WO2021186171A1

WO2021186171A1 - Microfluidic device and method

Info

Publication number: WO2021186171A1
Application number: PCT/GB2021/050663
Authority: WO
Inventors: Shawn BALDACCHINO
Original assignee: Applied Biotech Ltd
Priority date: 2020-03-17
Filing date: 2021-03-17
Publication date: 2021-09-23
Also published as: EP4136452A1; GB202003827D0

Abstract

A method and device for capturing and detecting extracellular vesicles is described. Flow of a sample fluid is initiated through a plurality of capture chambers and a distinct combination of capture reagents is present in each capture chamber. The capture reagents are retained in each capture chamber, as the sample fluid flows through each capture chamber. Detection reagents may be added following capture.

Description

Microfluidic Device and Method

This invention relates to devices, such as microfluidic devices, and method of using such devices, particularly for the detection of analytes in bodily fluids, such as extracellular vesicles. The invention also relates to methods for diagnosing diseases such as cancer.

Blood-based testing, also known as liquid biopsy, is expected to be significant in the improvement of cancer care. A sensitive and accurate blood test could drastically improve patient survival on all aspects of the cancer care pathway: earlier cancer detection; diagnostic; prognostic; predictive; tumour therapeutic response monitoring; enabling minimal residual disease detection for solid tumours; and surveillance to detect early relapse and/or metastasis.

Currently, early detection screening methods are only available for a small number of cancer types. Therapeutic response monitoring is limited by the number of tissue biopsies that can be sampled and does not provide adequate information on tumour mechanisms. Minimal residual disease determination is only possible in blood malignancies such as leukaemia, but is important to direct treatment decisions and preventing relapse.

Cells release extra-cellular vesicles (EVs) into the inter-cellular spaces and into the circulation. It is understood that tumours release specific EVs that modulate the microenvironment to promote growth and metastasis. Consequently, EVs are expected to be found in blood at early stages of the disease. EVs contain DNA, RNA (e.g. mRNA, miRNA) and proteins that are representative of the cells of origin. Measuring and profiling this information would allow detection of malignant signatures originating from a solid tumour that is shedding EVs.

A wide variety of EV isolation methods are currently employed to isolate EVs from bodily fluid. These include ultracentrifugation, size exclusion chromatorgraphy (SEC), filtration, precipitation and magnetic bead isolation [1] These methods are generally used primarily for isolation, such that downstream methods are required to characterise the isolated EVs. Conventional exosome detection and analysis methods include ELISA and western blot, flow cytometry, TRPS, and NTA & DLS.

More recently, microfluidic-based techniques for EV isolation and analysis have provided advantages over the more conventional methods, such as smaller sample volumes, shorter processing times and the capacity for integrating multiple functional modules and automation.

The inventor has developed an efficient and sensitive method and device that allows, inter alia, the selective capture and detection of EV populations, such as tumour-derived EV populations.

According to the invention, there is provided a method comprising initiating flow of a sample fluid through at least one capture chamber in a device, one or more capture reagent being present in the or each capture chamber; and retaining the one or more capture reagent in the or each capture chamber as the sample flows through the or each capture chamber.

Preferably, the device is a microfluidic device, a microfluidic chip, or a lab-on-a-chip device. The device may be a device of the invention, as described herein.

The method may comprise introducing or loading the one or more capture reagent into the or each capture chamber, prior to initiating the flow of sample fluid through the or each capture chamber. Alternatively, the or each capture chamber may be pre-loaded with the one or more capture reagent.

The capture reagent may be retained in the or each capture chamber by a semi-permeable physical barrier, such as a filter. Preferably, the one or more capture reagent comprises a magnetic component, such as a magnetic bead, and is retained in the or each capture chamber by a magnetic force. Passage of capture reagents between capture chambers may not be permitted, or may be prevented, as the sample flows through the or each capture chamber.

Preferably, the one or more capture reagent is mobile, such that it can move within the capture chamber. However, preferably the one more capture reagent cannot move between capture chambers as the sample flows through the or each capture chamber.

