The ETsy method presents a versatile nanodevice-based measurement platform that harnesses the combined power of a simple thermodynamic principle at the nanoscale and high-throughput large-area optical imaging to meet a variety of molecular analytical goals under native conditions in solution: (1) precise mass inference on globular molecules (2) size measurements (3) binding affinity measurements (4) coarse-grained shape readouts capable of informing atomistic molecular structural modelling in a top-down manner (5) low concentration / copy number detection reaching into the 10 fM regime for sensitive molecular counting in molecular diagnostics applications. Note that by lowering the salt concentration in solution to less than 50 mM the very same platform can be leveraged to operate in "charge mode" enabling readouts of molecular effective charge in conjunction with mass / size and shape, adding an additional powerful dimension to the existing multiparametric molecular property readout in the solution phase. Although we focus on simple molecular-shape spectra containing 3-4 species in the present study, machine learning approaches may in future support the construction of more complex spectra from single-molecule escape time data recorded on mixtures. Furthermore, although some of the examples discussed above use fluorescence detection, the fluorescent label in fact serves the beneficial function of conferring (bio)chemical specificity of the readout, which is desirable in applications focussing on the detection of species of interest in a complex mixture. Future improvements in the stability and longevity of the label will foster high precision measurements at the one-molecule level and therefore the ability to record highly resolved molecular spectra by permitting N -> 103-104per molecule. Finally, in purely analytical applications label-free optical detection may be leveraged to deliver the same information without the use of covalent labelling.

[0081] Figure 14 is a schematic side sectional view of an example of a variation on the embodiments described above in which the reservoir 6 comprises a further observation volume 104 in addition to the observation volume 4 described above. Thus, the sample may be provided in the observation volume 4 and, additionally, in the further observation volume 104. Except where explicitly stated otherwise, the further observation volume 104 may take any of the forms discussed above for the observation volume 4. A plurality of the further observation volumes 104 may be provided having the same or different properties relative to each other.

[0082] The further observation volume 104 is defined by a pair of further confinement surfaces 111, 112 of the reservoir 6 containing the sample. In the orientation of Figure 14, one of the further confinement surfaces I l l is substantially horizontal and faces vertically downwards and the other of the further confinement surfaces 112 is substantially horizontal and faces vertically upwards. The pair of further confinement surfaces 111, 112 face into the further observation volume 104 from opposite sides of the further observation volume 104. The confinement surfaces 11, 12 and the further confinement surfaces 111, 112 may be substantially planar and / or parallel to each other. In some embodiments, a plurality of the further observation volumes 104 are present. Each further observation volume has a respective pair of further confinement surfaces 111, 112. A plurality of the pairs of further confinement surfaces 111, 112 is thus present. The pairs may have different surface compositions relative to each other and / or different average separations outside of the further traps relative to each other.

[0083] A separation between the further confinement surfaces 111, 112 in the traps 114 is larger (in the example shown, due to the presence of respective indentations in the uppermost further confinement surface 111) than an average separation between the further confinement surfaces 111, 112 outside of the further traps 114. Providing multiple, separately observable confinement volumes allows multiplexed measurements to be made. Observation of target entities in different observation volumes (e.g., in the observation volume 4 and one or more further observation volumes 104) allows the target entities to be probed differently, for example to obtain information about different characteristics of the target entities and / or to vary relative sensitivities of the measurements to different characteristics. The observation volume 4 and the one or more further observation volumes 104 may be fluidically interconnected, which allows the same sample to be present in each of the different observation volumes 4, 104.

