Concentrating substances

Abstract

A system (2) for concentrating a substance dispersed in a fluid is provided. The system comprises a nanofluidic structure (14). The nanofluidic structure (14) comprises at least one dead-end nanochannel (18) extending in an extension direction from an entrance (22) to a dead-end (24). The dead-end nanochannel has a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width. The system further comprises an electrical apparatus (5) arranged to apply an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel, to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

Description

[0001] Concentrating substances

[0002] The present invention relates to structures, systems and methods for concentrating substances such as proteins.

[0003] Neurodegenerative diseases such as Alzheimer’s and Parkinson’s have been associated with the aggregation in the brain of a protein called Tau (more precisely, full-length hTau441). In the human brain, hTau441 takes many years to aggregate into amyloid fibrils. It is useful when researching these diseases to aggregate Tau in vitro under varying conditions, e.g. as part of anti-body and cell toxicity experiments.

[0004] The slow natural aggregation rate limits current research, as it can take many months to grow Tau amyloid fibrils in the lab without external chemical cues. Conventionally, heparin has been used as an aggregation agent to expedite aggregation. However, recently heparin has been shown to induce structural changes in Tau fibrils that make them different to those actually found in patients. This may limit the usefulness of research that relies on heparin-induced Tau aggregation. In vitro aggregation of other proteins also faces similar challenges. Existing approaches to protein aggregation also typically require expensive facilities and involve labour- intensive tasks.

[0005] An improved approach may be desired.

[0006] According to a first aspect of the present invention there is provided a system for concentrating a substance dispersed in a fluid, the system comprising: a nanofluidic structure comprising at least one dead-end nanochannel extending in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and an electrical apparatus arranged to apply an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel, to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

[0007] According to a second aspect of the present invention there is provided a method for concentrating a substance dispersed in a fluid, the method comprising: providing a sample comprising the substance dispersed in the fluid to a nanofluidic structure comprising a dead-end nanochannel extending in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and applying an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel so as to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

[0008] Thus, it will be appreciated by those skilled in the art that, exposing the fluid in the nanofluidic structure to the electric field can induce surface flows which cause the concentration of the substance dispersed in the fluid to increase in localised regions of the nanofluidic structure. For instance, electrostatic forces produced by the electric field may attract particles of the substance towards the dead-end of the dead-end nanochannel. The electric field may induce electroosmotic or electrophoretic flows in the nanofluidic structure. Particles of the substance may become trapped in a vortex or other flow trap formed at the entrance of the dead-end nanochannel (e.g. at or adjacent the interface between a microchannel and the dead-end nanochannel), producing a local increase in substance concentration. Thus the invention may provide a quick and simple method for concentrating substances that does not require chemical or mechanical intervention. The nanofluidic structure may also be relatively simple and inexpensive to produce.

[0009] In a set of embodiments, the nanofluidic structure comprises a fluid input for receiving a sample comprising the substance dispersed in the fluid; and a microchannel extending from the fluid input; wherein at least one dead-end nanochannel extends from the microchannel in the extension direction.

[0010] Providing the sample comprising the substance dispersed in the fluid to the nanofluidic structure may consist of inputting the sample to a fluid input of the nanofluidic structure, wherein the nanofluidic structure comprises a microchannel extending from the fluid input and the dead-end nanochannel extends from the microchannel in the extension direction.

[0011] According to a third aspect of the present invention there is provided a system for concentrating a substance dispersed in a fluid, the system comprising: a nanofluidic structure comprising: a fluid input for receiving a sample comprising the substance dispersed in the fluid; a microchannel extending from the fluid input; and at least one dead-end nanochannel extending from the microchannel in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and an electrical apparatus arranged to apply an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel, to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

[0012] According to a fourth aspect of the present invention there is provided a method for concentrating a substance dispersed in a fluid, the method comprising: inputting a sample comprising the substance dispersed in the fluid to a fluid input of a nanofluidic structure comprising a microchannel extending from the fluid input and a dead-end nanochannel extending from the microchannel in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and applying an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel so as to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

[0013] The substance may comprise any suitable substance for which it is desirable to produce local increases in concentration. In a set of embodiments the substance comprises a nucleic acid such as DNA or RNA (e.g. messenger RNA). Producing local increases in substance concentration within the nanofluidic structure may be useful in many applications including concentrating DNA or mRNA molecules for testing of aptamer binding to target specimens, concentrating nanoparticles and antibody-target binding assays.

[0014] In a set of embodiments, the system may be arranged to filter the fluid by inducing sedimentation of the substance. In such embodiments the local concentration increases may cause the substance to aggregate into larger particles, which then settle out of the fluid (e.g. due to gravity and / or the applied electric field). In some embodiments the method comprises filtering the fluid to isolate substance aggregates (e.g. amyloids) from the fluid.

[0015] The inventors have recognised that embodiments of the present invention may be particularly useful for concentrating proteins. In a set of embodiments, the substance comprises one or more proteins. The substance may comprise protein monomers, oligomers or polymers. In a set of embodiments, the substance comprises Tau protein (e.g. Tau monomers) such as full-length hTau441. Additionally or alternatively, the substance may comprise other amyloid proteins such as TDP-43, Alpha synuclein or Amyloid beta.