The one or more capture reagent may be in solution. Preferably, the one or more capture reagent is not immobilised, such as covalently immobilised, to the device. For example, the one or more capture reagent may not be immobilised to a wall of the or each capture chamber. Preferably, a plurality of capture reagents are in the or each capture chamber. The or each capture chamber may comprise a plurality of different or distinct capture reagents, such that more than one analyte or target can be captured in the or each capture chamber.

The capture reagents may be identifiable or distinguishable by fluorescence. So, distinct capture reagents may provide a distinct fluorescent signal. For example, capture reagents may be distinguishable by virtue of the ratio of two or more fluorescent dyes incorporated within. For example, different ratios of fluorescent dyes may be incorporated in magnetic beads of the capture reagents.

Preferably, there is a plurality of capture chambers. The one or more capture reagent in each capture chamber may thus be retained in its respective capture chamber as the sample flows through each capture chamber.

According to the invention, there is provided a method comprising: initiating flow of a sample fluid through at least one capture chamber in a device in which one or more capture reagent is present in the or each capture chamber; and retaining the one or more capture reagent in the or each capture chamber as the sample flows through the or each capture chamber, wherein the device comprises a plurality of capture chambers and the one or more capture reagent in each capture chamber is retained in its respective capture chamber as the sample fluid flows through each capture chamber.

The use of multiple capture chambers may allow capture of multiple analytes or multiple targets from a sample. Distinct analytes may be captured in each chamber. For example, specific EV types or species may be captured in each chamber. The analyte or analytes captured in each chamber may be specific to a particular condition or medical indication. For example, each chamber may capture an analyte or analytes indicative of a particular tumour type. The analyte or analytes in each chamber may then be analysed more easily or processed more efficiently. The, or each, analyte that is captured may include analytes that are not associated with a particular medical indication, such as normal, physiological EVs. Consequently, the remaining analyte or analytes in the sample that have not been captured may be the analyte or analytes of interest that are relevant to a particular medical indication.

When there is a plurality of capture chambers, each capture chamber may comprise a distinct capture reagent. This may permit distinct analytes to be captured in different capture chambers. Each capture chamber may comprise a plurality of different capture reagents, such that more than one analyte can be captured in each chamber. Each capture chamber may comprise a distinct combination, or profile, of capture reagents.

There may be at least two, at least three, or at least four capture chambers.

If there is a plurality of capture chambers, they are preferably arranged in series, such that the fluid passes through each capture chamber in sequence. Alternatively, the capture chambers could be arranged in parallel.

According to the invention, there is provided a method comprising: initiating flow of a sample fluid comprising extracellular vesicles through at least one capture chamber in a device, in which one or more capture reagent is present in the or each capture chamber; and retaining the one or more capture reagent in the or each capture chamber as the sample fluid flows through the or each capture chamber, wherein: a) the device comprises a plurality of capture chambers and in each capture chamber is a distinct capture reagent, or a distinct combination of capture reagents; and/or b) the device comprises a distinct combination of capture reagents in the or each capture chamber.

According to the invention, there is provided a method, preferably for capturing and detecting extracellular vesicles, comprising: initiating flow of a sample fluid comprising extracellular vesicles through a plurality of capture chambers in a device, in which a distinct combination of capture reagents is present each capture chamber; and retaining the capture reagents in each capture chamber, as the sample fluid flows through each capture chamber.

The method may comprise urging the capture reagent in the or each capture chamber to move independently of sample fluid flow. For example, the capture reagent may be urged to move across the direction of sample fluid flow, or against the direction of fluid flow, whilst the sample fluid is flowing through the or each capture chamber. For example, the capture reagent may be urged to reciprocate, oscillate or move back and forth within the capture chamber when the sample fluid is in the capture chamber. The capture reagent may move, preferably repeatedly, from one side, or edge, of the capture chamber, to an opposing side, or edge, of the capture chamber when the sample is in the capture chamber. This principle is exemplified in Figures 2 and 3. As exemplified in Figure 2, the capture reagent in the or each capture chamber may be urged to move laterally relative to the direction of fluid flow. As exemplified in Figure 3, the capture reagent may be urged to move in a direction which is against, or opposed to, the direction of fluid flow. The movement of the capture reagent may be independent of any movement caused by turbulent flow of the sample fluid. The movement of the capture reagent within the or each capture chamber can improve mixing and increase exposure of the capture reagents to the sample, resulting in improved analyte capture.

