Device for near-field molecule detection

This application claims priority from US provisional application no 63/346994 filed 30 May 2022, the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention

The present invention relates to an integrated photonic device for the near-field detection and measurement of samples, in particular biological samples.

Background

A system has previously been developed to measure molecular charge using an electrostatic fluidic trap. The theory behind this technique is described as follows. A physical structure is created comprising an array of wells (pockets). Consider a charged molecule that is placed inside an ionic solution and contained in one of the wells. It has been previously shown [1] that the interaction between surface charge on the well, the solution, and the molecule, results in an electrostatic potential landscape that can spatially confine the molecule within the well. The well therefore acts like an electrostatic trap, with the molecule preferentially residing within the trap. Under certain conditions (e.g. temperature, molecular charge, buffer conditions), the molecule will “hop” to another well. The movement of that molecule will be a combination of random diffusion and electrostatic/dynamic interaction between the molecule and the surrounding structure and solution. In essence, the charge of the molecule determines how strongly it is trapped in the structure, and hence how long one would expect the molecule to remain within a trapping structure (the resonance time). Therefore, by observing how long a molecule remains trapped, one can estimate the charge on the molecule [2],

Ensemble measurements of the position and movement of the molecule with respect to the structure can allow accurate determination of the charge of the molecule to a high accuracy. In previous systems, the molecule’s position has been measured using a standard optical microscope. However, standard microscope systems are large (typically taking up a full bench top) and relatively expensive. The large space required intrinsically limits the throughput of the technique. Further, changes in environment and temperature can cause the components to drift out of alignment. Consequently, existing microscope systems require regular realignment of optics.

Therefore, there is a need for an improved system which is more compact, stable, and reliable, while also allowing high-precision detection and characterisation of individual molecules.

The present invention has been devised in light of the above considerations. Summary of the Invention

At its most general, the present invention provides a device for detecting and measuring a sample. The device integrates a structure for holding a sample under measurement; and a light delivery mechanism for creating evanescent light to illuminate the sample.

Accordingly, in one aspect the present invention provides a device having a sample-containment layer for holding a sample; and an optical structure configured to receive light from a light source and create an evanescent light field within the sample-containment layer, to illuminate said sample held within the sample-containment layer in use; wherein the sample-containment layer and optical structure are formed as part of a single integrated structure.

The optical structure is configured to receive light from a light source. From the received light, the optical structure is configured to create (generate) evanescent illumination at the sample-containment layer. The optical structure may also be referred to herein as an “optical layer”, “illumination structure/layer” or “light delivery structure/layer”.

As used herein, the term “evanescent illumination”, “evanescence light” and so on takes its normal meaning in the art, referring to a non-propagating light field whose strength decays rapidly away from its point of origin. This may also be described as providing “near-field” illumination. In contrast, previous devices have relied on wide-field illumination, in which a propagating light field is produced that is transmitted far (e.g. many wavelengths) from the light source.

Sample contained within the device may be measured using a detector (e.g. camera or microscope). The present invention envisages two different implementations for the detector, described below as the “first implementation” and “second implementation”.

The evanescent field helps to minimise the amount of light that would leak toward the detector from the optical structure. Additionally, since the evanescent field does not propagate strongly towards the detector (and any other layers), it reduces the amount of light that is unnecessarily lost in the system. This reduces the requirements on the light source, which may consequently only be a small I low-power light source. This helps to make the device smaller, cheaper, and less power-hungry. The samplecontainment layer and optical structure are very close to each other, within the same device. Accordingly, the sample-containment layer may be located within the near-field of the optical structure, e.g. within 300 nm, optionally within 200 nm, optionally within 20 nm of the optical structure. In some cases, a larger distance can be used (e.g. up to 200 pm, optionally up to 100 pm, optionally up to 10 pm), e.g. if there are adhesives or aligning/bonding layers between the optical structure and sample-containment layer.

First implementation

According to a first implementation of the invention, there is provided an integrated photonic device for detecting molecules in a sample, the device comprising: a sample-containment layer for holding a sample; an optical structure configured to receive light from a light source and create an evanescent light field within the sample-containment layer, to illuminate said sample held within the sample-containment layer in use; and a detector configured to detect light from the sample-containment layer; wherein the sample-containment layer, optical structure, and detector form a single integrated structure. Advantageously, by providing a single device that integrates the above functionalities, the device can be used to detect/measure molecules while overcoming many of the obstacles typically encountered by large-scale microscope systems. For example, the device can be made significantly smaller (e.g. 3 to 4 orders of magnitude smaller) than a system based around the use of a typical microscope. By integrating the sample-containment layer into the same device as the detector, the sample can be brought closer to the detector, avoiding the need for lenses between the sample and detector, and meaning that each pixel detects a greater fraction of the light scattered/emitted from objects in the sample (i.e. boosting the photon collection efficiency of the system). However, integrating both the sample-containment layer and detector layer into the same device also creates challenges, which the inventors have had to overcome. In particular, including the detector layer as part of the integrated photonic device means it is not straightforward to deliver light to the sample. Illumination light cannot be sent to the sample “up” through the detector. Conversely, providing illumination light “down” onto the sample towards the detector can mean that the illumination light swamps signal from objects of interest in the sample, necessitating the use of filter layers which could act to decrease collection efficiency (due to increasing the distance from sample to detector layer) and increase manufacturing complexity and cost. Advantageously, the integrated optical device of the present invention includes an optical layer configured to illuminate the sample using near field light without requiring light to pass through the detector, limiting or avoiding the delivery of illumination light to the detector, and thereby boosting the signal to noise of the system.

Additionally, the integrated arrangement advantageously reduces the possibility of components becoming misaligned over time, and is therefore more robust than the prior art systems, since no separate imaging optics are required. Such an arrangement may also allow the detector to have a greater field of view across the sample than the wide-field arrangements of the prior art.

The integrated photonic device includes multiple layers/structures (e.g. sample-containment layer, optical structure, and detector) which are attached to one another to form a single integrated structure. The device may also be referred to herein as a “chip”, “integrated circuit”, or “monolithic integrated circuit”, or “photonic integrated circuit” (PIC). These terms take their normal meaning in the art, relating to a monolithic structure having circuitry integrated with a substantially flat piece of material (e.g. semiconductor material).

The detector may comprise any suitable optical sensor for detecting light from the sample-containment layer. For example, the detector may comprise an image sensor, such as a (lensless) CMOS or CCD detector. Advantageously, CMOS or CCD detectors can be easily miniaturised and are relatively inexpensive. Accordingly, such detectors can be made cheaply and reliably, e.g. as part of a disposable photonic device. The detector may also be referred to herein as a “detector layer”, “sensor”, or “sensor layer”.

The various layers of the device may be attached in any suitable manner. For example, two or more layers of the device may be attached to each other by direct chemical deposition, one or more adhesive layers, mechanical locking, anodic bonding, plasma binding, or any other means. The various layers (e.g. sample-containment layer and/or optical structure) may be formed directly on the detector (e.g. by chemical deposition), or may be formed separately and subsequently attached together (e.g. by an adhesive layer). Suitably, samples suitable for use with the integrated photonic device of the invention are provided in the form of a liquid. The sample contains one or more objects of interest, to be detected and measured.

The sample may be, for example, a liquid containing a molecule of interest. For example, the molecule may be a biomolecule, such as a protein, a nucleotide, a peptide, DNA (either single or double stranded), RNA (either single or double stranded), a carbohydrate, a lipid, or a metabolite.

Optionally, the molecule may be an individual molecule.

The sample may be, for example, a liquid containing an assembly of atoms or molecules. For example, the object of interest may be a complex, an aggregate, a particle, a micelle, a vesicle, a liposome. Suitably particles may be, for example, polymeric particles, metal particles (e.g. gold particles), quantum dots or magnetic particles.

Alternatively, the object of interest may be a cell or a virion.

The object of interest may be detectable by fluorescence. The object of interest may inherently be fluorescent, e.g. due to having one or more inherent fluorophores. Additionally or alternatively, the object of interest may be labelled with one or more fluorophores. The object of interest may be a fluorophore (i.e. a fluorescent molecule), or may be a non-fluorescent molecule that is tagged with a fluorescent marker. After being illuminated, fluorescent molecules/markers will re-emit fluorescence, which can then be detected by the detector. Advantageously, fluorescence imaging helps to distinguish the light which is to be detected (fluorescent light) from the light which is for illuminating the sample, since these are at different wavelengths due to the Stokes shift of the emitted light. The integrated photonic device may include an optical filter layer, to preferentially allow transmittance of the fluorescence emission instead of the illumination light.

The object of interest may be detectable by scattering. For example, in some embodiments, the detector may instead detect a particle by virtue of its absorption or scattering of light. The device may include an optical filter to assist with this detection, as discussed further below.

Second implementation

In another implementation of the first aspect of the present invention, the device may be part of a system for detecting and measuring a sample. The system may comprise:

(i) a sample holder, comprising: a. a sample-containment layer for holding a sample; and b. an optical structure configured to receive light from a light source and create an evanescent light field within the sample-containment layer, to illuminate said sample held within the sample-containment layer in use; wherein the sample-containment layer and optical structure form a single integrated structure; and

(ii) a microscope, configured to detect light from the wells of the sample-containment layer.

Thus, this second implementation differs from the first in that the integrated detector of the first implementation is replaced by a microscope configured to detect light from the sample-containment layer. This implementation retains the benefits of being able to probe the sample using an evanescent light field, whilst allowing use with conventional microscopy equipment.