[0016] Concentrating proteins may be useful because it can increase the propensity of the proteins to interact, for instance to undergo a phase transition and / or aggregate (e.g. into oligomers or amyloid fibrils). In a set of embodiments, the system is arranged to induce aggregation of the substance (e.g. the proteins). Inducing protein aggregation can be very useful for medical research, as explained above. In general, the more proteins present in an area the higher the likelihood of nucleation and protein aggregate formulation. Embodiments of the invention may reduce the time needed for substantive protein aggregation from hours or days to minutes or seconds. Moreover, because effective concentration of the proteins may be achieved with only the electrically-induced surface flows, the need for chemical stimuli (e.g. heparin) to encourage protein aggregation may be reduced or entirely eliminated. Thus embodiments of the invention may facilitate accelerated production of aggregated proteins without risking undesirable chemical changes. In other words, the invention may be used to produce protein aggregates which are more representative of naturally-occurring aggregated proteins than those produced with conventional approaches, without requiring impractically long aggregation times.

[0017] Some embodiments of the present invention may usefully be employed in screening applications. For example, the system may be used to encourage concentration of a substance under various different conditions, to determine the relative impact of the different conditions on concentration behaviour (or concentration-induced effects such as aggregation). For instance, a protein may be concentrated in the presence of different potential aggregation inhibitors, and a resulting degree and / or type of aggregation then analysed to assess the relative effectiveness of the potential inhibitors (i.e. to screen the potential inhibitors). Because embodiments of the invention can induce substantive aggregation within short timescales (e.g. minutes / seconds), this assessment can be performed quickly to enable rapid identification of effective inhibitors from a potentially large set of candidates (e.g. a chemical library).

[0018] The results of concentrating the substance (e.g. protein aggregates) may be advantageously used in one or more subsequent processes. For instance, it may be useful to introduce aggregated proteins produced in the nanofluidic structure to cell cultures (e.g. neural cell cultures). In some embodiments, the cell cultures may be held in the nanofluidic structure itself (e.g. in the microfluidic channel(s)), so that the proteins aggregate in their immediate vicinity. Additionally or alternatively the nanofluidic structure may be arranged for fluid connection to a cell culture apparatus, e.g. via one or more additional microchannels or nanochannels or via additional tubing. For instance, a fluid input or output of the nanofluidic structure may be suitable for connecting to a cell culture apparatus, e.g. to allow the result of substance concentration to be drained directly into the cell culture apparatus.

[0019] The substance may be dissolved in the fluid, i.e. such that the fluid comprises a solution of the substance and a suitable solvent. However this is not essential and in other embodiments the substance may simply be mixed with and dispersed within the fluid (e.g. substantially homogeneously). The substance is not necessarily soluble.

[0020] The effectiveness with which the substance is concentrated may be optimised through appropriate design of the shape and / or dimensions of the nanofluidic structure. In some embodiments, the dead-end nanochannel and / or the microchannel has a cuboid shape. A nanochannel will be understood to have a smallest dimension (e.g. a height or width of a cuboid channel or a width (i.e. diameter) of a cylindrical channel) that is on the nanometre or nearnanometre scale, i.e. 5 pm or less, 2 pm or less, 1 pm or less, 500 nm or less or 100 nm or less. In contrast, a microchannel will be understood to have a smallest dimension (e.g. a height or width of a cuboid channel or a width (i.e. diameter) of a cylindrical channel) that is on the micrometre or near-micrometre scale, i.e., 2 mm or less, 1000 pm or less, 500 pm or less, 250 pm or less, 100 pm or less, 50 pm or less, 10 pm or less, 5 pm or less or 2 pm or less.. The fluid input may also be of a micrometre or near-micrometre scale. In some embodiments, the fluid input is large enough for a human user to interact with (e.g. to add the sample with a pipette).

[0021] The shape and / or dimensions of the nanofluidic structure may be tuned to optimise concentration of a target substance, i.e. to optimise entrance aggregation or dead-end aggregation of a particle of the substance. For instance, in a set of embodiments a shape and / or dimension of the nanofluidic structure is arranged for use with an expected particle shape and / or dimension of the target substance. For instance, in embodiments where the substance comprises Tau protein monomers (with a molecular weight of approximately 45.8 kDa and a hydrodynamic radius of approximately 3 nm), the dead-end nanochannel may be a cuboid channel with a width of approximately 1.5 pm, a height of approximately 2 pm and a length of approximately 40 pm.

[0022] The way in which the dead-end nanochannel extends from the microchannel impacts the flow of the fluid when the electric field is applied. In a set of embodiments the dead-end nanochannel and the microchannel are arranged to induce the formation of a flow trap (e.g. a vortex) at the entrance of the dead-end nanochannel. In a set of embodiments, the dead-end nanochannel extends substantially perpendicular to the microchannel. In other words, in some embodiments the microchannel extends in a direction substantially perpendicular (e.g. making an angle of between 70° and 110°, between 80° and 100°, between 85° and 95° or between 88° and 92°) to the extension direction of the dead-end nanochannel. Having a sharp change of direction from the microchannel into the dead-end nanochannel may promote the formation of an effective flow trap for the substance at the entrance to the dead-end nanochannel, improving the concentration increase at the entrance to the dead-end nanochannel.

[0023] As explained above, particles of the substance may be trapped in localised regions of the nanofluidic structure (e.g. through the balance of electro-osmotic flows and hydrodynamic behaviour of the fluid and the substance). The effectiveness with which a vortex or other flow trap is generated at or adjacent the entrance to the dead-end nanochannel (i.e. at the nanomicro interface) may depend at least in part on the length of the dead end nanochannel. In a set of embodiments, the length of the dead-end nanochannel is at least two times the width of the dead-end nanochannel, at least five times the width of the dead-end nanochannel, at least ten times the width of the dead-end nanochannel, at least fifty times the width of the dead-end nanochannel or at least 100 times the width of the dead-end nanochannel. The length of the dead-end nanochannel may be 5 pm or more, 10 pm or more, 25 pm or more, or 50 pm or more.