According to the invention, there is provided a method comprising: initiating flow of a sample fluid through at least one capture chamber in a device in which one or more capture reagent is present in the or each capture chamber; and retaining the capture reagent in the or each capture chamber as the sample flows through the or each capture chamber, wherein the method comprises urging the one or more capture reagent in the or each capture chamber to move independently of sample fluid flow.

The oscillation of the capture reagent may be achieved by altering the magnetic force exerted by a magnet or component that can generate a magnetic force.

A magnet or a component that can generate a magnetic force may be positioned adjacent to the or each capture chamber such that it can exert a magnetic force on reagents in the or each chamber.

There may be a first magnet or component and a second magnet or component positioned on opposing sides of the or each capture chamber. The first and second magnets/components could be permanent magnets which alter their position relative to the capture chamber. For example, each magnet/component could move away from, or closer to, each side of the capture chamber. Alternatively, electromagnets could be employed, and the strength of the magnetic force could be adjusted electrically. The relative strength of the magnetic force generated on reagents in the or each capture chamber, by the first and second magnets/components, may be variable and may alternate.

Preferably, the method comprises recirculating the sample through the or each capture chamber. This may maximise the efficiency of analyte capture. If there is a plurality of capture chambers, the sample could be recirculated through a portion of the available capture chambers. For example, the sample could be recirculated through one of the plurality of capture chambers. Thus, recirculation may be excluded from certain chambers at particular stages of the method. According to the invention, there is provided a method comprising: initiating flow of a sample fluid through at least one capture chamber in a device in which one or more capture reagent is present in the or each capture chamber; and retaining the capture reagent in the or each capture chamber as the sample flows through the or each capture chamber, wherein the method comprises recirculating the sample fluid through the or each capture chamber

The method of the invention may be for capturing or isolating one or more analytes from a sample. Preferably, the method is for capturing or isolating extracellular vesicles, such as exosomes, microvesicles and/or apoptotic bodies. Alternatively, or additionally, the method may be for capturing cell-free DNA or circulating free DNA (cfDNA).

The one or more capture reagents preferably comprises a carrier complexed with, or conjugated to, a capture moiety that has a binding specificity for an analyte in a sample. In one example, the capture moiety is covalently bound to the carrier. In another embodiment, the capture moiety is biotinylated and the carrier is modified with streptavidin, such that the capture moiety can be conjugated to the carrier.

The capture moiety may be a protein or ligand. In a particularly preferred embodiment, the capture moiety that binds to the analyte is an antibody or a fragment or derivative thereof that retains the ability to bind the target analyte, such as Fab, F(ab) , Fv, scFv, scFv-Fc, nanobody or diabody. Alternatively, the capture reagent may be an oligonucleotide. The capture reagent may be an aptamer.

Preferably, the carrier is a bead, more preferably a magnetic bead. Examples of suitable beads include xMAP™ beads (Luminex™), Dynabeads™ (Thermofisher™) and MACSPlex™ (Miltenyi™).

Preferably, there are at least 500 or at least 1000 beads of a particular capture reagent in the or each capture chamber. Preferably, there are 5000 beads or less of a particular capture reagent in the or each capture chamber. A lower bead count may be preferable because this can result in higher sensitivity.

An exemplary capture reagent and a mechanism of capturing exosomes is illustrated in Figure 4a.

The method may comprise detection of the one or more analyte. Following capture and before detection, the one or more capture reagents (which may be bound to analyte) may be washed. Washing may be achieved by initiating flow of a cleaning fluid, such as an aqueous buffer (e.g. phosphate-buffered saline (PBS)), through the or each capture chamber.

The method may comprise exposing the one or more capture reagent (which may be bound to analyte) to one or more detection reagent. The method may comprise introducing a detection reagent to the device, following capture of the one or more analyte. The detection reagent may comprise a detectable label, such as a fluorophore or a dye. In one example, the label may be conjugated to a moiety with a binding specificity for the analyte. This may include a ligand or protein, such as an antibody, or a derivative or fragment thereof that is able to bind to the analyte. Fluorophores may be conjugated to the moiety, for example via avidin or a polymer binding protein. An exemplary detection reagent and a method of detecting exosomes is illustrated in Figure 4b.