[0084] In an embodiment, the liquid sample, the confinement surfaces 11, 12 in the observation volume 4, and the further confinement surfaces 111, 112 in the further observation volume 104, are configured such that the residence times of the target entities in the traps 14 of the observation volume 4 are influenced by electrostatic effects to a different extent than the residence times of the target entities in the further traps 114 of the further observation volume 104. This allows multiplexed measurements of target entities to be made with different measurement sensitivities to their electrostatic properties. For example, the residence times of the target entities in the traps 14 of the observation volume 4 may be arranged to be predominantly determined by non-electrostatic effects while the residence times of the target entities in the further traps 114 of the further observation volume 104 are arranged to be predominantly determined by electrostatic effects. This may be achieved for example by using an intermediate salt concentration that suppresses electrostatic effects less strongly than higher salt concentrations and then arranging for the separation between the confinement surfaces 11, 12 to be large enough to allow electrostatic effects to be suppressed and arranging for the separation between the further confinement surfaces 111, 112 to be small enough that electrostatic effects become important despite the presence of the salt. Thus, the average separation between the confinement surfaces 11, 12 in the observation volume 4 may be larger than the average separation between the further confinement surfaces 111, 112 in the further observation volume 104, and the liquid sample may comprise a salt concentration that suppresses electrostatic effects such that the residence times of the target entities are predominantly determined by non-electrostatic effects in the traps 14 of the observation volume 4 and are predominantly determined by electrostatic effects in the further traps 114 of the further observation volume 104. The intermediate salt concentration may typically be less than about 45mM, optionally about 40mM.

[0085] Alternatively or additionally, the confinement surfaces 11, 12 of the observation volume 4 may have a different surface composition compared to the further confinement surfaces 111, 112 of the further observation volume 104. The surface compositions are configured such that the residence times of the target entities are predominantly determined by non-electrostatic effects in the traps 14 of the observation volume 4 and are predominantly determined by electrostatic effects in the further traps 114 of the further observation volume 104. The surface composition of the confinement surfaces 11, 12 of the observation volume 4 may, for example, comprise an electrostatics-suppressing coating such as a polymer, optionally polyethylene glycol (PEG).

[0086] In multiplexing implementations such as those discussed above, residence times of target entities in the further traps 114 are measured separately from residence times of target entities in the traps 14. Thus, in addition to the steps discussed above with reference to Figures 1 to 5, the method may further comprise obtaining information about residence times of the target entities in the further traps 114 and determining information about the target entities from the obtained information about residence times of the target entities in the further traps 114. The information about the target entities determined from the obtained information about residence times of the target entities in the further traps 114 (in the further observation volume 104) may be different from the information about the target entities determined from the obtained information about residence times of the target entities in the traps 14 (in the observation volume 4).Embodiments of the disclosure are defined in following numbered clauses.

[0087] 1. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: a ratio of the average separation between the confinement surfaces outside of the traps in the observation volume and a maximum dimension of each target entity is equal to or less than 50.

[0088] 2. The method of clause 1, wherein the confinement surfaces are substantially parallel to each other outside of the traps in the observation volume, such that the separation between the confinement surfaces is substantially uniform outside of the traps in the observation volume.

[0089] 3. The method of clause 1 or 2, wherein an average separation between the confinement surfaces in the traps is at least 2 times larger than the average separation between the confinement surfaces outside of the traps in the observation volume.

[0090] 4. The method of any of clauses 1-3, wherein the separation between the confinement surfaces in the traps is substantially the same for at least a majority of the traps. 5. The method of any of clauses 1-4, wherein the number of traps in the observation volume is at least 50.

[0091] 6. The method of any of clauses 1-5, wherein the liquid sample and / or the confinement surfaces in the observation volume are configured such that the residence times of the target entities are predominantly determined by non-electrostatic effects.

[0092] 7. The method of clause 6, wherein the liquid sample comprises a salt concentration configured to suppress electrostatic effects such that the residence times of the target entities are predominantly determined by non-electrostatic effects, the salt concentration optionally being at least 50mM.

[0093] 8. The method of clause 6 or 7, wherein the confinement surfaces in the observation volume are defined by an electrostatics-suppressing coating such as a polymer, such as polyethylene glycol, PEG.