[0024] The sample may be added to the nanofluidic structure manually (e.g. by a user with a pipette) or automatically (e.g. as part of an automated screening application).

[0025] In some embodiments, the nanofluidic structure comprises a fluid output for draining a fluid from the nanofluidic structure. The fluid output may extend from the microchannel. Additionally or alternatively, a single interface may comprise a fluid input and a fluid output (i.e. the same physical port may be used for both receiving and draining fluid from the nanofluidic structure). In some embodiments, it may not be necessary to drain fluid from the nanofluidic structure (e.g. the nanofluidic structure may be designed to be single-use). In some embodiments, a fluid output of the nanofluidic structure is connected to a fluid input of the nanofluidic structure, e.g. to allow a fluid to be re-circulated after passing through the nanofluidic structure. This may facilitate the filtration of a sample, e.g. by re-circulating a sample through the nanofluidic structure to increase a proportion of the substance that aggregates and is filtered out of the fluid by sedimentation. For instance, in a set of embodiments, cell media comprising an aggregation- prone protein species may be provided in the fluid input. The application of the electric field causes the proteins to concentrate, aggregate and settle out of the fluid. The fluid may then be drained from a fluid output before being returned to the cell media at the input for re-circulation and re-filtration. This process may be used to measure or detect the presence of the protein in the cell media and / or to remove the protein from the cell media.

[0026] As explained above, local increases in substance concentration may be caused by flow patterns induced by the electric field. However in some applications it may be beneficial to induce additional flow through the nanofluidic structure beyond that achieved by the electric field. In a set of embodiments the fluid input and / or fluid output is arranged for connection to a pumping apparatus for inducing fluid flow in the nanofluidic structure. The pumping apparatus may be part of the system. Flow induced by the pumping apparatus may enhance local concentration increases caused by the electric field. The pumping apparatus may additionally or alternatively be operable to add or remove fluid from the nanofluidic structure, e.g. before and / or after use. This may expedite operation and / or facilitate automation of the system.

[0027] Effective concentration may be achieved with only a single dead-end nanochannel extending from the microchannel. However, in a set of embodiments the nanofluidic structure comprises a plurality of dead-end nanochannels extending from the microchannel. The dead-end nanochannels may be arranged in an array. Each of the plurality of dead-end nanochannels may extend in the extension direction from respective entrances to respective dead-ends (i.e. the dead-end nanochannels may be arranged in parallel). Each of the of the plurality of deadend nanochannels may have a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width. In some embodiments the plurality of dead-end nanochannels are substantially identical. However, in some other embodiments the plurality of dead-end nanochannels includes at least two different dead-end nanochannels (e.g. having different dimensions).

[0028] In a set of embodiments, the nanofluidic structure comprises a second microchannel. The nanofluidic structure may comprise a second fluid input from which the second microchannel extends. The nanofluidic structure may comprise a fluid output (e.g. a second fluid output) extending from the second microchannel. The nanofluidic structure may comprise one or more dead-end nanochannels extending from the (first) microchannel towards (but not all the way to) the second microchannel. The nanofluidic structure may comprise one or more dead-end nanochannels extending from the second microchannel (e.g. towards the first microchannel). These may extend parallel to dead-end nanochannels extending from the first microchannel (e.g. in an opposite direction to the extension direction). The one or more dead-end nanochannels extending from the second microchannel in the opposite direction to the deadend nanochannels extending from the first microchannel may provide additional locations for concentrating the substance. For instance, appropriate control of the electric field may cause a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel(s) extending from the second microchannel (e.g. reversing a direction of the electric field). This may allow a user to concentrate substance selectively in the first or second microchannel.

[0029] The electric field may extend in the same direction as the extension direction, or in the opposite direction (i.e. the potential difference may be positive or negative in the extension direction).

[0030] The electrical apparatus may comprise any suitable components for applying the electric field over the nanofluidic structure. The electrical apparatus may be arranged to generate the electric field by producing a potential difference between different regions of fluid in the nanofluidic structure. The electrical apparatus may comprise one or more electrodes (e.g. platinum electrodes) configured to apply a voltage to fluid in the nanofluidic structure. For instance, the electrical apparatus may comprise an electrode arranged to be placed in the microchannel or the fluid input or a fluid output (i.e. in electrical contact with fluid). The electrical apparatus may comprise a second electrode arranged to be placed in a microchannel (e.g. a second microchannel) a fluid input or a fluid output. The electrical apparatus may comprise a voltage generator for applying a voltage to one or both electrodes. The electrical apparatus may be arranged to produce a potential difference between different regions of fluid in the nanofluidic structure of 1 V or more, 5 V or more, 10 V or more, 20 V or more or 50 V or more.

[0031] As explained above, the flow patterns in and at the entrance of the dead-end nanochannel(s) extending from the microchannel can produced localised increases in substance concentration when subject to the electric field. However, in some embodiments these surface flows may be further improved by providing a flow path from the microchannel that is not a dead-end. In a set of embodiments, the nanofluidic structure comprises a through channel which extends from the first microchannel to the second microchannel. The through channel allows the fluid to circulate and flow from the first microchannel to the second microchannel, e.g. by electro-osmosis due to the electric filed. The through channel(s) may extend parallel to the extension direction (i.e. parallel to one or more dead-end nanochannels). The through-channel may be a nanochannel or a microchannel.