The detection reagent may comprise a primary detection moiety (e.g. a primary antibody) which binds to the analyte (e.g. EV). A secondary detection moiety (e.g. a secondary antibody) bound to a label (e.g. fluorophore or dye) may bind to the primary detection moiety. Detection may utilise ELISA. The detection reagent may comprise a lipophilic dye. The detection reagent may comprise an antibody conjugated to a fluorophore. The detection reagent may comprise a detection antibody with biotin, which can be detected using a streptavidin-fluorophore conjugate.

The detection reagent may be added to the or each capture chamber. A different detection reagent may be added to each capture chamber, if there is a plurality of capture chambers.

A plurality of different capture reagents may be introduced into the or each capture chamber. If there is a plurality of capture chambers, each capture chamber may comprise a distinct combination of capture reagents.

In some embodiments, the same detection reagent or detection reagents may be introduced into each capture chamber, or the same marker or markers may be detected in each capture chamber.

Following addition of a detection reagent, a complex may be formed which includes the capture reagent, analyte and detection reagent. This may be referred to as a capture reagent-analyte-detection reagent complex. Such a complex is exemplified in Figure 3b. If the analyte is an EV, such as an exosome, this may be referred to as an exosome detection complex, or EV detection complex.

In one example, the chambers on the device could be visualised, e.g. using a microscope, such as a fluorescent microscope, or a detector or camera, for the presence of an analyte, or to determine the amount of a particular analyte. The amount of a particular analyte on the or capture reagent may be determined.

Capture reagent-analyte-detection reagent complexes could be extracted from the chip for further analysis. For example, they could be analysed using a flow cytometer or Luminex™ analysers (e.g. if using xMAP™ beads). If capture reagents comprising magnetic beads are used, three parameters may be measured: the bead type or species, bead count, and/or signal on bead (indicating, for example, detection antibody / fluorophore presence). Thus surface marker expression information can be obtained providing an indication of the analyte (e.g. EV species) present in each chamber. For example, a bead conjugated with an anti-HEFt2 antibody giving a fluorescence signal off a secondary anti-CD63 antibody could be differentiated from a bead conjugated with an anti-CDH1 antibody giving a fluorescence signal off the same secondary anti-CD63 antibody.

In some embodiments, the one or more detection reagent may be introduced into the or each capture chamber simultaneously with, or at the same time as, the sample. For instance, the one or more detection reagents may be added to the sample prior to capture. This may enable detection in real time.

Alternatively, or additionally, the one or more detection reagents may be introduced following capture.

The captured analyte or analytes from each chamber could also be processed for other downstream applications. For example, analytes such as EVs, could be released from the capture reagent and/or the detection reagent. This may be achieved by chemical cleavage of a spacer between the carrier and the capture moiety to extract analytes. The extracted analytes may include intact EVs. EVs could also be lysed to extract DNA, RNA and/or proteins for analysis. Lysates could be collected from each capture chamber individually, which may therefore result in the collection of lysates from specific EVs.

Methods of the invention may capture targets, such as EVs, or cells. The targets may thus be a biological structure comprising a plurality of various molecules or markers. The method may involve analysis of analytes extracted from the targets (e.g. following lysis). The analytes may include, nucleotides, proteins, or other molecules.

In a preferred example, the method and devices of the invention are for capturing and/or detecting EVs, preferably for cancer detection. Capture reagents and/or detection reagents may thus comprise moieties, such as antibodies, that are specific for one or more of the targets or markers. Methods of the invention may comprise capturing or detecting one or more of the targets or markers listed in Table 1 . The markers may include protein biomarkers (such as surface proteins or internal proteins, DNA biomarkers and/or RNA biomarkers), which may be detected following lysis. Table 1 : List of candidate capture and profiling targets for EV isolation and characterization

The sample fluid may be any bodily fluid, such as plasma, serum, cerebrospinal fluid, pleural fluid, urine or bronchial lavages. Other potential fluids include ascitic fluid, saliva, sputum, stool, breast milk, tears. Preferably, the bodily fluid is plasma. Preferably the fluid is extracted from a human or animal.

The sample fluid may be diluted, for example diluted with buffer, prior to analysis.