[0094] 9. The method of any of clauses 1-8, wherein the sample is additionally provided in a further observation volume, the further observation volume being defined by a pair of further confinement surfaces of the reservoir containing the sample, wherein the pair of further confinement surfaces face into the further observation volume from opposite sides of the further observation volume, and wherein the further confinement surfaces define a plurality of further traps in which a separation between the further confinement surfaces is larger than an average separation between the further confinement surfaces outside of the further traps in the further observation volume.

[0095] 10. The method of clause 9, wherein the liquid sample, the confinement surfaces in the observation volume, and the further confinement surfaces in the further observation volume, are configured such that the residence times of the target entities in the traps of the observation volume are influenced by electrostatic effects to a different extent than the residence times of the target entities in the further traps of the further observation volume.

[0096] 11. The method of clause 10, wherein: the residence times of the target entities in the traps of the observation volume are predominantly determined by non-electrostatic effects; and / or the residence times of the target entities in the further traps of the further observation volume are predominantly determined by electrostatic effects. 12. The method of clause 10 or 11, wherein: the average separation between the confinement surfaces in the observation volume is larger than the average separation between the further confinement surfaces in the further observation volume; and the liquid sample comprises a salt concentration configured to suppress electrostatic effects such that the residence times of the target entities are predominantly determined by non-electrostatic effects in the traps of the observation volume and are predominantly determined by electrostatic effects in the further traps of the further observation volume, optionally the salt concentration being less than about 45mM.

[0097] 13. The method of any of clauses 10 to 12, wherein the confinement surfaces of the observation volume have a different surface composition compared to the further confinement surfaces of the further observation volume.

[0098] 14. The method of clause 13, wherein the surface compositions are configured such that the residence times of the target entities are predominantly determined by nonelectrostatic effects in the traps of the observation volume and are predominantly determined by electrostatic effects in the further traps of the further observation volume.

[0099] 15. The method of clause 13 or 14, wherein the surface composition of the confinement surfaces of the observation volume comprises an electrostatics-suppressing coating such as a polymer, optionally polyethylene glycol, PEG.

[0100] 16. The method of any of clauses 9 to 15, wherein the observation volume and the further observation volume are fluidically interconnected.

[0101] 17. The method of any of clauses 9 to 16, wherein the method comprises: obtaining information about residence times of the target entities in the further traps; and determining information about the target entities from the obtained information about residence times of the target entities in the further traps, wherein the information about the target entities determined from the obtained information about residence times of the target entities in the further traps is different from the information about the target entities determined from the obtained information about residence times of the target entities in the traps.

[0102] 18. The method of any of clauses 9 to 17, wherein: the confinement surfaces and the further confinement surfaces are substantially planar and / or parallel to each other; and / or a plurality of the further observation volumes are provided, comprising a corresponding plurality of pairs of further confinement surfaces, the pairs having different surface compositions relative to each other and / or different average separations outside of the further traps relative to each other.

[0103] 19. The method of any of clauses 1-18, wherein the maximum dimension of the target entity is defined as the diameter of the smallest notional bounding sphere that can contain the target entity.

[0104] 20. The method of any of clauses 1-19, wherein an aspect ratio of the target entity is at least 2.

[0105] 21. The method of any of clauses 1-20, wherein the target entity comprises one or more of the following: an elongate molecule, such as a DNA molecule or portion of a DNA molecule; a protein molecule or portion of a protein molecule; an RNA molecule or a portion of an RNA molecule; a lipid nanoparticle; a virus; a biological molecule; a colloidal particle; a nanoparticle; a quantum dot; and a polymer.

[0106] 22. The method of any of clauses 1-21, wherein the obtaining information about residence times comprises: obtaining image data or video data by performing optical measurements through one or both of the confinement surfaces and: analysing the obtained image data or video data to measure residence times; and / or visually inspecting the obtained image or video data to obtain information about residence times; and / or visually inspecting the observation volume through one of the confinement surfaces.