[0032] In addition to concentration, the kinetics of protein aggregation may also depend on rely other parameters such as pH, ionic strength and temperature. In a set of embodiments the system comprises a temperature control apparatus arranged to control a temperature of the nanofluidic structure. The temperature of the nanofluidic structure may be controlled to optimise aggregation of a target protein. The nanofluidic structure may comprise a substrate, e.g. comprising or consisting of silicon, glass or a polymer. The substrate may define a boundary (e.g. a lower boundary) of the microchannel(s) and / or nanochannel(s) of the nanofluidic structure (i.e. the substrate may provide a floor of the channels).

[0033] The nanofluidic structure may comprise a structure layer adjacent the substrate. The structure layer may be bonded (e.g. plasma bonded) to the substrate. The structure layer may comprise or consist of a polymer (e.g. polydimethylsiloxane (PDMS)). The structure layer may define one or more boundaries of the microchannel(s) and / or nanochannel(s) of the nanofluidic structure (e.g. one or more lateral and / or upper boundaries). The structure layer may define the shapes / dimensions of the channels and / or the directions in which the channels extend.

[0034] The structure layer may be relatively thick compared to the scale of the nanofluidic structure (e.g. to improve robustness, facilitate handling and / or aid manufacture). The structure layer may have a thickness of 100 pm or more, 200 pm or more, 500 pm or more, 1 mm or more, 5 mm or more, 8 mm or more or even 10 mm or more. The structure layer may have a thickness of less than 15 mm, less than 10 mm, less than 5 mm or less than 1 mm. The fluid input(s) and / or fluid outputs may be defined by hole(s) in the structure layer, e.g. extending from part of the structure layer defining the microchannel. The fluid input(s) and / or fluid output(s) may provide fluid reservoir(s), e.g. with side walls defined by the hole in the structure layer.

[0035] The nanofluidic structure may be provided on a nanofluidic chip. In other words, the system may comprise a nanofluidic chip comprising the nanofluidic structure. The nanofluidic chip may comprise a plurality of nanofluidic structures. In some such embodiments, the plurality of nanofluidic structures may share a common substrate and / or structure layer. Some or all of the nanofluidic structures may comprise a microchannel and a dead-end nanochannel as described herein.

[0036] In a set of embodiments, the structure layer is transparent to at least some wavelengths of light (e.g. to one or more visible or IR wavelength ranges). This may facilitate imaging of the sample whilst it is in the nanofluidic structure. The system may be used for concentrating a fluorescent substance, and in some such embodiments the structure layer is transparent to one or more fluorescence wavelengths of said substance.

[0037] Being able to image the contents of the nanofluidic structure may be of particular interest when the system is used for aggregating proteins, because the aggregation process and / or the subsequent behaviour of aggregates can then be observed and analysed. Moreover, the fact that the concentration of the substance (e.g. the protein monomers) occurs in predictable areas of a nanofluidic structure (i.e. in relatively confined regions) means that high quality imaging may be achieved with relative convenience.

[0038] In a set of embodiments, the behaviour of the substance in the nanofluidic structure may be analysed. The present disclosure extends to a method of analysing a substance dispersed in a fluid, the method comprising: concentrating the substance using the method disclosed herein; and determining one or more properties of the substance.

[0039] Determining one or more properties of the concentrated substance may comprise imaging the substance in the nanofluidic structure before and / or during and / or after the electric field is applied, and analysing one or more resulting images. Determining one or more properties of the substance may comprise one or more of: determining a size of one or more substance particles (e.g. single-molecule sizing ; determining a rate at which the substance concentrates after application of the electric field (e.g. measuring a gradient of concentration increase or a time taken to reach a concentration threshold); determining a rate at which the substance disperses after application of the electric field (e.g. measuring a gradient of concentration decrease or a time taken to reach a lower concentration threshold); analysing diffusion behaviour after concentration (e.g. diffusion patterns and / or time profiles). The rate at which a substance of interest concentrates / disperses may be correlated with a size and charge of particles of the substance. For instance, larger aggregates tend to diffuse more slowly than smaller aggregates. In one set of embodiments, the method comprises determining a size of one or more substance particles by determining a rate at which the substance concentrates on application of the electric field and / or a rate at which the substance disperses after application of the electric field.

[0040] In all of these approaches, the size, position and / or concentration of substance particles may be measured indirectly using optical means such as fluorescence, autofluorescence, scattering or quantitative phase microscopy (e.g. by measuring an intensity of scattered and / or fluorescence light from substance particles).

[0041] In a set of embodiments, the system comprises imaging apparatus for imaging the nanofluidic structure (i.e. the contents of the nanofluidic structure). The imaging apparatus may comprise a microscope (e.g. a total internal reflection fluorescence microscope). The imaging apparatus may comprise a processing device for controlling the microscope and / or analysing image data produced by the microscope. In a set of embodiments the same processing device controls the imaging apparatus and the electrical apparatus. The imaging apparatus may comprise a moveable stage for holding the nanofluidic structure e.g. for holding a nanofluidic chip comprising the nanofluidic structure. This may allow the microscope to image different parts of the nanofluidic structure and / or on different nanofluidic structures as required.

[0042] The nanofluidic structure may be fabricated using one or more lithographic techniques such as photolithography (e.g. UV lithography), electron beam lithography or two-photon lithography (also known as two-photon direct laser writing). In a set of embodiments, one or more micrometre-scale elements of the nanofluidic structure (e.g. the microchannel and / or the fluid input) may be formed using UV lithography. Additionally or alternatively one or more nanometrescale elements of the nanofluidic structure (e.g. the dead-end nanochannel) may be formed using two-photon lithography.