The sample-containment layer and optical structure in the second implementation may have any of the optional or preferred features of the first implementation (discussed below). In particular, it may comprise a one-dimensional waveguide or two-dimensional waveguide, such as a plurality of apertures sized to create an evanescent light field.

The microscope may comprise, for example, a lens to collect light from the sample-containment layer, a detector (such as an CCD camera, or CMOS camera), imaging optics to direct collected light to the detector, and optionally one or more optical filters.

The features discussed below may be provided in combination with either the first and/or second implementation, unless discussed otherwise.

Optionally (in the first and/or second implementation), the sample-containment layer comprises an array of fluidically-interconnected wells configured to electrostatically trap molecules of the sample in use.

The array of wells may also be referred to as a “confinement zone", “measurement zone” or “microfluidic zone” of the sample-containment layer. The array of wells are fluidically interconnected to enable a molecule from the sample to travel (or “hop") from one well to another. Each well functions as an electrostatic fluidic trap, in a similar manner as described above with respect to the prior art. The time that a molecule spends in a given well can therefore be measured in order to conduct an assay on the molecule (e.g. to determine its molecular charge).

In the first implementation, the detector may have an array of pixels configured to detect light from the wells. Since the wells are integrated into the same device as the optical structure and detector, the depth of the sample-containment layer (e.g. the depth of the wells) can be configured to bring the molecules even closer to the detector than in the prior art, thereby further improving the device’s ability to detect molecules of the sample. Further, due to the integrated arrangement of the device, the wells can also be located much closer together than in the prior art, and the detector can still resolve those wells from each other. Therefore, the space required for performing an assay on a single molecule (i.e. the size of a single array of wells) can be significantly smaller than in the prior art. This can also be referred to herein as the device having a higher “measurement density” than the devices of the prior art.

The wells may alternatively be referred to herein as “pockets", “microfluidic wells”, “electrostatic traps”, “electrostatic fluidic traps”. The device may comprise one or more microfluidic passageways (microfluidic channels/layers/regions) which interconnect the wells. The dimensions of the microfluidic passageways (including the wells) may be configured to balance several factors, e.g, the properties of the sample (e.g. its ionic charge / pH, viscosity, temperature); any coatings or passivation used in the channels; the pressures in the channels and around the device; the rates for capillary action; and the molecular specimen being quantified (charge, size, weight/mass).

The sample-containment layer may comprise a substrate with a trapping layer provided thereon, the trapping layer having a series of walls defining the sides of the wells. The substrate may form a base of the sample-containment layer. The substrate is preferably transparent to enable light from within the well (e.g. emitted from a fluorophore within the well) to travel towards the detector. For example, the substrate may be formed of glass.

The trapping layer may be formed of a semiconductor material (e.g. silicon) and may be formed directly on the substrate, e.g. by silicon lithography. The trapping layer may take the form of a grid of intersecting walls, positioned on the substrate. The grid may have any suitable shape, but is preferably provided such that the intersecting walls form a square or rectangular grid (akin to a grate, gridiron or waffle).

The array of wells may be defined at an upper boundary thereof by a further transparent substrate (e.g. a glass slide), or by the optical structure itself (which can therefore be directly in contact with the sample).

As used herein, vertical directions such as “upper” and “lower” may be used to refer to a direction which is perpendicular to the plane of the detector. An “upper” direction may refer to a region that is away from the detector, while a “lower” (“base”, etc) direction may refer to a region that is closer to the detector. Further, the term “lateral” may refer to regions across a plane of any layer of the device. The lateral directions are perpendicular to the vertical (upper and lower) directions.

The wells are fluidically-interconnected to enable the molecule to travel between wells within a given confinement zone (array of wells). However, the trapping layer may also comprise walls at its lateral edge(s) which extend fully from the base substrate to an upper boundary of the microfluidic channel, in order to retain the sample within the array of wells.

The array of pixels of the detector (under the first implementation) and/or the microscope (under the second implementation) may be configured in any suitable manner to detect light from the array of wells. For example, under the first implementation, the wells may correspond in shape and/or size to the pixels of the detector. The plurality of wells may be arranged in a rectangular (e.g. square) array, to align with a corresponding rectangular (e.g. square) array of pixels of the detector. The lateral dimensions of the sample containment layer may correspond to (e.g. match) the lateral dimensions of the detector. Each well may have a rectangular (e.g. square) shape. Each well may be dimensioned to have a size that is less than the pixel size of the detector. For example, optionally, each pixel may have a width (measured laterally across the pixel) of at least 1 pm up to 10 pm. For example, each pixel may have a width of at least 2 pm up to 9 pm, optionally at least 3 pm up to 8 pm, optionally at least 4 pm up to 7 pm.

The size of each well may be chosen to find a compromise between the amount of light collected by the pixels below the well, and the required measurement size (confinement zone) density. The depth of the wells can be chosen to bring a molecule closer to the detector.

The well width w, well depth D, and microfluidic channel depth S can be chosen to introduce a particular level of confinement, e.g. depending on the size of a particle of interest. For example, the width w and/or depth D of each well can be at least 1 nm up to 10 pm. For example, the well width w and/or well depth D may be at least 2 nm, optionally at least 10 nm, optionally at least 50 nm, optionally at least 100 nm, optionally at least 1 pm, optionally at least 5 pm. The well width w and/or well depth D may be up to 10 pm, optionally up to 8 pm, optionally up to 5 pm, optionally up to 3 pm. Preferably, the well width w is greater than the well depth D. By reducing the well depth, the particle can be brought closer to the detector. Subtracting the well depth D from the microfluidic channel depth S gives the depth h of the channels (or “slit”) between wells, i.e. S = D + h. Optionally, this inter-well channel depth h may be at least 5 nm up to 20 pm. This is advantageous because these dimensions are large enough to allow particles to move between wells (through the channels), whilst not being so large as to affect the trapping capabilities of the wells. Similarly to the dimensions w, D, and S, the inter-well channel depth h may be selected in dependence on the properties (in particular the size) of the objects of interest to be trapped. For example, larger dimensions may be appropriate in the case of trapping polymer beads compared to trapping of individual molecules. Optionally, the depth h may be at least 10 nm up to 15 pm, optionally at least 20 nm up to 10 pm, optionally at least 50 nm up to 1 pm, optionally at least 100 nm up to 500 nm.

Suitably, the array of pixels are configured to be registered with (overlap/align with) the array of wells. For example, the array of pixels may have a pattern having a periodicity that corresponds with (e.g. is equal to or is a fraction/multiple of) a periodicity of a pattern of the wells.

Optionally, underthe first implementation, each well of the sample-containment layer is aligned with a respective pixel of the detector. By aligning each well with a respective pixel, the photons from a single well (e g. a single fluorophore within the well) can be collected predominantly by a single pixel. Since the wells are aligned with respective pixels, only the illumination of individual pixels needs to be measured. This helps improve the resolution compared to the prior art, since it is not essential for the detector to have a good spatial resolution in order to detect a target molecule. Instead, the detector only requires single-pixel resolution, since each pixel corresponds to a single well. Therefore, the detector may be significantly cheaper than the detectors in conventional devices.

Optionally, each well may be located in a centre of a respective pixel.

In variant embodiments, which are not illustrated, the array of pixels may be positioned differently with respect to the array of wells. For example, a well could be registered (aligned) with a plurality of pixels, i.e. so that a plurality of pixels can detect the light from said well, for redundancy. Alternatively, a pixel could be registered (aligned) with a plurality of wells. For example, the device may comprise a 4x4 array of pixels that overlap with an 8x8 array of wells, with multiple wells per pixel. The pixels may therefore be used in combination to infer changes in well location, by detecting changes in the distribution of the detected signal (e.g. changes in the centre of mass of the detected signal across the pixels).

During manufacture, the wells may be aligned with respective pixels using any suitable method. For example, suitable alignment techniques include using microscopes, alignment features, precision alignment stages, and computer vision systems.

The sample may be injected or extracted from the sample-containment layer in any suitable manner. Optionally, the sample-containment layer further comprises a fluid channel having an opening (aperture, hole, port) for transferring (injecting or extracting) the sample into or from the device.

Optionally, the detector may have one or more pixels that are not aligned with (overlapping with, registered with) a well. For example, one or more pixels may instead be vertically aligned with a fluid port or channel. Such pixels may optionally be disregarded in measurements, or may optionally be used to perform system diagnostics such as flow detection. The fluid channel may extend into or across the sample-containment layer in any suitable manner.

Optionally, the fluid channel extends vertically into the array of wells (e.g. aligned with a single pixel of the detector, if present). Alternatively, the fluid channel may extend laterally alongside the array of wells.

This can allow the fluid channel to be in communication with a plurality of wells at once, thereby improving fluid flow to/from the wells.

Optionally, the opening of the fluid channel is located at a lateral edge of the sample-containment layer (e g. a lateral edge of the integrated photonic device). This enables convenient connection of the fluid channel to an external fluid line.

Optionally, the opening of the fluid channel extends vertically through one or more layers of the photonic device. The opening may be located vertically above the sample-containment layer. A vertical opening facilitates convenient (e.g. top-down) injection or extraction of fluid to/from the device. Optionally, the optical structure may be located above the sample-containment layer, and the opening may be formed within the optical structure. The fluid channel may then extend vertically downwards directly into the array of wells, or may extend laterally alongside the array of wells.