[0107] 23. The method of any of clauses 1-22, wherein the obtaining information about residence times comprises determining a proportion of the traps that contain a target entity in an image, in a plurality of images, and / or during a predetermined observation time, optionally such that the determination of information about the target entities from the obtained information about residence times comprises determining a measure of a number or concentration of target entities in the sample that have a characteristic that promotes trapping of the target entities in the traps.

[0108] 24. The method of any of clauses 1-23, wherein the target entities are configured to fluoresce.

[0109] 25. The method of any of clauses 22 to 24, wherein the obtaining information about the residence times comprises simultaneously observing at least 50 of the traps.

[0110] 26. The method of any of clauses 1-25, wherein the determining information about the target entities comprises determining information about the size and / or shape of the target entities based on the obtained information about the residence times.

[0111] 27. The method of clause 26, wherein the determining information comprises detecting changes in size and / or shape of the target entities and / or detecting the size and / or shape of the target entities.

[0112] 28. The method of clause 26 or 27, comprising distinguishing between two or more different types of target entity based predominantly on differences in shape between the two or more different types of target entity.

[0113] 29. The method of clause 28, wherein the different types of target entity are substantially identical in respect of one or more of the following: mass, charge, and hydrodynamic radius.

[0114] 30. The method of any of clauses 1-29, wherein the sample contains first target entities and second target entities and the determining of information about the target entities comprises determining information about binding of the first target entities to the second target entities to form third target entities, optionally the determining of information comprising determining variations in an amount of the third target entity in the sample as a function of time.

[0115] 31. The method of clause 30, wherein: the first target entities and the second target entities are identical monomers; or the first target entities and the second target entities have different compositions relative to each other.

[0116] 32. The method of clause 31, wherein the determining of information comprises determining variations in an amount of the third target entity in the sample as a function of time.

[0117] 33. The method of any preceding clause, wherein the sample contains a plurality of monomers and the target entities are multimers of the monomers.

[0118] 34. The method of clause 33, wherein the determining of information comprises determining information about the size, shape and / or abundance of each of one or more multimer orders of the multimers.

[0119] 35. The method of clause 33 or 34, wherein an optical marker is attached to each of a minority of the monomers in the sample to formed labelled monomers, optionally such that a ratio of the labelled monomers to unlabelled monomers in the sample is less than 1: 103, optionally less than 1: 104.

[0120] 36. The method of any of clauses 33-35, wherein the monomers are proteins, optionally monomeric insulin.

[0121] 37. The method of any of clauses 1-36, further comprising driving flow of the sample through the observation volume.

[0122] 38. The method of any of clauses 1-37, wherein the target entity comprises a complex of an optical marker bound to a predetermined binding target of interest, optionally wherein the liquid sample contains a plurality of optical markers that are optically distinguishable from each other and configured to bind to a corresponding plurality of different binding targets.

[0123] 39. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the number of traps in the observation volume is at least 50.

[0124] 40. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample contains first target entities and second target entities and the determining of information about the target entities comprises determining information about binding of the first target entities to the second target entities to form third target entities, optionally the determining of information comprising determining variations in an amount of the third target entity in the sample as a function of time.

[0125] 41. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample contains a plurality of monomers and the target entities are multimers of the monomers.

[0126] 42. The method of clause 41, wherein the determining of information comprises determining information about the size, shape and / or abundance of each of one or more multimer orders of the multimers.

[0127] 43. The method of clause 41 or 42, wherein an optical marker is attached to each of a minority of the monomers in the sample to formed labelled monomers, optionally such that a ratio of the labelled monomers to unlabelled monomers in the sample is less than 1: 103, optionally less than 1: 104.

[0128] 44. The method of any of clauses 41-43, wherein the monomers are proteins, optionally monomeric insulin.