[0043] The nanofluidic structure of the system described herein is itself considered to be independently inventive and thus according to a fifth aspect of the present invention there is provided a nanofluidic structure comprising at least one dead-end nanochannel extending in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width.

[0044] The fifth aspect extends to a nanofluidic chip comprising the nanofluidic structure. The nanofluidic chip may comprise a plurality of nanofluidic structures, e.g. sharing a common substrate and / or structure layer.

[0045] According to a sixth aspect of the present invention there is provided a nanofluidic structure comprising: a fluid input for receiving a sample comprising a substance dispersed in a fluid; a microchannel extending from the fluid input; and at least one dead-end nanochannel extending from the microchannel in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width.

[0046] The sixth aspect extends to a nanofluidic chip comprising the nanofluidic structure. The nanofluidic chip may comprise a plurality of nanofluidic structures, e.g. sharing a common substrate and / or structure layer. Some or all of the nanofluidic structures may comprise a microchannel and a dead-end nanochannel as described herein. As explained above, at least one dead-end nanochannel of the nanofluidic structure may extend from a microchannel which in turn extends from a fluid input. However, it is possible to provide a fluid sample to the nanochannel in a different way. In a set of embodiments, the nanofluidic structure comprises a main body with an outer surface arranged to be put in contact with a fluid sample comprising a substance dispersed in a fluid (e.g. placed into a bulk container such as a beaker or a flask containing the sample). In some such embodiments the entrance of the at least one dead-end nanochannel is located on the outer surface (i.e. to be in fluid communication with the fluid reservoir), i.e. the at least one dead-end nanochannel extends from the outer surface into the main body in the extension direction. In other words, in such embodiments, the extension direction of the dead-end nanochannel has a component which is normal to the outer surface at the entrance to the dead-end nanochannel. In a set of embodiments, the dead-end nanochannel extends substantially perpendicular to the outer surface at the entrance to the dead-end nanochannel (e.g. making an angle of between 70° and 110°, between 80° and 100°, between 85° and 95° or between 88° and 92°).

[0047] The main body may comprise or consist of a polymer, e.g. polydimethylsiloxane (PDMS) or a UV-curable photo resin.

[0048] According to a seventh aspect of the present invention there is provided a system for concentrating a substance dispersed in a fluid, the system comprising: a nanofluidic structure comprising: a main body having an outer surface arranged to be put in contact with a fluid sample comprising a substance dispersed in a fluid; and at least one dead-end nanochannel extending from the outer surface into the main body in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and an electrical apparatus arranged to apply an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel, to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

[0049] According to an eighth aspect of the present invention there is provided a method for concentrating a substance dispersed in a fluid, the method comprising: providing a sample comprising the substance dispersed in the fluid to an outer surface of a nanofluidic structure comprising a dead-end nanochannel extending from the outer surface into a main body of the nanofluidic structure in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and applying an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel so as to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

[0050] In embodiments of the seventh and eighth aspects of the invention, the electric field may be applied with an electrical apparatus comprising at least one electrode positioned within the main body of the nanofluidic structure, e.g. at a position towards which the dead-end nanochannel(s) extend from the outer surface. The electrode may extend in a perpendicular direction to the extension direction of the dead-end nanochannel(s). One or more second electrodes may be positioned within the fluid sample away from the outer surface, to establish the electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel.

[0051] Providing the sample comprising the substance dispersed in the fluid to the outer surface may comprise immersing part or all of the outer surface in a container containing the sample (e.g. a beaker or flask).

[0052] According to a ninth aspect of the present invention there is provided a nanofluidic structure comprising: a main body having an outer surface arranged to be put in contact with a fluid sample comprising a substance dispersed in a fluid; and at least one dead-end nanochannel extending from the outer surface in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width.

[0053] The ninth aspect extends to a nanofluidic chip comprising the nanofluidic structure. The nanofluidic chip may comprise a plurality of nanofluidic structures, e.g. sharing a common main body and outer surface.

[0054] In a set of embodiments the nanofluidic structure comprises a plurality of dead-end nanochannels extending from the outer surface. The dead-end nanochannels may be arranged in an array. Each of the plurality of dead-end nanochannels may extend in the extension direction from respective entrances to respective dead-ends (i.e. the dead-end nanochannels may be arranged in parallel). The nanofluidic structure may comprise multiple sets of dead-end nanochannels, e.g. with the dead-end nanochannels of each set being arranged in parallel.

[0055] The outer surface of the main body may have several parts (e.g. sides of a cuboid main body). The nanofluidic structure may comprise multiple sets of dead-end nanochannels, with each set extending from a different part of the outer surface. Each set of nanochannels may extend in a different direction, e.g. depending on the orientation of each part of the outer surface. The deadend nanochannels of different sets may all extend towards a common electrode (e.g. located centrally in the main body). In a set of embodiments, the nanofluidic structure comprises two sets of dead-end nanochannels extending from opposing parts of the outer surface, e.g. extending in opposite directions from their entrances to their dead-ends. The arrangement of dead-end nanochannels may be symmetric.