The method of the invention is preferably a multiplex assay method that captures and/or detects multiple analytes simultaneously. So, the method may enable multiplex capture and multiplex detection. According to the invention there is provided an in vitro or ex vivo method for diagnosing cancer in a human or animal. The method may comprise capturing and/or detecting EVs from a sample, for example using a capture and/or detection method as described herein. The sample may be a bodily fluid sample extracted from a human or animal. The method may comprise capturing and/or detecting one or more of the targets or markers listed in Table 1. The method may be for diagnosis of breast, ovarian, prostate, lung, colorectal and/or brain cancer. The method of the invention may also be used for disease prognosis, such as cancer prognosis. The method of the invention may also be used for therapeutic response prediction, such as companion diagnostics.

According to the invention, there is provided a device comprising: at least one capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through the or each capture chamber; and a retainer that can retain the capture reagent in the or each capture chamber as the fluid flows, in use, through the or each capture chamber. The device is preferably for capturing one or more analyte from a sample fluid and/or detecting one or more analyte in the fluid sample.

The device is preferably a microfluidic device, microfluidic chip or a lab-on-a-chip.

The device may comprise a reagent inlet for introducing a capture reagent into the or each capture chamber. The, or each, capture chamber may be connected to its own reagent inlet, such that, for example, different capture reagents or different combinations of capture reagents can be added to each capture chamber.

Alternatively, the device may be pre-loaded with one or more capture probes or capture reagents, such that one or more capture probes, or capture reagents, do not have to be introduced into the device.

The device may be for carrying out a method of the invention, as described herein.

The retainer preferably comprises a magnet or a component that can exert a magnetic force, such as an electromagnet.

In one embodiment, there are a plurality of capture chambers. Preferably, the plurality of capture chambers are arranged in series. Consequently, in use, the sample fluid would pass through each chamber consecutively, or in sequence. Within each capture chamber may be one or more capture reagent. A different capture reagent, or a different combination of capture reagent, may be in each capture chamber.

According to the invention, there is provided a device for capturing one or more analyte from a sample fluid, the device comprising: at least one capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through the or each capture chamber; and a retainer that can retain the capture reagent in the or each capture chamber as the fluid flows, in use, through the or each capture chamber, wherein the at least one capture chamber comprises a plurality of capture chambers, and a different capture reagent or a different combination of capture reagents, is in each capture chamber.

According to the invention there is provided a device for capturing one or more analytes from a fluid sample, the device comprising: at least one capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through the or each capture chamber; and a retainer for, in use, retaining a capture reagent in the or each capture chamber as the fluid sample flows through the or each capture chamber, wherein: a) the at least one capture chamber comprises a plurality of capture chambers, and a different capture reagent or a different combination of capture reagents, is in each capture chamber; or b) the at least one capture chamber comprises a plurality of capture reagents in the or each capture chamber, and a distinct combination of the capture reagents is in the or each capture chamber

According to the invention, there is provided a device, preferably a microfluidic device, for capturing extracellular vesicles from a fluid sample, the device comprising: a plurality of capture chambers; a distinct combination of capture reagents in each capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through each capture chamber; and a retainer for, in use, retaining the capture reagents in each capture chamber as the fluid sample flows through each capture chamber.

Preferably, the device comprises a means for urging the capture reagent to reciprocate or oscillate in a transverse direction relative to, in use, the direction of sample fluid flow, as already described herein.

According to the invention, there is provided a device for capturing one or more analyte from a sample fluid, the device comprising: at least one capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through the or each capture chamber; and a retainer that can retain the capture reagent in the or each capture chamber as the fluid flows, in use, through the or each capture chamber, and wherein the device comprises a means for urging the capture reagent to reciprocate or oscillate in a transverse direction relative to, in use, the direction of sample fluid flow.

The means for urging the capture reagent to reciprocate or oscillate is preferably a magnet or a component that can exert a magnetic force. The means for urging the capture reagent to reciprocate or oscillate may also be the retainer. Preferably the magnetic force exerted by the magnet or component can vary or alternate. The magnet or the component may comprise a first part and a second part, and the first and second parts may be positioned at opposing sides of the or each capture chamber. The magnetic force that can be applied to the or each capture chamber, by the first and second parts, is preferably variable. The variability in magnetic strength may be achievable as described herein. The device preferably comprises a channel or conduit for recirculating the sample through the or each capture chamber. Circulation through the or each capture chamber may be assisted by means of a switch or a valve. The device may be configured or configurable to enable recirculation through a proportion or a selection of capture chambers.