[0129] 45. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample is additionally provided in a further observation volume, the further observation volume being defined by a pair of further confinement surfaces of the reservoir containing the sample, wherein the pair of further confinement surfaces face into the further observation volume from opposite sides of the further observation volume, and wherein the further confinement surfaces define a plurality of further traps in which a separation between the further confinement surfaces is larger than an average separation between the further confinement surfaces outside of the further traps in the further observation volume; and the liquid sample, the confinement surfaces in the observation volume, and the further confinement surfaces in the further observation volume, are configured such that the residence times of the target entities in the traps of the observation volume are influenced by electrostatic effects to a different extent than the residence times of the target entities in the further traps of the further observation volume.

[0130] 46. The method of clause 45, wherein: the residence times of the target entities in the traps of the observation volume are predominantly determined by non-electrostatic effects; and / or the residence times of the target entities in the further traps of the further observation volume are predominantly determined by electrostatic effects.

[0131] 47. The method of clause 45 or 46, wherein: the average separation between the confinement surfaces in the observation volume is larger than the average separation between the further confinement surfaces in the further observation volume; and the liquid sample comprises a salt concentration configured to suppress electrostatic effects such that the residence times of the target entities are predominantly determined by non-electrostatic effects in the traps of the observation volume and are predominantly determined by electrostatic effects in the further traps of the further observation volume, optionally the salt concentration being less than about 45mM.

[0132] 48. The method of any of clauses 45 to 47, wherein the confinement surfaces of the observation volume have a different surface composition compared to the further confinement surfaces of the further observation volume.

[0133] 49. The method of clause 48, wherein the surface compositions are configured such that the residence times of the target entities are predominantly determined by nonelectrostatic effects in the traps of the observation volume and are predominantly determined by electrostatic effects in the further traps of the further observation volume.

[0134] 50. The method of clause 48 or 49, wherein the surface composition of the confinement surfaces of the observation volume comprises an electrostatics-suppressing coating such as a polymer, optionally polyethylene glycol, PEG.

[0135] 51. The method of any of clauses 45 to 50, wherein the observation volume and the further observation volume are fluidically interconnected. 52. The method of any of clauses 45 to 51, wherein the method comprises: obtaining information about residence times of the target entities in the further traps; and determining information about the target entities from the obtained information about residence times of the target entities in the further traps, wherein the information about the target entities determined from the obtained information about residence times of the target entities in the further traps is different from the information about the target entities determined from the obtained information about residence times of the target entities in the traps.

[0136] 53. The method of any of clauses 45 to 52, wherein: the confinement surfaces and the further confinement surfaces are substantially planar and / or parallel to each other; and / or a plurality of the further observation volumes are provided, comprising a corresponding plurality of pairs of further confinement surfaces, the pairs having different surface compositions relative to each other and / or different average separations outside of the further traps relative to each other.

[0137] Cross reference to related applications

[0138] This application claims priority from GB2401051.4 filed on 26 January 2024, the contents of which are hereby incorporated by reference.

Claims

CLAIMS1. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein a ratio of the average separation between the confinement surfaces outside of the traps in the observation volume and a maximum dimension of each target entity is equal to or less than 50.

2. The method of claim 1, wherein the confinement surfaces are substantially parallel to each other outside of the traps in the observation volume, such that the separation between the confinement surfaces is substantially uniform outside of the traps in the observation volume.

3. The method of claim 1 or 2, wherein an average separation between the confinement surfaces in the traps is at least 2 times larger than the average separation between the confinement surfaces outside of the traps in the observation volume.

4. The method of any preceding claim, wherein the separation between the confinement surfaces in the traps is substantially the same for at least a majority of the traps.

5. The method of any preceding claim, wherein the number of traps in the observation volume is at least 50.

6. The method of any preceding claim, wherein the liquid sample and / or the confinement surfaces in the observation volume are configured such that the residence times of the target entities are predominantly determined by non-electrostatic effects, wherein, optionally: the liquid sample comprises a salt concentration configured to suppress electrostatic effects such that the residence times of the target entities are predominantly determined by non-electrostatic effects, the salt concentration optionally being at least 50mM; and / or the confinement surfaces in the observation volume are defined by an electrostatics-suppressing coating such as a polymer, such as polyethylene glycol, PEG.