Optionally, a fluid manifold layer may be arranged in fluid connection with the vertical opening. The fluid manifold layer may extend across an upper layer (e.g. uppermost layer) of the integrated photonic device, e.g. above the optical structure, to direct fluid towards the array of wells.

Optionally, the sample-containment layer comprises a plurality of said fluid channels. The plurality of channels may thus be used to provide different functionalities. For example, one fluid channel may be used for the injection of a sample (and may thus be referred to as a “fluid injection channel”) whereas another fluid channel may be used for the extraction of the sample after measurements are complete (and may thus be referred to as a “fluid extraction channel”). The channels may also be useful to permit washing I cleaning of the confinement zone for reuse.

The plurality of fluid channels may have the same or different configurations. For example, two channels may each extend laterally across the device, or one channel may extend laterally while the other channel extends vertically.

Optionally, the sample-containment layer comprises a plurality of confinement zones, each confinement zone having an array of said fluidically-interconnected wells configured to electrostatically trap molecules of a sample. This enables a single device to be used for conducting multiple assays, optionally in parallel. Since the array of wells within a given confinement zone are fluidically interconnected with each other, a molecule can move between multiple wells within an individual confinement zone. The resonance time within a well can therefore be measured to perform an assay on a sample in that specific zone.

Accordingly, the sample-containment layer may comprise a first confinement zone and a second confinement zone, wherein the first confinement zone comprises a first array of wells which are fluidically interconnected with each other, and the second confinement zone comprises a second array of wells which are fluidically interconnected with each other. Preferably, the confinement zones are separated by a boundary (partition; fluid barrier) that is configured to inhibit a molecule that is within a first well in a first confinement zone travelling directly to a second well in a second confinement zone. Therefore, each confinement zone represents an independent region for measuring molecule resonance time. For example, the boundary may be formed by a wall which forms a fluid barrier between the two confinement zones. Therefore, in such embodiments, there is no direct fluid connection between wells of different confinement zones. Rather, the wells are only fluidically connected by an indirect / convoluted path which has a greater pathlength than the direct distance between those wells.

Each confinement zone (and thus each array of wells, and each well) may be configured in any suitable manner, as discussed herein. The confinement zones may have the same or different configurations to each other.

Optionally, the plurality of confinement zones are fluidically isolated from each other. Thus, the sample in a first confinement zone is prevented from flowing into a second confinement zone, and vice versa. This enables measurements of different species, or different ionic solution conditions, in parallel, and without cross-contamination between different confinement zones. In such embodiments, each confinement zone may have its own dedicated fluid channel(s).

Alternatively, the plurality of confinement zones may be fluidically connected by one or more fluid channels. A given fluid channel can therefore be used to inject the same sample into multiple confinement zones, thereby enabling multiple measurements of a single sample to be conducted in parallel without requiring each confinement zone to have its own dedicated fluid channel. The fluid channel is therefore a fluid pathway connecting wells of different confinement zones. However, a given molecule is still prevented from directly travelling (hopping) from a well in a first confinement zone to a well in a second confinement zone, due to the length of this fluid pathway and fluid barriers (partitions, walls) which separate adjacent wells that are in different zones.

In embodiments having multiple confinement zones, the optical structure may be configured to create (generate) evanescent light at wells of each of the confinement zones, and the detector may be configured to detect light from wells of each of the confinement zones.

The optical structure may be configured in any suitable manner to receive light from a light source (which may be external from the photonic device), and create an evanescent field from the received light at the sample-containment layer. In some embodiments, the optical structure may be located above the sample-containment layer, such that the sample-containment layer is positioned between the optical structure and the detector. In other embodiments, the optical structure may be located below the samplecontainment layer, e.g. as a waveguide sandwiched between the sample-containment layer and the detector.

Optionally, the integrated photonic device may include the light source for transmitting light to the optical structure. The light source may be integrated into the photonic device, e.g. at a lateral or upper surface thereof. The light source may comprise a laser or LED.

Optionally, the optical structure comprises a waveguide. A waveguide enables illumination by total internal reflection fluorescence (TIRF) microscopy. As the light travels through the waveguide, the waveguide causes the light to generate evanescent fields at the wells. Advantageously, light can be “piped” into the waveguide side-on, from a light source at a lateral edge of the device, thereby minimising the amount of light entering the detectors directly from the light source. For example, light can be injected into the waveguide by fibre coupling or by butt coupling a laser diode directly to a lateral edge of the integrated photonic device. Optionally, the device may further comprise a beam-dump (beam trap, beam stop) for absorbing any remaining light that reaches a terminal end of the waveguide.

The waveguide may comprise two or more layers of different refractive indices. In particular, the waveguide may comprise a core (or “primary”) layer and one or more cladding (or “secondary”) layers. As used herein, the term “core layer” refers to the layer within which light is “piped”, while the term “cladding layer” refers to an adjacent layer which has a lower refractive index than a refractive index of the core layer, in order to ensure that light is guided through the core layer.

In some embodiments, the waveguide may comprise two cladding layers, with the core layer being sandwiched between the two cladding layers. The refractive indices of the two cladding layers may be equal or different to each other.

The refractive index of the core and cladding layers will determine the penetration depth of the evanescent wave. Accordingly, the refractive indices and the distance between the optical structure and sample can be selected to ensure good illumination of the sample.

In variant embodiments, the waveguide may comprise only a single cladding layer, such that the core layer is not sandwiched between two layers. For example, the waveguide may comprise a core layer which is located below the cladding layer, with the core layer being directly in contact with the confinement zone.

The dimensions and materials of the waveguide may be configured in various different manners, e.g. to alter the strength of the evanescent field. Increasing the strength of the evanescent field may help ensure that the light is sufficient to reach all molecules that are trapped in wells, and excite them (if fluorescent).

For example, suitable materials for the core layer may include: silicon nitride (SislSk, which has a refractive index n of 2.037, 2.021 , or 2.007 at 488 nm, 561 nm, and 660 nm respectively); tantalum pentoxide (Ta2Os, which has a refractive index of approximately 2.12); or titanium dioxide (TiCh, which has a refractive index of approximately 2.6). A suitable material for the cladding may be silicon dioxide (SiO2, which has a refractive index n of 1 .482, 1 .479, or 1 .475 at 488 nm, 561 nm, and 660 nm respectively). Optionally, the waveguide may be formed on a substrate, e.g. a silicon-on-insulator (SOI) substrate. The core may be SisN4 and the cladding may be SiOz.

Optionally, the optical structure may comprise a metallisation layer (e.g. aluminium or a metal alloy) for light field enhancement.

Optionally, the waveguide may be silicone oxynitride (SiON) waveguide. Such a waveguide may be formed from SiO2 with SisiX dielectrics (which have refractive indices of 1 .45 and 2.01 respectively). A SiON film may be deposited on the dielectrics, e.g. using plasma-enhanced CVD. For example, the SisN4 may have a thickness of 150 nm, and may be ribbed to provide monomode illumination. This arrangement may provide an evanescent field having a penetration depth of approximately 150 nm, and may have propagation losses along the waveguide of less than 0.2 dB/cm.

Optionally, the waveguide may be configured as a single mode waveguide (e.g. a rib waveguide). This may help provide uniform illumination to the sample. Alternatively, the waveguide may be a multimode waveguide. Any interference may then be averaged out of the exposure time (or sequence of measurements) of the detector. The waveguide may be configured in any suitable manner to illuminate each of the wells. For example, the waveguide may be configured as a one-dimensional waveguide. This may also be referred to herein as a slab waveguide or planar waveguide. A one-dimensional waveguide can confine light in one direction. The one-dimensional (slab/planar) waveguide may fill a substantially continuous (whole, uninterrupted) planar area that corresponds to (e.g. covers) a majority (e.g. substantially an entirety) of the sample-containment layer. For example, the waveguide may be a square or rectangular shape, extending entirely over the array(s) of wells. The waveguide may have some discontinuities e.g. to include openings that provide fluid inlets/outlets vertically through the waveguide, while still covering a remaining majority of the sample-containment layer. The one-dimensional (slab/planar) waveguide may be configured to provide even illumination across the sample containment layer.

Alternatively, the waveguide may be configured as a two-dimensional waveguide. A two-dimensional waveguide can confine light in two directions. For example, the two-dimensional waveguide may be a channel waveguide (also referred to as a strip waveguide), rib waveguide, or ridge waveguide. The two- dimensional waveguide can be significantly smaller than a one-dimensional waveguide, since the channel waveguide can be configured to cover a smaller portion of the device. The two-dimensional waveguide may be configured to illuminate the sample-containment layer in an uneven manner. For example, the two-dimensional waveguide may follow a non-linear (e.g. serpentine) path (pattern/shape) across the sample-containment layer.

In embodiments where the sample-containment layer comprises wells, the waveguide may be aligned with the wells in any suitable manner. For example, each well may be directly aligned with the waveguide, i.e. so that a given region of the waveguide illuminates only a single well. Alternatively, a given region of the waveguide may be configured to illuminate two wells at once. For example, each well may be laterally offset from the waveguide, e.g. with the waveguide being equidistant between two adjacent wells. This arrangement can allow the lateral spacing between adjacent portions of the waveguide to be increased. This enables a bend radius of the waveguide (e.g. a two-dimensional waveguide following a serpentine path) to be increased, which in turn reduces light losses at the bends of the waveguide.