[0056] According to a tenth aspect of the present invention there is provide a method of manufacturing a nanofluidic chip comprising the nanofluidic structure comprising: placing an electrode within or near to a stamp comprising at least one nanopillar which extends in an extension direction, so that the electrode extends in a perpendicular direction to the extension direction, said nanopillar having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; at least partially immersing the electrode and the nanopillars with a liquid polymer; and curing the liquid polymer to produce a nanofluidic structure comprising: a main body formed from cured polymer having an outer surface; and at least one dead-end nanochannel corresponding to the at least one nanopillar, extending from the outer surface into the main body in the extension direction from an entrance to a dead-end.

[0057] In some embodiments, the liquid polymer comprises PDMS. In such embodiments, curing the liquid polymer may comprise temperature treatment. In a set of embodiments the liquid polymer comprises UV curable photo resin. In such embodiments, curing the liquid polymer may comprise exposing the polymer to UV light.

[0058] The nanopillar(s) may be cuboid or cylindrical. A nanopillar will be understood to have a smallest dimension (e.g. a height or width of a cuboid channel or a width (i.e. diameter) of a cylindrical channel) that is on the nanometre or near-nanometre scale, i.e. 5 pm or less, 2 pm or less, 1 pm or less, 500 nm or less or 100 nm or less. Features of any aspect or embodiment described herein may, wherever appropriate, be applied to any other aspect or embodiment described herein. Where reference is made to different embodiments, it should be understood that these are not necessarily distinct but may overlap. It will be appreciated that all of the preferred features of the nanofluidic structure described with reference to embodiments of the first to fourth aspects described above may also apply to embodiments of the other aspects of the invention, and vice versa.

[0059] One or more non-limiting examples will now be described, by way of example only, and with reference to the accompanying figures in which:

[0060] Figure 1 is a schematic view of a system according to an embodiment of the present invention;

[0061] Figure 2 is a schematic view of the nanofluidic chip of the system;

[0062] Figure 3 is a schematic view of part of the nanofluidic structure of the system;

[0063] Figure 4 is a schematic view of a system according to an embodiment of the invention;

[0064] Figure 5 is a schematic view of a nanofluidic chip according to an embodiment of the invention;

[0065] Figures 6-11 are schematic views of a method of manufacturing a nanofluidic chip according to an embodiment of the invention;

[0066] Figure 12 is a schematic view of another system according to an embodiment of the present invention; and

[0067] Figure 13 is a schematic view of a method of making the system of Figure 12.

[0068] Figure 1 shows a system 2 for concentrating tau protein monomers to expedite the aggregation of tau aggregates. The system 2 may also be used to concentrate other substances dispersed in a fluid, e.g. to facilitate aggregation of other proteins.

[0069] The system 2 comprises a nanofluidic chip 4 and an electrical apparatus 5. The nanofluidic chip 4, which is also shown in Figure 2, comprises a silicon substrate 6 and a structure layer 8 adjacent the substrate 6 and formed from polydimethylsiloxane (PDMS).

[0070] The nanofluidic chip 4 comprises a nanofluidic structure 14 defined by the substrate 6 and the structure layer 8. The nanofluidic structure 14 comprises several fluid-carrying regions. The structure layer 8 (illustrated in Figure 1) comprises a layer of PDMS which defines the sides and top boundaries of the fluid-carrying regions. The bottom boundaries of the fluid-carrying regions are defined by the substrate 6.. The nanofluidic structure 14 comprises four fluid inputs 10 aligned with holes in the structure layer 8, two microchannels 12, a through nanochannel 16 and a plurality of dead-end nanochannels 18. The fluid inputs 10 are used to introduce fluid into the nanofluidic structure 14 and drain fluid from the nanofluidic structure 14.

[0071] The nanochannels 16, 18 are shown in more detail in Figure 3. The through nanochannel 16 extends from between the two microchannels 12. Each of the dead-end nanochannels 18 extends from its respective microchannel 12 from an entrance 22 to a dead-end 24, along an extension direction that is perpendicular to the microchannels 12. The dead-end nanochannels 18 are longer in the extension direction than they are wide.

[0072] The electrical apparatus 6 comprises a voltage generator 26 and two electrodes 28. The electrodes 28 are positioned in fluid inputs 10 extending from opposing microchannels 12.

[0073] In use, a user adds fluid in which Tau protein monomers are dispersed to the fluid reservoirs 20 (e.g. using a pipette). The user then controls the electrical apparatus 6 to generate a voltage with the voltage generator 26 which is applied via the electrodes 28 to generate an electric field over the nanofluidic chip 4.

[0074] The electric field is aligned with the extension direction of the dead-end nanochannels 18. The electric field induces surface flow in the fluid within the nanofluidic chip 4. Tau protein monomers flow along the microchannels 12 and into the nanochannels 16, 18. The flow also produces vortices at or adjacent the micro-nano interface at the entrances 22 of the dead-end nanochannels 18.

[0075] The electric field thus causes local increases in Tau protein monomer concentration at the dead-ends 24 of the dead-end nanochannels 18 as well as in the vortices at the entrances 22 to the dead-end nanochannels 18. This localised concentration increase significantly accelerates the aggregation of the protein monomers into oligomers and fibrils.

[0076] The structure layer 8 is optically transparent. This means that the contents of the nanofluidic structure 14 can be observed, e.g. the aggregation of the Tau protein monomers can be observed in real time. Figure 4 shows an embodiment of the system which includes imaging apparatus 100 for imaging the contents of the nanofluidic structure 14.

[0077] The imaging apparatus 100 includes a sample stage 102, a microscope 104 and a processing device 106. The nanofluidic chip 4 is positioned on the stage, which can be moved around to image different parts of the nanofluidic chip 4. The microscope 104 is used to image a desired section of the nanofluidic chip 4 (e.g. the entrance 22 of a dead-end nanochannel 18). The processing device 106 receives and stores image data from the microscope 104.