7. The method of any preceding claim, wherein the sample is additionally provided in a further observation volume, the further observation volume being defined by a pair of further confinement surfaces of the reservoir containing the sample, wherein the pair of further confinement surfaces face into the further observation volume from opposite sides of the further observation volume, wherein the further confinement surfaces define a plurality of further traps in which a separation between the further confinement surfaces is larger than an average separation between the further confinement surfaces outside of the further traps in the further observation volume, and wherein the observation volume and the further observation volume are optionally fluidically interconnected.

8. The method of claim 7, wherein the liquid sample, the confinement surfaces in the observation volume, and the further confinement surfaces in the further observation volume, are configured such that the residence times of the target entities in the traps of the observation volume are influenced by electrostatic effects to a different extent than the residence times of the target entities in the further traps of the further observation volume.

9. The method of claim 8, wherein: the residence times of the target entities in the traps of the observation volume are predominantly determined by non-electrostatic effects; and / or the residence times of the target entities in the further traps of the furtherobservation volume are predominantly determined by electrostatic effects.

10. The method of claim 8 or 9, wherein: the average separation between the confinement surfaces in the observation volume is larger than the average separation between the further confinement surfaces in the further observation volume; and the liquid sample comprises a salt concentration configured to suppress electrostatic effects such that the residence times of the target entities are predominantly determined by non-electrostatic effects in the traps of the observation volume and are predominantly determined by electrostatic effects in the further traps of the further observation volume, optionally the salt concentration being less than about 45mM.

11. The method of any of claims 8 to 10, wherein the confinement surfaces of the observation volume have a different surface composition compared to the further confinement surfaces of the further observation volume.

12. The method of claim 11, wherein the surface compositions are configured such that the residence times of the target entities are predominantly determined by non-electrostatic effects in the traps of the observation volume and are predominantly determined by electrostatic effects in the further traps of the further observation volume.

13. The method of claim 11 or 12, wherein the surface composition of the confinement surfaces of the observation volume comprises an electrostatics-suppressing coating such as a polymer, optionally polyethylene glycol, PEG.

14. The method of any of claims 7 to 13, wherein the method comprises: obtaining information about residence times of the target entities in the further traps; and determining information about the target entities from the obtained information about residence times of the target entities in the further traps, wherein the information about the target entities determined from the obtainedinformation about residence times of the target entities in the further traps is different from the information about the target entities determined from the obtained information about residence times of the target entities in the traps.

15. The method of any of claims 7 to 14, wherein: the confinement surfaces and the further confinement surfaces are substantially planar and / or parallel to each other; and / or a plurality of the further observation volumes are provided, comprising a corresponding plurality of pairs of further confinement surfaces, the pairs having different surface compositions relative to each other and / or different average separations outside of the further traps relative to each other.

16. The method of any preceding claim, wherein an aspect ratio of the target entity is at least 2.

17. The method of any preceding claim, wherein the obtaining information about residence times comprises: obtaining image data or video data by performing optical measurements through one or both of the confinement surfaces and: analysing the obtained image data or video data to measure residence times; and / or visually inspecting the obtained image or video data to obtain information about residence times; and / or visually inspecting the observation volume through one of the confinement surfaces.

18. The method of any preceding claim, wherein the obtaining information about residence times comprises determining a proportion of the traps that contain a target entity in an image, in a plurality of images, and / or during a predetermined observation time, optionally such that the determination of information about the target entities from the obtained information about residence times comprises determining a measure of a number or concentration of target entities in the sample that have a characteristic that promotes trapping of the target entities in the traps.