In variant embodiments, the optical structure might not be configured as a waveguide. For example, optionally, the optical structure comprises a plurality of apertures sized to create the evanescent light field. In order to create the evanescent light field, the apertures have a size that is less than the wavelength of light received from the light source. The apertures may also be referred to as “subwavelength apertures”, “near-field apertures”, or “nanoapertures”. This arrangement enables top-down illumination of the photonic device, since a light source may be positioned above the optical structure. The intensity of light transmitted through a subwavelength aperture falls off with distance from the aperture [3][4][5]. A volume around each aperture (e.g. at a substrate of the optical structure) will not create evanescent light. Therefore the light source may be positioned above the optical structure, without in turn causing excessive illumination at the detector.

The sub-wavelength apertures may be created in several ways. The optical structure may include a substrate (e.g. a transparent and/or dielectric substrate) and a metallised layer (e.g. gold plating) formed on the substrate. The apertures may be formed vertically through the metallised layer. The metallised layer may be plated directly on the substrate, and may be etched to form the sub-wavelength apertures. Alternatively, lithographic techniques may be used to create the apertures. The apertures may have a diameter and/or depth optionally 10000 nm or less, optionally 1000 nm or less, optionally 800 nm or less, optionally 500 nm or less, optionally 100 nm or less, optionally 10 nm or less, optionally 1 nm.

Each sub-wavelength aperture may be aligned (e.g. directly aligned) with a respective well.

The thickness of the substrate and the diameter of the apertures can be tuned to optimize the intensity of light in the well.

Optionally, the device further comprises a transparent layer (e.g. glass slide) which separates the optical structure from the confinement zone. This can be particularly useful, for example, in combination with an optical structure that comprises sub-wavelength apertures, in order to prevent the sample from entering the apertures.

Alternatively, in some embodiments, there is no additional physical barrier (e.g. glass slide) separating the optical structure from the sample. Therefore, optionally, the optical structure is configured to directly contact the sample within the sample containment layer. This arrangement helps to improve the coupling of the evanescent field into the sample.

Optionally, the optical structure may include a liquid crystal. A liquid crystal may help to control the extent to which the evanescent field extends into the sample, and may help to direct emission light (from a fluorescent sample) to the detector. The liquid crystal may be incorporated into the waveguide, e.g. as a cladding layer of the waveguide.

Optionally, the device may further include an optical filter layer to filter light before it reaches the detector. Optionally, the device may include a mirror (e.g. polychroic mirror) to reflect light before it reaches the detector. The filter and/or mirror help to reduce noise at the detector. The filter and/or mirror may be provided between the optical structure and the detector, and/or between the sample-containment layer and the detector. The optical filter layer (which may also be referred to herein simply as an “optical filter”) may be configured to (at least partially) filter light between the optical structure and the detector. For example, the optical filter may comprise a single or multi band pass/stop filter, short pass filter, or long pass filter. The optical filter may be positioned between the sample-containment layer and the detector. The optical filter may be configured in any suitable manner (based on the wavelength of the light source and the molecule being assessed) in order to improve the detection of the molecule.

For example, the optical filter may be configured to (at least partially) prevent light from the illumination optics (light source and/or optical structure) reaching the detector. This helps reduce noise so that the detector can better detect the molecule itself. This is particularly useful for arrangements detecting the fluorescence of a molecule/marker.

Alternatively, the optical filter may be configured to allow light from the illumination optics to reach the detector (or, the device may have no optical filter at all). This is particularly advantageous for using the device to detect the shadow or absence of light that is caused by the molecule.

Alternatively, in some embodiments, the device is configured to detect the interference between scattered light from the object of interest and transmitted (coherent) light. The optical filter may therefore be configured to reduce the amount of transmitted light reaching the detector. Optionally, the system includes an optical trap (optical tweezer), configured to provide an attractive or repulsive force on an object held in the sample containment layer by virtue of difference in the relative refractive index between the object and surrounding medium. Such an “optical trap" may alternatively be referred to as an optical tweezer, optoelectronic tweezer, or “OET”. Optical traps enable non-invasive, optically controlled manipulation of objects, including cells and particles.

In such embodiments, the optical trap is preferably implemented as part of the integrated photonic device. For example, the integrated photonic device can include one or more layers to achieve optical trapping of the object of interest. The one or more layers may be referred to as an optical trapping structure.

In use, as light passes through the optical trapping structure, the optical trapping structure creates a localised electric field which generates a force on the object of interest in the sample containment layer (e.g. in a well of the sample containment layer). The force induced by the optical trapping structure can be in a direction opposite to the electrostatic trapping force from well confinement. In order to measure the object of interest, the electric field at the optical trapping layer can be varied until it reaches a point where the object of interest is released from the confinement of the well. When the object of interest is lifted from the well, the signal at the detector decreases. The energy required to lift the object of interest from the well confinement is proportional to the charge of the particle and the electrostatic potential of the well. The electrostatic potential of the well is dependent on the dimensions of the confinement and an ionic buffer on which the particle is suspended.

By measuring the energy required to lift the object from the confinement of the well, the object’s charge can be calculated. The method of measuring charge by using an optical trapping layer is different to that which is done in prior art, which instead uses 3D Brownian simulations of the escape of the particle from the well. By using an optical trapping layer, the measurements in this embodiment are advantageously faster than those of the prior art.

The optical trapping structure may have any suitable configuration. For example, in one implementation the optical trap is configured to hold a sample over a photoconductive layer, where the sample and photoconductive layer are sandwiched between two electrode layers. The illumination of the photoconductive layer with a patterned light (e.g. provided by a Digital Micromirror Device, of “DMD”) generates changes in the conductivity within the photoconductive layer. When an alternating current (AC) bias is applied between the first electrode layer and the second electrode layer, this results in changes in the electric field between the non-illuminated regions of the photoconductive layer and the illuminated regions of the photoconductive layer. In other words, gradients in the electric field are generated which apply a force to the sample for moving the sample. The illuminated regions of the photoconductive layer are sometimes called virtual electrodes in view of the above functioning of the OETs.

To achieve this implementation, the optical trapping structure may comprise a first electrode layer; a second electrode layer; and a transparent or semi-transparent photoconductive layer positioned between the first electrode layer and the second electrode layer. The sample-containment layer may be sandwiched between the electrode layers.

Alternatively, the optical trapping structure may be positioned between the sample-containment layer and the optical structure, so that the sample-containment layer is positioned on only one side of the optical trapping structure. In other words, such a device may have an optical tweezer comprising a first electrode layer; a second electrode layer; and a transparent or semi-transparent layer sandwiched between the first electrode layer and the second electrode layer, wherein the sample containment layer is positioned on (only) one side of the optical tweezer, e.g. on one side of the second electrode layer.

The inventors have recognized that, whilst positioning the sample such that it is not sandwiched between the electrode layers of the OET improves versatility, this positioning limits the magnitude of the DEP force that can be exerted on the sample. To address this finding, in some embodiments, one or both of the electrodes or electrode layers of the optical trapping structure are formed of a conductive material having one or more non-conductive regions. When the photoconductive layer is illuminated, the change between conductive and non-conductive regions in the electrode layer(s) enable the exertion of a greater DEP force on the sample.

The inventors have further recognized that the force applied to the sample can be increased if the first electrode layer and/or the second electrode layer is patterned by providing electrically conductive and electrically nonconductive regions. The electrically conductive region(s) and the electrically nonconductive region(s) can form the first and/or second electrode layers, respectively. For example, the electrically conductive region(s) and the electrically nonconductive region(s) may have the same thickness which corresponds to the thickness of the respective electrode layer.

The first electrode layer and/or the second electrode layer each include an array of electrically nonconductive regions in a matrix of electrically conductive material. The electrically nonconductive regions and/or the electrically conductive region can form an array or grid having various configurations. For example, the conductive regions and/or the non-conductive regions have a square, rectangular, or circular shape in a top view (a view perpendicular to the second external surface). The conductive regions and/or the non-conductive regions may have a periodicity or pitch in a first direction and/or a second direction perpendicular to the first direction (the first and/or second directions extend within the electrode layers). The periodicity or pitch may be in the range between 0.5 pm to 500 pm, optionally between 5 pm to 20 pm. In other words, a distance between two periodically arranged conductive regions and/or the non-conductive regions may be 5 pm, 10 pm, or 15 pm. A width, breath, or diameter of a conductive region and/or the non-conductive region may be in the range between 0.1 pm to 1000 pm, optionally between 1 pm to 15 pm, e.g. 1 pm, 2 pm, 3 pm, 5pm, 10 pm. The conductive regions and/or the nonconductive regions may have the shape of lines or a grid of lines.

The photoconductive layer as described herein can be transparent or semi-transparent. In some embodiments, “transparent” means at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.5%, at least 99.8%, or 100% of the intensity of impinging light passes the component described as transparent. The term “transparent” may relate to a particular wavelength or wavelength range (e.g. visible light and/or infrared light). For example, transparent (e.g. as described above) can refer to transmittance in the range between 200 nm and 2500 nm and/or in one or more sub-ranges (e.g. having width of 50 nm, 100 nm, 150 nm, 200 nm) within the range between 200 nm to 2500 nm. In some embodiments, the above optional definitions of “transparent” equally apply for the term “semi-transparent” except that “semi-transparent” means at least 20%, at least 25%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65, or at least 70% of the intensity of impinging light passes the component described as semi-transparent. Preferably, the optical trapping structure itself is transparent.