[0078] It may be advantageous to provide more than one nanofluidic structure on a single nanofluidic chip (e.g. to allow for convenient imaging of several nanofluidic structures in quick succession). Figure 5 shows a nanofluidic chip 200 according to an embodiment of the present invention which comprises a plurality of different nanofluidic structures 202.

[0079] The nanofluidic structures 202 share a common substrate, a structure layer and a common sealing layer. The nanofluidic structures 202 may be identical (e.g. for conveniently repeating the same concentration procedure multiple times) or they may be different (e.g. for performing concentration procedures on different samples). For instance, the nanofluidic structures 202 may have different nanofluidic features (i.e. defined by different patterns in the structure layer), such as different arrangements and / or shapes and / or dimensions of nanochannels and / or microchannels.

[0080] A method of manufacturing a nanofluidic chip according to the present invention will now be desired with reference to Figures 6-11.

[0081] First, a lithography master 500 for forming the nanofluidic structure is produced by UV mask lithography and two-photon direct laser writing. As shown in Figure 6, a thin layer of Sll-8 photoresist 502 is spin coated onto a silicon substrate 504, and the result is exposed to UV light 506 through a mask 508 to cross-link sections 510 of the photoresist 502 that will ultimately define micrometre-scale parts of the nanofluidic structure in the finished chip (e.g. microchannels and fluid inputs).

[0082] Then, as shown in Figure 7, two-photon direct laser writing is used to cross-link sections 512 of the photoresist 502 that will ultimately define nanometre-scale parts of the nanofluidic structure of the nanofluidic chip (e.g. through nanochannels and dead-end nanochannels)

[0083] The photoresist 502 is then developed to produce the lithography master 500shown in Figure 8.

[0084] Next, as shown in Figure 9, PDMS is applied to the master 500 and cured to form a transparent structure layer 516 with an integral nanofluidic structure imprinted by the nanometre and micrometre-scale features of the master 500. As shown in Figure 10, the structure layer 516 is then removed from the master 500 and plasma bonded to a glass substrate 517 to form a finished nonfluidic chip 524. The glass substrate 517 forms the lower boundary of the nanofluidic structure in the finished chip 524.

[0085] Finally, a cutting tool 518 is used to cut holes 520 into the PDMS structure layer 516 aligned with the fluid inputs of the nanofluidic structure. These holes 520 provide access for a user to add fluid to and remove fluid from the nanofluidic structure, e.g. using a pipette 522 as shown in Figure 11.

[0086] Figure 12 shows another system 602 for concentrating substances dispersed in a fluid, e.g. tau protein monomers. The system 602 comprises a nanofluidic chip 604 and an electrical apparatus 605. The nanofluidic chip 604 is shown immersed in a fluid container 611 containing a fluid sample 613 with a substance dispersed therein.

[0087] The nanofluidic chip 604 comprises a main body 608 formed from polydimethylsiloxane (PDMS) with space for a first electrode 628 centrally in the main body 608. The main body 608 extends from the first electrode 628 to an outer surface 609. The outer surface 609 is in contact with the fluid sample 613. The main body 608 may be cuboid or cylindrical. The outer surface 609 can be divided into two parts, one on either side of the main body 608 (i.e. the left and right side as illustrated in Figure 12).

[0088] The nanofluidic chip 604 comprises a nanofluidic structure 614 defined by the main body 608. The nanofluidic structure 614 comprises a plurality of dead-end nanochannels 618 defined in the main body 608, which extend from entrances at the outer surface 609 towards dead-ends deeper in the body, nearer to the central electrode 628. The nanofluidic structure 614 comprises two sets of dead-end nanochannels 618 which extend from the two parts of the outer surface 609 respectively.

[0089] The electrical apparatus 6 comprises a voltage generator 626, a first electrode 628 and two second electrodes 629. The first electrode 628 is positioned centrally in the main body 608 and the second electrodes 629 are located in the fluid sample 613 on either side of the nanofluidic chip 604. The electrodes 628 may be cuboid (e.g. when the main body 609 is cuboid), or cylindrical (e.g. when the main body 609 is cylindrical).

[0090] In use, a user puts the nanofluidic chip 604 into a container 611 containing a fluid sample 613 and controls the electrical apparatus 605 to generate a voltage with the voltage generator 626 between the first and second electrodes 628, 629. This generates an electric field over the nanofluidic chip 604.

[0091] The electric field is aligned with the extension direction of the dead-end nanochannels 618. The electric field induces flow in the fluid, causing the concentration of the substance of interest dispersed in the fluid sample 613 to increase at the dead-ends of the dead-end nanochannels 618 as well as at the entrances to the dead-end nanochannels 618.

[0092] As illustrated in Figure 13, the nanofluidic chip 604 may be manufactured by placing a metal electrode 628 (e.g. of approximately 500 urn, 1 mm, 2 mm or 5mm length) within to a micro- lithographically structured stamp 702. The stamp 702 contains nanopillars 704 (nano-scale pillars) which extend perpendicular to the long axis of the electrode 628.

[0093] The electrode 628 and the stamp 702 are then immersed in liquid PDMS 706 (or another polymer such as UV curable photo resin). The PDMS 706 is cured via temperature treatment (UV exposure may be for UV curable photo resin), which forms the dead-end nanochannels 618 in the finished nanofluidic chip 604.