19. The method of any preceding claim, wherein the determining information about the target entities comprises determining information about the size and / or shape of the target entities based on the obtained information about the residence times, wherein, optionally: the determining information comprises detecting changes in size and / or shape of the target entities and / or detecting the size and / or shape of the target entities; the method comprises distinguishing between two or more different types of target entity based predominantly on differences in shape between the two or more different types of target entity; and / or the different types of target entity are substantially identical in respect of one or more of the following: mass, charge, and hydrodynamic radius.

20. The method of any preceding claim, wherein the sample contains first target entities and second target entities and the determining of information about the target entities comprises determining information about binding of the first target entities to the second target entities to form third target entities.

21. The method of claim 20, wherein: the first target entities and the second target entities are identical monomers; or the first target entities and the second target entities have different compositions relative to each other.

22. The method of claim 21, wherein the determining of information comprises determining variations in an amount of the third target entity in the sample as a function of time.

23. The method of any preceding claim, wherein the sample contains a plurality of monomers and the target entities are multimers of the monomers.

24. The method of claim 23, wherein the determining of information comprises determining information about the size, shape and / or abundance of each of one or more multimer orders of the multimers.

25. The method of claim 23 or 24, wherein an optical marker is attached to each of a minority of the monomers in the sample to formed labelled monomers, optionally such that a ratio of the labelled monomers to unlabelled monomers in the sample is less than 1: 103, optionally less than 1: 104.

26. The method of any of claims 23-25, wherein the monomers are proteins, optionally monomeric insulin.

27. The method of any preceding claim, further comprising driving flow of the sample through the observation volume.

28. The method of any preceding claim, wherein the target entity comprises a complex of an optical marker bound to a predetermined binding target of interest, optionally wherein the liquid sample contains a plurality of optical markers that are optically distinguishable from each other and configured to bind to a corresponding plurality of different binding targets.

29. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein:the number of traps in the observation volume is at least 50.

30. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample contains first target entities and second target entities and the determining of information about the target entities comprises determining information about binding of the first target entities to the second target entities to form third target entities, optionally the determining of information comprising determining variations in an amount of the third target entity in the sample as a function of time.

31. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample contains a plurality of monomers and the target entities are multimers of the monomers.

32. The method of claim 31, wherein the determining of information comprises determining information about the size, shape and / or abundance of each of one or more multimer orders of the multimers.

33. The method of claim 31 or 32, wherein an optical marker is attached to each of a minority of the monomers in the sample to formed labelled monomers, optionally such that a ratio of the labelled monomers to unlabelled monomers in the sample is less than 1: 103, optionally less than 1: 104.

34. The method of any of claims 31-33, wherein the monomers are proteins, optionally monomeric insulin.

35. A method of obtaining information about entities in a liquid sample, comprising: providing the sample in an observation volume, the observation volume being defined by a pair of confinement surfaces of a reservoir containing the sample, the pair of confinement surfaces facing into the observation volume from opposite sides of the observation volume, wherein the confinement surfaces define a plurality of traps in which a separation between the confinement surfaces is larger than an average separation between the confinement surfaces outside of the traps in the observation volume; obtaining information about residence times of target entities in the traps; and determining information about the target entities from the obtained information about residence times of target entities in the traps, wherein: the sample is additionally provided in a further observation volume, the further observation volume being defined by a pair of further confinement surfaces of the reservoir containing the sample, wherein the pair of further confinement surfaces face into the further observation volume from opposite sides of the further observation volume, and wherein the further confinement surfaces define a plurality of further traps in which a separation between the further confinement surfaces is larger than an average separation between the further confinement surfaces outside of the further traps in the further observation volume; and the liquid sample, the confinement surfaces in the observation volume, and thefurther confinement surfaces in the further observation volume, are configured such that the residence times of the target entities in the traps of the observation volume are influenced by electrostatic effects to a different extent than the residence times of the target entities in the further traps of the further observation volume.

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