The term “non-conducting” as used herein refers to materials that are electrically non-conductive but optionally includes materials that are less conductive than the material of the materials described as conductive. So, materials that are described as “non-conducting” or “non-conductive” can be electrically conductive however less conductive that materials that are described as “conductive” or “conducting”. For example, the conductivity of “non-conductive” materials is 5%, 10%, 20%, 30%, 40%, or 50% of the conductivity of the materials described as conductive. The term “electrically conductive” may be understood as describing a material, portion, and/or component that is electrically more conductive as a material, portion, and/or component that is described as “electrically non-conducting”.

Preferably, the system may include an optical trapping light source (which may also be referred to as an optical tweezer light source) for the optical trapping structure (optical tweezers), and an excitation light source for generating the evanescent field. Preferably, the optical trapping light source and the excitation light source are at different wavelengths. Preferably, the optical trapping light source provides a patterned illumination, e.g. through the use of a DMD. For example, in instances where the optical structure is in the form of a waveguide, the excitation light source may provide light into one end of the waveguide, and the optical trapping light source may be provided in a direction orthogonal to the waveguide.

Devices utilising optical trapping structures may have an optical structure comprising a waveguide or subwavelength apertures, as discussed above.

Further details of suitable optical trapping layers may be found in GB patent application number 2304864.8, which is incorporated herein by reference in its entirety.

In view of the advantages above, the present invention also provides a method of manipulating a sample comprising providing a sample to the sample containment layer of the integrated photonic device of the invention, wherein the sample containment layer comprises said polarity of fluidically-interconnected wells, and manipulating an object of interest (e.g. a molecule or particle) in at least one of said wells using an optical tweezer. Optionally, said manipulation may be using the optical tweezer to hold the object in said well. Alternatively, said manipulation may be using the optical tweezer to push the object from the well. The settings of the optical tweezers required to hold/move the object in the well may be used to determine characteristics of the object and/or the well. For example, the force applied may be used to characterise the electrostatic potential of the wells.

Any of the layers discussed herein (e.g. sample-containment layer, optical structure/layer, detector, filter, glass slide(s), optical trapping layer) may be attached to each other to form the integrated/monolithic photonic device (chip).

Optionally, the sample-containment layer includes a chemically-functionalised surface, referred to herein as simply a “functionalised surface”. The functionalised surface bears chemical moieties or molecules at the surface, which are generally introduced through a surface treatment of the sample-containment layer. In embodiments in which the sample-containment layer comprises fluidically-interconnected wells, an inner surface of the wells may be surface-functionalised. Optionally, the functionalised surface is proximate to the optical structure. Advantageously, positioning the functionalised surface proximate to the optical structure helps to maximise the intensity of the evanescent light field on the functionalised surface, which can be useful to interrogate interaction between the sample and the functionalised surface.

The functionalised surface may bear functional groups, such as hydroxyl groups or aldehyde groups. Such functional groups may be introduced by, for example, a chemical treatment (e.g. treatment with Piranha solution) or plasma treatment.

Additionally or alternatively, the functionalised surface may be functionalised with a surface coating. The surface coating may comprise or consist of molecules bound to the surface. The molecules may be adsorbed to the surface, or alternatively may be bonded to the surface via a chemical bond. For example, the functionalised surface may comprise a coating covalently bonded to the surface, e.g. molecules covalently bonded to the surface.

Optionally, the functionalised surface is functionalised with an agent which limits or prevents binding of components of the sample to the sample-containment layer. For example, the functionalised surface may be functionalised with a passivation agent. The passivation agent helps to prevent non-specific binding of components of the sample to the sample-containment layer. Suitable passivation agents may be, for example, a protein (e.g. bovine serum albumin (BSA) or human serum albumin (HSA)), a nonionic surfactant (such as polysorbate 20 (e.g. Tween®-20), Triton X-100, or poloxamer 407 (e.g. Pluronic™ F127)), polyethylene glycol (PEG) (optionally in the form of an ester), or any mixture thereof.

Alternatively, the functionalised surface is functionalised with a binding agent. The binding agent is suitably a molecule which interacts with a known or putative target (binding partner) within the sample. The target may be, for example, a biomolecule such as DNA, RNA, a lipid, a protein, or a metabolite.

The binding agent may be, for example, an antibody, aptamer, nucleic acid, polypeptide, or a purified or synthetic ligand, such as a drug candidate or an antigen. The antibody may be, for example, a natural or engineering antibody or fragment thereof, such as a monoclonal antibody, a polyclonal antibody, or an antibody fragment such as a F(ab’)2, F(ab)2, Fab’, Fab, variable fragment (Fv), single chain variable fragment (scFv), diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

Optionally, the functionalised surface may be functionalised with both a binding agent and a passivation agent.

In instances where the surface containment layer is a modular part formed from multiple components, different components may have different surface functionalisation. For example, when the samplecontainment layer comprises a substrate with a trapping layer provided thereon (as described above), the substrate may bear one form of surface functionalisation and the trapping layer may bear a different form of, or no, surface functionalisation. For example, the substrate may be functionalised with a binding agent, which is absent from the trapping layer. This may be achieved, for example, by carrying out surface functionalisation prior to assembly of the modular surface containment layer.

Alternatively, surface functionalisation may be carried out after assembly of a modular surface containment layer and/or after assembly of the components of the integrated photonic device as a whole. For example, the surface functionalisation may be achieved by passing a suitable functionalising solution through the sample-containment layer (e.g. a solution containing said binding agent and/or passivation agent). Optionally, treatment with the functionalising solution may be followed by treatment with a flushing solution, to remove unbound components of the functionalising solution from the samplecontainment layer. The sample-containment layer may be dried after formation of the functionalised surface. Alternatively, the sample-containment layer may be kept wet after formation of the functionalised surface. Advantageously, the latter approach can avoid deposition of unwanted impurities within the sample-containment layer, such as salt crystals.

In view of the above, the present invention also provides (as a separate aspect) the step of providing an integrated photonic device as described above, passing a functionalising solution through the samplecontainment layer to produce a functionalised surface within the sample-containment layer, and optionally passing a flushing solution through the sample-containment layer to remove unbound components of the functionalising solution. A further aspect of the invention also comprises a kit of parts for carrying out such a method, comprising an integrated photonic device of the invention, a functionalising solution, and optionally a flushing solution.

By detecting the hopping movement of an object of interest between wells (and in particular measuring the resonance time of the molecule within a well), the device may be used to conduct various assays.

Typically, the methods rely on characterising the resonance time of the object of interest. The resonance time is calculated as the amount of time that the object of interest remains in a well. For example, in the first implementation, this may correspond to the amount of time that a pixel of the detector (aligned with a well) is receiving an illumination signal.

The resonance time may be obtained by observing the ligand hopping between different wells using a device according to the invention, and calculating a suitable statistic to characterise the hopping behaviour of the ligand. The statistic may be, for example, the average residence time of the object of interest in a well before moving to another well. The statistic may be calculated by observing, for example, the behaviour over n different hops. The number n may be, for example, at least 3, at least 5, at least 10 or at least 20 hops. The average may be, for example, the mean average or median average.

For example, the device may be used to measure the charge of an object of interest with an extremely high accuracy, e.g. less than one electron charge. Optionally, the device can also be used to distinguish between molecules of different charge, by measuring multiple molecules and comparing their measured charges.

In view of this capability, the device can be used for a range of different assays, including the following:

Determining modification of biomolecules

The sensitivity of the device can be such as to identify/distinguish between variants of the same biomolecules, such as different protein variants. Accordingly, the present invention extends to methods of identifying/distinguishing between variants of a biomolecule using the device of the present invention.

For example, the device may be used in a method of identifying or distinguishing between biomolecules which have undergone different modifications, based on measuring the resonance time of the biomolecules. For example, the device may be used in a method of identifying/distinguishing between different protein variants, such as proteins which have undergone post-translational modifications. This is because different modifications will affect the charge characteristics of the protein in different ways. For example, the device may be used in a method of identifying/distinguishing proteins having different levels of phosphorylation, sulfation, glycosylation, or methylation.

Additionally, or alternatively, the device may be used in a method of identifying or distinguishing between proteins having different protein folding geometries and/or conformations. Different geometries/conformations will affect the relative “exposure” of different residues to their surroundings, and thereby alter the charge characteristics of the protein.

Ligand binding assays

The present invention also extends to use of a device according to the present invention to carry out a ligand binding assay.

For example, the present invention may provide a method of identifying and/or quantifying the interaction between a ligand and a binding partner, comprising measuring the resonance time of the ligand in the presence of the binding partner, and comparing this against the resonance time of the ligand between wells in the absence of the binding partner.

The ligand binding assay may be a qualitative assay - used to identify the interaction between a ligand and a known or putative binding partner.

Additionally, or alternatively, the ligand binding assay may be a (at least semi-) quantitative assay. For example, the assay may be used to assess the relative or absolute binding strength between a ligand and a binding partner. In the latter, case, the binding strength between the ligand and binding partner may be assessed through a titration experiment, in which the relative amount of ligand and binding partner is varied, and the effect on the resonance time is measured. Alternatively, the binding strength may be assessed through a competitive binding assay, in which a sample containing said ligand, binding partner and a competitor molecule (the competitor molecule being capable of competitively binding said ligand or binding partner) is compared against a control solution containing the ligand and binding partner but lacking the competitor molecule. For example, in the latter case, if the resonance time is relatively unaffected between the test and control experiment, this would suggest that the binding strength between ligand and binding partner is relatively weaker than that between the competitor molecule and the ligand/ binding partner.