[0094] While the invention has been described in detail in connection with only a limited number of embodiments, it should be readily understood that the invention is not limited to such disclosed embodiments. Rather, the invention can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the scope of the invention. Additionally, while various embodiments of the invention have been described, it is to be understood that aspects of the invention may include only some of the described embodiments. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.

Claims

Claims1. A system for concentrating a substance dispersed in a fluid, the system comprising: a nanofluidic structure comprising at least one dead-end nanochannel extending in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and an electrical apparatus arranged to apply an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel, to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

2. The system of claim 1 , wherein the substance comprises one or more proteins.

3. The system of claim 2, wherein the substance comprises Tau protein.

4. The system of any preceding claim, arranged to induce aggregation of the substance.

5. The system of any preceding claim, arranged to filter the fluid by inducing sedimentation of the substance.

6. The system of any preceding claim, wherein the nanofluidic structure is arranged for fluid connection to a cell culture apparatus.

7. The system of any preceding claim, wherein a shape and / or dimension of the nanofluidic structure is arranged for use with an expected particle shape and / or dimension of the substance.

8. The system of any preceding claim, wherein the length of the dead-end nanochannel is at least ten times the width of the dead-end nanochannel.

9. The system of any preceding claim, wherein the nanofluidic structure comprises: a fluid input for receiving a sample comprising the substance dispersed in the fluid; and a microchannel extending from the fluid input; wherein at least one dead-end nanochannel extends from the microchannel in the extension direction.

10. The system of claim 9, wherein the nanofluidic structure comprises a fluid output for draining a fluid from the nanofluidic structure.

11. The system of claim 10, wherein the fluid output of the nanofluidic structure is connected to the fluid input of the nanofluidic structure, to allow a fluid to be re-circulated after passing through the nanofluidic structure.

12. The system of any of claims 9-11 , wherein the fluid input is arranged for connection to a pumping apparatus for inducing fluid flow in the nanofluidic structure.

13. The system of any of claims 9-12, wherein the dead-end nanochannel and the microchannel are arranged to induce the formation of a flow trap at the entrance of the deadend nanochannel.

14. The system of any of claims 9-13, wherein the dead-end nanochannel extends substantially perpendicular to the microchannel.

15. The system of any of claims 9-14, wherein the nanofluidic structure comprises a plurality of dead-end nanochannels extending from the microchannel in the extension direction from respective entrances to respective dead-ends.

16. The system of any of claims 9-15, wherein the nanofluidic structure comprises a second microchannel and a through channel which extends from the first microchannel to the second microchannel.

17. The system of claim 16, wherein the through-channel is a nanochannel.

18. The system of any of claims 9-17, wherein the nanofluidic structure comprises a substrate and a structure layer adjacent the substrate, the structure layer defining one or more boundaries of the microchannel(s) and / or nanochannel(s) of the nanofluidic structure.

19. The system of claim 18, wherein the structure layer is transparent to at least some wavelengths of light.

20. The system of any of claims 1-8, wherein the nanofluidic structure comprises a main body with an outer surface arranged to be put in contact with a fluid sample comprising asubstance dispersed in a fluid and the at least one dead-end nanochannel extends from the outer surface into the main body in the extension direction.

21. The system of any preceding claim, comprising a nanofluidic chip comprising the nanofluidic structure.

22. The system of claim 21 , wherein the nanofluidic chip comprises a plurality of nanofluidic structures.

23. The system of any preceding claim, comprising imaging apparatus for imaging the nanofluidic structure.

24. The system of claim 23, wherein the imaging apparatus comprises a microscope such as a total internal reflection fluorescence microscope.

25. A method for concentrating a substance dispersed in a fluid, the method comprising: providing a sample comprising the substance dispersed in the fluid to a nanofluidic structure comprising a dead-end nanochannel extending in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width; and applying an electric field over the nanofluidic structure which is at least partially aligned with the extension direction of the dead-end nanochannel so as to encourage a concentration of the substance to increase at the entrance and / or the dead-end of the dead-end nanochannel.

26. The method of claim 25, wherein providing the sample comprising the substance dispersed in the fluid to the nanofluidic structure consists of inputting the sample to a fluid input of the nanofluidic structure, wherein the nanofluidic structure comprises a microchannel extending from the fluid input and the dead-end nanochannel extends from the microchannel in the extension direction.

27. The method of claim 25, comprising providing the sample comprising the substance dispersed in the fluid to an outer surface of the nanofluidic structure into a main body of the nanofluidic structure, wherein the dead-end nanochannel extends from the outer surface in the extension direction.

28. A method of analysing a substance dispersed in a fluid, the method comprising:concentrating the substance using the method of any of claims 25-27; and determining a size of one or more substance particles by determining a rate at which the substance concentrates on application of the electric field and / or a rate at which the substance disperses after application of the electric field.

29. A nanofluidic structure comprising at least one dead-end nanochannel extending in an extension direction from an entrance to a dead-end, said dead-end nanochannel having a length in the extension direction and a width perpendicular to the extension direction, the length being greater than the width.

30. The nanofluidic structure of claim 29, comprising: a fluid input for receiving a sample comprising the substance dispersed in the fluid; and a microchannel extending from the fluid input; wherein at least one dead-end nanochannel extends from the microchannel in the extension direction.

31. The nanofluidic structure of claim 29, comprising a main body with an outer surface arranged to be put in contact with a fluid sample comprising a substance dispersed in a fluid, wherein the the at least one dead-end nanochannel extends from the outer surface into the main body.

32. A nanofluidic chip comprising the nanofluidic structure of any of claims 29-31.

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