In one implementation, the assay is a liquid-phase ligand binding assay, examining the interaction of the ligand with a known or putative binding partner where both the ligand and binding partner are in solution. In such assays, the different resonance characteristics of the free ligand compared to the bound ligand may be used to identify binding. For example, in some implementations an experiment is carried out observing the resonance time of a fluorescently-labelled ligand in the presence of a (known or putative) binding partner, and compared against the resonance time for the same fluorescently-labelled ligand in a control experiment. The resonance time of the fluorescently-labelled ligand may be different when free, as compared to when it is attached to its binding partner.

In other implementations, the assay is a solid-phase ligand binding assay, examining the interaction of the ligand with a binding partner, wherein either the ligand or the binding partner is part of the functionalised surface of the sample-containment layer. In such assays, the different resonance characteristics of the ligand when bound to the functionalised surface versus when it is not bound to the surface can be used to probe the interaction of the ligand with the test molecule.

In some implementations, the ligand is an antigen and the binding partner is an antibody. In such instances, the assay may be analogous to a single-molecule ELISA assay. For example, the assay may be used to measure the binding strength of an antibody to a particular biomarker. The component bonded to the surface may be directly attached, or may be attached via a secondary capture molecule, such as an antibody specific to that component. For example, the assay may be analogous to a sandwich ELISA assay.

In some implementations, the binding partner is a receptor molecule. For example, the assay may be used to measure the binding strength of a receptor to a particular ligand. The test ligand may be, for example, a known or putative inhibitor to the receptor, an activator of the receptor.

In some implementations, the ligand may be a signalling lipid or a neurotransmitter, and the binding partner may be a known or putative receptor for the signalling lipid or neurotransmitter.

In some implementations, the ligand is a drug and the binding partner is a drug target. For example, in one implementation, the present invention provides a method of screening a drug candidate for activity against a given drug target, the method comprising immobilising the drug target in the wells of the surface-containment layer of the device of the invention, and observing the resonance time of a (e.g. fluorescently-labelled) drug candidate as it hops between the wells. Alternatively, the drug screening method may be inverted, such that the method comprises immobilising the drug candidate in the wells of the surface-containment layer of the device of the invention, and observing the resonance time of a (e.g. fluorescently-labelled) drug target as it hops between the wells. The appropriate methodology may be determined, for example, by the likely impact of an identification label (e.g. fluorescent probe) on the drug target versus the drug candidate.

Protein sequencing

In some implementations, the device may be used for protein sequencing.

Protein sequencing may be achieved by (i) a degradation step, comprising removing an amino acid residue from a protein or protein fragment to create a liberated amino acid or derivative thereof, and then (ii) measuring the resonance time of the liberated amino acid or derivative thereof; and (iii) using the resonance time to determine the identity of the amino acid.

The step (i) of removing the amino acid from the protein or protein fragment may be carried out using techniques known in the art. For example, the method may use Edman degradation, in which treatment of the protein with phenyl isothiocyanate removes a single amino acid from the N-terminus in the form of a phenylthiohydantoin-amino acid derivative.

Optionally, the method may involve (i-a) a fragmentation step, comprising cleaving the protein into multiple protein fragments prior to the degradation step. This step may be included when the protein exceeds a certain threshold of amino acids, since the efficacy of step (I) reduces as the size of the protein increases. For example, the fragmentation step may be carried out when the protein exceeds 60 residues in size, 50 residues in size, 40 residues in size, or preferably 30 residues in size. The fragmentation step may be carried out using known cleaving agents, such as thrombin, cyanogen bromide, trypsin, chymotrypsin, and enzymes.

Step (iii) may comprise comparing the resonance time data against reference data, wherein the reference data corresponds to resonance time data obtained for known amino acids or derivatives thereof.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

FIG 1 shows a cross-sectional side view of an embodiment integrated photonic device according to the first implementation of the invention;

FIG 2 shows a close-up view of the device of FIG 1 , showing a single pixel and a single well;

FIG 3 shows a top-down view of the arrangement of FIG 2, as viewed from within a microfluidic channel above the well;

FIG 4 shows a top-down view of a plurality of confinement zones of an embodiment device;

FIG 5 shows a top-down view of a plurality of confinement zones and laterally-extending fluid channels;

FIG 6 shows a cross-sectional side view of a laterally-extending fluid channel that has a vertical opening through multiple layers of the device;

FIG 7 shows a top-down view of a plurality of confinement zones that have the same type of fluid channels shown in FIG 6;

FIG 8 shows a top-down view of a plurality of confinement zones which each have vertical fluid ports;

FIG 9 shows a cross-sectional side view of the arrangement of FIG 2, in which the optical structure is a waveguide;

FIG 10 shows a top-down view of the arrangement of FIG 5, further including a channel waveguide optical structure;

FIGs 11A-11 B are cross-sectional side views of an arrangement having a rib waveguide, looking alongside and into the waveguide, respectively;

FIG 12 is a cross-sectional side view of a device having a rib waveguide, viewed looking into the waveguide, in which the waveguide is laterally offset from the wells;

FIG 13 is a cross-sectional side view of the arrangement of FIG 2, in which the optical structure comprises a sub-wavelength aperture at each well;

FIG 14 is a cross-sectional side view showing a section of a device in which the optical structure is configured as a waveguide located between the sample-containment layer and the detector; FIG 15 shows a cross-sectional side view of an embodiment integrated photonic device according to the second implementation of the invention;

FIG 16 shows a cross-sectional side view of an embodiment integrated photonic device according to the first implementation of the invention, further including an optical trapping structure;

FIG 17 shows a cross-sectional side view of the device of FIG 16, in which the sample containment layer is sandwiched within the optical trapping structure; and

FIG 18 shows a cross-sectional side view of an alternative arrangement of the device of FIG 16, in which the optical trapping structure is only on one side of the sample containment layer.

Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

FIG. 1 shows a cross-sectional view of an embodiment integrated photonic device 106, which comprises a series of layers formed on top of a detector 104. FIG. 2 shows a cross-sectional close-up view of a sub-unit (sub-region) 100a of the integrated photonic device at a single pixel 102 of the detector 104.

At an upper surface of the device 106, opposite the pixels 102a-d, there is an optical structure 108 which is configured to receive light from a light source and to generate an evanescent light field from the received light. The light source (not shown) may optionally be integrally attached to the photonic device. The optical structure 108 is attached at its base to a transparent layer/substrate (e.g. glass slide) 110 which forms a barrier between the optical structure 108 and a sample-containment layer 112 for holding a sample.

As is best shown in FIG. 1 , in an embodiment device 106, the sample-containment layer 112 is integrally attached at its lateral edges to the transparent layer 110. The transparent layer 110 and a samplecontainment layer 112 together define a microfluidic channel 111 therebetween. In variant embodiments, the transparent layer 110 may be omitted, and so the optical structure itself may define an upper boundary of the microfluidic channel and may thus be in direct contact with the sample.

In this embodiment, the sample-containment layer 112 comprises a transparent substrate (e.g. glass slide) 114 and a trapping layer 116 which extends vertically upwardly from the substrate 114. The trapping layer 116 has a plurality of wells 118 formed therein across the device 106. In this embodiment, each well 118 is aligned with a single pixel 102.

The transparent substrate 114 may be configured similarly to the upper transparent layer 110, or these substrates may differ, e.g. by providing a biofunctionalized coating on one of the substrates (e.g. the lower transparent substrate 114). The transparent substrates 110 and 114 are separated by a distance S, which defines the maximum depth of the microfluidic channel 111 , i.e. at the location of the well. At regions laterally between the wells, the channel has a smaller depth h (shown in FIG 2), which is dimensioned to allow a particle to move between wells whilst also still enabling a trapping effect to occur at the wells themselves. The trapping layer 116 may be formed by silicon lithography on the transparent substrate 114. FIG. 3 shows a top-down view of a sub-region (sub-unit) of the trapping layer 116 as viewed from within the microfluidic channel 111 looking downwards through a well 118.

As shown in FIGs. 2-3, the pixel 102 (including its substrate 120) below the well 118 forms a rectangular (e.g. square) zone having width HZ. The width H/of each pixel sub-unit sets the separation between adjacent wells 118. This dimension can be made on the order of a pixel size of a pixel of the detector, i.e. the width of a pixel 102 including a detector substrate 120 on which the pixel is attached. For example, if the detector is a CMOS image sensor, the width W could be on the order of 2.5 pm to 10 pm. The size 1/1/ may be chosen to find a compromise with the amount of light collected by the pixel and the required density of the pixels/wells (also referred to herein as a measurement density).

The well 118 for electrostatically trapping one or more molecules of the sample has a diameter or width w. The height D of each partition (which may also be referred to as the well depth), the depth S of the microfluidic channel 111 , the inter-well depth h, and/or the well width w ean be tuned to change the trapping energy landscape which influences the trapping and dwell time of the molecules. This can be further understood in view of reference [1 ], Example values of suitable dimensions are discussed in the Summary above.

The well “hopping” mechanism that forms the basis of the charge sensing may dictate the acceptable ranges of the separation l/l/ between adjacent wells. Ranges of ~2.2 pm have been demonstrated [2] though other ranges would be possible. For example, the well separation can be set large enough such that the dynamics in each well are independent of one-another.

Returning to FIGs 1 and 2, the transparent substrate 114 of the sample containment layer 112 may be attached, at its base, to an optical filter 122. The optical filter 122 may be a single or multi band pass/stop filter, short pass filter, or long pass filter.

The optical filter 122 is attached, at its base, to the detector 104 which comprises the detector substrate 120 and a light-sensitive area in each sub-unit 100a. In this embodiment, the detector 104 comprises a CMOS or CCD device, and the light-sensitive area comprises a single pixel 102 of said detector.

In some embodiments, the optical filter 122 may be replaced by or combined with a polychroic mirror.

As shown in FIG 1 , the particular sub-unit (sub-region) 100a of FIG 2 is located away from the lateral edges of the device 106 (towards a centre/interior of the device). Conversely, the lateral edges of the photonic device 106 have sub-units 100b which differ in that their trapping layer 116 comprises a barrier (partition, wall) 124 that extends fully across the depth S of the microfluidic channel, from the lower transparent substrate 114 to the upper transparent layer 110. These walls 124 retain the sample within the microchannel 111.

The plurality of sub-units 100a, 100b together define an array of fluidically-interconnected wells 118a-d, and a corresponding array of pixels 102a-d that are configured to detect light from the wells. Each of the wells 118a-d are configured to electrostatically trap a molecule of a sample in use.

In use, an ionic medium containing the molecules of interest is introduced into the microfluidic channel 111. As shown in FIG. 1 , through random diffusion and electrostatic interactions, a molecule 126 may enter a well 118b (e.g. from a previous position in well 118c, as shown by the dashed curve in FIG 1) and may dwell in the well 118b for a length of time that depends on its properties (e.g. charge). Light is supplied from an external light source (not shown) to the optical structure 108, which generates an evanescent light field 128, e.g. by total internal reflection or by using sub-wavelength apertures. The evanescent light field 128 illuminates a short (near-field) distance through the glass substrate 110 and into the well 118b.

In this example, the molecule of interest 126 is tagged with a fluorescent marker. Therefore, after absorbing light from the evanescent light field 128, the molecule 126 will later re-emit fluorescence 130, some of which will travel downwards through the glass substrate 114 and optical filter 122, to be collected by the pixel 102b of the detector 104, resulting in an electrical readout.

To improve the ability of the pixel 102b to detect fluorescence, the optical filter 122 may be configured to block light that comes directly from the optical structure 108 and/or light source.

In other embodiments, the molecule may be non-fluorescent, and the optical filter 122 may be configured to let through light 128 from the optical structure, in which case the signal of interest at the pixel 102b may be a shadow/absence of light which is instead scattered by the molecule. In yet further embodiments, the signal of interest at the pixel 102b may be an interference of light between the scattered light from the molecule and the transmitted (coherent) light from the optical structure 108.

It will be appreciated that, although FIG 1 only shows a cross-section of the photonic device along a single dimension, the photonic device may include a 2-dimensional array of these sub-units. Each subunit may be built on top of a pixel of a lensless CMOS or CCD camera, and thus any suitable number N subunits may be stacked side-by-side in a 2D array which is limited only by the size of the underlying CMOS or CCD camera. For example, a grid size of 4032 x 3024 is currently available in the Sony IMX557 sensor where each pixel, and therefore subunit size W, would be 1 .8 microns.

FIG 4 shows a top-down view of a portion of an integrated photonic device 206 according to another embodiment of the invention. Features in common with previous embodiments are given corresponding reference numerals. The device 206 of FIG 4 is similar to the device 106 of FIG 3, but differs in that the sample-containment layer 112 of the device 206 has a plurality of confinement zones 132a-d. Each confinement zone 132 has an array of fluidically-interconnected wells 118 configured to electrostatically trap molecules of a sample. Each confinement zone 132 is configured similarly to the device 106 shown in FIG 1 . The confinement zones 132a-d are fluid ically isolated from each other by walls 124 formed by the trapping layer (similarly to the walls 124 of FIG 3), to prevent cross-contamination of samples between different confinement zones 132a-d.

As shown in FIG 4, each confinement zone 132a-d defines an array of wells 118 having dimensions PxQ. P and Q can be chosen to be larger than the expected random walk distance of the molecule of interest within a measurement time (so that the hopping measurements are not influenced by the boundary effects of the measurement region size), but small enough to provide a desired throughput of the device. The maximum size may be determined to ensure that the well height S (shown in FIG 1) across the array is substantially constant, i.e. to limit the risk that a large device could bend (e.g. at layers 110, 114) to cause a variation in well height S.

FIGs 5 to 8 show various mechanisms for introducing the sample into the device and/or for washing the device between uses to eliminate cross-contamination. FIG 5 shows a top-down view of a portion of a device 306 that has a plurality of confinement zones 132a- d. The device is similar to that of FIG 4, but differs in that the device 306 of FIG 5 further includes a plurality of fluid channels 134 and 136 for transferring a sample into or from the device. The arrows shown in FIG 5 depict the paths of respective molecules within a fluid sample. These paths also show the molecule’s movement and dwell times within wells of the sample-containment layer.

Each fluid channel 134, 136 extends laterally alongside an array of wells of a respective confinement zone. In this embodiment, each fluid channel 134, 136 extends laterally across the sample-containment layer 112, alongside a plurality of arrays of wells 118 (i.e. a plurality of confinement zones 132a-d). Each fluid channel 134, 136 has an opening 138 which is located at a lateral edge of the sample-containment layer. The openings 138 may be connected with external fluid lines (not shown). This arrangement enables each fluid channel to provide a sample to a plurality of confinement zones, so that multiple assays can be performed in parallel on the same sample. In variant embodiments, the confinement zones could each differ in their configuration/geometry to enable parallel measurements under different conditions.

FIG 5 shows four confinement zones 132a-c and four fluid channels 134, 136. However, any suitable number of zones and channels may be provided. It will be appreciated that FIG 5 only shows a section of the device, and that the pattern shown in FIG 5 may repeat so that each confinement zone 134a-d is fluidically connected to two channels including a first (inlet) channel 134 on one side thereof, and a second (outlet) channel 136 on another side thereof. Thus, one channel may be used for injecting a solution/molecules/wash and the other channel may be used for extraction of the solution/molecules/wash after measurements or cleaning is complete. Fluid channels which are adjacent to each other may be separated by a barrier (e.g. similarly to the walls 124) to prevent cross-contamination between different confinement zones.

FIGs 6 and 7 show a cutaway side view showing a portion of an embodiment device 406 having a different arrangement of fluid channels. In this embodiment, the fluid channels 134, 136 have openings 140 (inlets/outlets) which extend vertically through the photonic device 406, e.g. through the glass layer 110 and optical structure 108. The device 406 further includes a fluidic manifold 142 attached to an upper surface of the device (e.g. to an upper surface of the optical layer 108) to help direct fluid to/from the openings 140.

As shown in FIG 7, this top-down fluid transfer method enables further multiplexing compared to the device 306 of FIG 5, since the device 406 can be split lengthways and widthways into various fluidically isolated regions 144a-d, each of which comprise their own confinement zone(s). Therefore, this arrangement does not require a single channel to provide the same sample to all confinement zones e.g. across an entire width of the device. As shown in FIG 7, some of the confinement zones (e.g. zones 132b and 132c) are fluidically isolated from each other, whereas some confinement zones (e.g. zones 132a and 132b) are fluidically connected by a fluid channel 134.

FIG 8 shows a top-down view of another embodiment device 506 having a different fluid transfer mechanism. In this arrangement, single sub-units (e.g. single wells/pixels) of the device are sacrificed to act as vertical fluid injection/extraction ports 140. These ports extend vertically through one or more layers through of the photonic device in a similar manner as previously discussed. In this embodiment, the fluid channels do not extend laterally alongside the wells, and instead extend vertically into the fluid confinement zones. Similarly to the other embodiments, the fluid channels 134, 136 are located at opposite lateral sides of a given confinement zone 132, to promote fluid flow throughout the entire confinement zone.

FIGs 9 to 14 show various embodiments having different optical structures for providing evanescent illumination to the sample.

FIG 9 shows an arrangement which is similar to that of FIG 2, but in which the optical structure 108 is configured as a waveguide. The waveguide has a plurality of layers including a core layer 146 sandwiched between two cladding layers 148 and 150. The materials of the core and cladding layers are selected so that the core layer 146 has a greater refractive index than the refractive indices of the cladding layers 148, 150.

In use, light 152 from a light source is injected along the waveguide. The waveguide generates an evanescent light field 128 which leaks through the glass layer 110 and into the well. The evanescent field can then interact with the molecule 126, e.g. by exciting a fluorescent dye on the molecule, which then generates fluorescence 130 that is detected by the detector pixel 102. The dimensions and materials of the photonic device may be selected such that the evanescent field 128 is strong enough to reach all the molecules in the well 118 and excite them. Other examples of waveguides for fluorescence microscopy may be further understood in light of Reference [7].

In this embodiment, the optical filter 122 is configured to block light coming directly from the waveguide and only to let through fluorescence from the molecule, in a similar manner as previously discussed in relation to FIGs 1 and 2.

In some embodiments, the waveguide may be configured as a slab (e.g. rectangle or square) that extends across substantially an entirety of an array of wells. For example, the waveguide may cover substantially an entirety of the sample-confinement layer 112, e.g. with the exception of any openings for fluid pathways. The light source may therefore couple into the waveguide from one or multiple locations at a lateral edge of the photonic device.

Alternatively, the waveguide may be a 2D waveguide (e.g. a channel waveguide). FIG 10 shows such an arrangement, in which the optical structure 108 is a channel waveguide configured to overlie an array of wells 118 across a plurality of confinement zones 132a-d. The waveguide follows a serpentine path, snaking around the chip to deliver light (via the evanescent field) to each of the wells 118. The waveguide may be positioned so that it passes directly over the position of a well in each sub-unit 100a, b that it crosses. A beam dump 154 is located at a terminal end of the waveguide.

FIGs 11 A and 11 B show side views and front views, respectively, of an embodiment device having a waveguide which is directly connected to (i.e. forms a barrier of) the microfluidic channel 111. FIG 11A shows a side view alongside the waveguide, whereas FIG 11 B shows a front view looking into the waveguide. This embodiment excludes the upper glass layer 110 which was present in FIGs 1 and 2, so that the waveguide may be in direct contact with a sample. Since this could change the charge and motion dynamics of the molecules, this embodiment may have a different geometry from that of FIGs 1 and 2. As shown in FIG 11 B, the waveguide is configured as a rib waveguide, with a rib protruding towards the well 108. However, in variant embodiments, the waveguide may have other configurations, e.g. as a ridge waveguide or otherwise.

FIG 12 shows a front view of an embodiment which is similar to that of FIG 11 B, but in which a single section of the waveguide is configured to illuminate to adjacent sub-units (i.e. two adjacent wells). In this embodiment, the waveguide is a rib waveguide, and is oriented such that each well 118a-b is laterally offset from the waveguide, e.g. with the waveguide being substantially equidistant between two adjacent wells. The waveguide of FIG 12 may therefore be designed to generate an evanescent field 128 that has a greater strength than that of FIGs 10-11 B, so that the evanescent field is sufficient to excite molecules in both wells 118a-b.

FIG 13 shows a side view of an embodiment in which the optical structure 108 is not configured as a waveguide. Instead, the optical structure 108 comprises a plurality of sub-wavelength apertures 156 which are sized (relative to the wavelength of the light source) to create the evanescent light field 128. The optical structure 108 may therefore be illuminated from above.

In this embodiment, the optical structure 108 comprises a transparent substrate (e.g. glass slide) 158 having a metallised layer (e.g. gold plating) 160 formed thereon. The metallised layer 160 has the subwavelength apertures 156 formed vertically therethrough. Since the apertures have a size that is less than the wavelength of the source illumination, they transmit light that decays away from the hole, i.e. creating an evanescent light field at the well 118.

The metallised layer has a thickness T and the nanoapertures have a diameter D. The dimensions T and D can be selected to optimise the intensity of light in the channel. For example, if conducting an assay by detecting fluorescent molecules, the dimensions should be chosen to create sufficient light to excite the molecules such that their fluorescence can be detected.

FIG 14 shows an embodiment which is similar to that of FIG 9, but differs in that the waveguide of FIG 13 is located between the sample-containment layer 112 and the detector 104. The waveguide may therefore be fabricated directly on the sensor, with evanescent waves being sent upwardly through the transparent layer 110 and the waveguide’s cladding layer 150 (if present).

FIG 15 shows a device of the present invention according to the second implementation taught above. In this instance, integrated chip 606 is identical to integrated chip 108 from FIG 13, but lacks the filter layer 122 and integrated detector components. Instead, in this instance, fluorescence 602 emitted by a molecule held within the well is collected by separate detection apparatus 601 . Detection apparatus 601 comprises objective lens 603 which directs the emission towards camera 605 via an optical filter and lens assembly 607 (in this case including a longpass filter and a tube lens). Data from camera 605 is then fed to computer 609 for further processing. Although not depicted in FIG 15, the detection apparatus 601 is configured such that the camera is able to operate a plurality (all) of the wells incorporated into integrated chip 606.

The system of FIG 15 can permit flexibility in terms of the illumination approach and detection optics. For example, whilst FIG 15 depicts optical structure 158 and 160 and illumination from above the detector, it is possible for the optical structure to be incorporated between the sample-containment layer apparatus with illumination provided from underneath the integrated chip via objective lens 603. Alternatively, the optical structure can take the form of the alternative structures, such as those depicted in FIGs 9, 11 a, 11b, 12 and 13. In addition, the lens assembly 607 can allow multi-colour imaging, through the use of dichroic mirror and shortpass/longpass/bandpass filters to separate emission of different colours.

FIGs 16 to 18 show devices 706, 806, and 906 of the present invention, according to the first implementation taught above. The device 706 is similar to the device 106, but differs in that the device 706 further includes an optical trapping structure (optical tweezer) 708, i.e. for achieving optical trapping of an object of interest within a well 118.

FIGs 17 and 18 show different embodiments for the optical trapping structure 708 in more detail. In FIG 17, the optical trapping structure (e.g. GET) has a double-sided architecture, so that the sample containment layer 112 is sandwiched between different layers of the optical trapping structure. As shown in FIG 17, the optical trapping structure comprises a photoconductive layer 710, first dielectric layer 712a, second dielectric layer 712b, first electrode layer 714a, and second electrode layer 714b. The first electrode layer and/or second electrode layer may be transparent or semi-transparent, and/or may include indium tin oxide (ITO).

The first dielectric layer 712a may provide electrical isolation between the photoconductive layer 710 and the first electrode layer 714a. The second dielectric layer 712b may provide electrical isolation between the second electrode layer 714b and the sample.

A power source is electrically connected to the first electrode layer 714a and the second electrode layer 714b and configured to apply an electrical bias between the first electrode layer and the second electrode layer. In use, the generation of power by the power source generates a varying electrical field between the first electrode layer 714a and the second electrode layer 714b.

FIG 18 shows an optical trapping structure which has similar features to that of FIG 17, but which differs in that the optical trapping structure of FIG 18 has a single-sided architecture, so that it is arranged on only one side of the sample containment layer 112 (e.g. between the sample containment layer and the optical structure 108). The device of 906 has an optical trapping structure comprising a first electrode layer 714, first dielectric layer 712a, photoconductive layer 710, second dielectric layer 712b, and second electrode layer 716. In this embodiment, the second electrode layer 716 comprises a patterned layer of ITO.

FIG 18 also shows two illumination beams from different sources (an optical trapping light source and excitation light source). Excitation beam 152 provides evanescent illumination of an object of interest within the sample containment layer, in a similar manner as discussed above. Optical trapping beam 153 provides patterned illumination from an optical trapping light source (e.g. a DMD) to enable the optical trapping structure to provide an attractive or repulsive force on the object held in the sample containment layer. The beams 152 and 153 are at different wavelengths, e.g. so that the optical trapping beam 153 does not cause excitation of the object of interest in use.

The embodiment devices described herein may be used for a variety of purposes. These devices may enable a compact, fast, and accurate measurement of molecules (e.g. molecule charge). The devices may also be used to perform such measurements simultaneously under different conditions, e.g. by using a plurality of confinement zones to perform multiplexed measurements. The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

1 . “Geometry-induced electrostatic trapping of nanometric objects in a fluid”, Krishnan et al., 2010, Nature, https://www. nature. com/articles/nature09404?message-global=remove&page=4

2. “Single-molecule electrometry”, Ruggeri et al., 2017, Nature Nanotechnology, htps://www.nature.com/articles/nnano.2017.26

3. “Light passing through subwavelength apertures”, Weiner, 2009, htps://iopscience.iop.Org/article/10.1088/0034-4885/72/6/064 401

4. “Probing the negative permittivity perfect lens at optical frequencies using near-field optics and single molecule detection”, Moerland et al., 2005, https://opg.optica.org/oe/fulltext.cfm7urFoe- 13-5-1604&id=82913 5. “Single Molecules Observed by Near-Field Scanning Optical Microscopy”, Betzig and Chichester, 1993, https://pubmed.ncbi.nlm.nih.gov/17736823/

6. “Waveguide excitation fluorescence microscopy: A new tool for sensing and imaging the biointerface", Grandin et al., 2006, https://pubmed.ncbi.nlm.nih.gov/16137877/

7. “Evanescent-wave fluorescence microscopy using symmetric planar waveguides”, Agnarsson et al., 2009, https://opg.optica.org/oe/fulltext.cfm7urFoe-17-7-5075&i d=177351

Reference numerals

106, 206, 306, 406, 506, 606, 706, 806, 906 photonic device

100a,b sub-unit

104 detector

102 pixel

120 substrate

122 optical filter

108 optical structure

146 core layer

148, 150 cladding layers

154 beam dump

158 transparent substrate

160 metallised layer

156 sub-wavelength apertures

110 transparent layer

112 sample-containment layer

114 transparent substrate

116 trapping layer

118 well

124 wall

144a-d fluidically isolated regions

132a-d confinement zones

111 microfluidic channel

134, 136 fluid channel

138 lateral opening

140 vertical opening

142 fluidic manifold

708 optical trapping structure

710 photoconductive layer

712a, b dielectric layers

714a, b, 716 electrode layers

126 molecule

128 light field

130 fluorescence

152 received light (from light source) detection apparatus

603 objective lens

605 camera

607 optical filter and lens assembly emitted light computer