According to the invention, there is provided a device for capturing one or more analyte from a sample fluid, the device comprising: at least one capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through the or each capture chamber; and a retainer that can retain the capture reagent in the or each capture chamber as the fluid flows, in use, through the or each capture chamber, wherein the device comprises a channel for recirculating the sample through the or each capture chamber.

In one embodiment the device may comprise chambers or compartments downstream of the capture chambers for processing. For example, the device may comprise a lysis chamber for lysing the analyte, e.g. an EV. The device may comprise a chamber or compartment, which may be downstream of the lysis chamber, for analysing DNA, RNA or proteins. The device may comprise a chamber or compartment that is able to capture and detect cfDNA, using a Magnetic ExTRactiOn procedure (METRO) [8], for example. A schematic diagram of an exemplary device according to the invention is shown in Figure 3.

The invention will now be described, with reference to specific embodiments, in which:

Figure 1 is a schematic diagram of a TEVIPLEX microfluidic chip having three distinct bead set chambers;

Figure 2 is a schematic diagram of a capture chamber of the TEVIPLEX chip comprising two different types of magnetic bead-based capture reagents;

Fig 3 is a schematic diagram of a capture chamber of an alternative example of a TEVIPLEX chip comprising two different types of magnetic bead-based capture reagents;

Figure 4a is a schematic diagram of a process of exosome capture suitable for use in the TEVIPLEX chip;

Figure 4b is a schematic diagram of a process of exosome detection suitable for use in the TEVIPLEX chip; Figure 5 is a schematic diagram of a TEVIPLEX chip including means for downstream processing; and

Figure 6 is a schematic diagram of an alternative TEVIPLEX chip arrangement.

The TEVIPLEX microfluidic system is for capturing EVs, such as exosomes, from bodily fluids by means of antibody-magnetic bead complexes (immunobeads). A schematic diagram of the TEVIPLEX chip is shown in Figure 1 . The chip 1 has a first capture chamber 3, a second capture chamber 5 and a third capture chamber 7 arranged in series. A first reagent input channel 9 is connected to the first chamber, a second reagent input channel 11 is connected to the second chamber and a third reagent input channel 13 is connected to the third chamber. The chip also has a sample input channel 15, a waste output channel 17 and a recirculation channel 19.

A schematic diagram of each capture chamber is shown in Figure 2. Each capture chamber is flanked by a first magnet 21 on one side of the capture chamber and a second magnet 23 on the other side of the capture chamber. The first and second magnets are each able to move closer to or further away from their respective sides of the capture chamber. Alternatively, the magnets may be replaced by electromagnets of which the magnetic strength of each can be varied.

An alternative arrangement of capture chamber is shown in Figure 3.

In use, distinct combinations of immunobeads are added to the first chamber 3, second chamber 5 and third chamber 7 through their respective reagent input channels 9, 11 , 13. Each combination of immunobeads is specific for a particular tumour type. A sample fluid is added to the chip 1 through the sample input channel 15 and the sample flows through the first capture chamber, into the second capture chamber and into the third capture chamber, in series. The immunobeads in each chamber are retained in in their respective chambers by the magnets as the sample fluid flows through each chamber. The relative magnetic strength of the first magnet 21 and the second magnet 23 is varied such that the immunobeads in each chamber reciprocate or oscillate from one side of the chamber to the other, in a transverse direction relative to the direction the fluid flow, as shown in Figures 2 and 3. This increases the exposure of the immunobeads to the exosomes (or other EVs) in the sample. Figure 4a is a schematic representation of the capture of exosomes from the sample which occurs in each capture chamber 3, 5, 7. Figure 4a shows two capture reagents, each capture reagent comprising a magnetic bead conjugated to antibodies, the antibodies being specific for markers on the surface of exosomes. When the capture reagents are exposed to a bodily fluid, they bind and capture exosomes displaying the markers and form exosome capture complexes.

The sample fluid is recirculated through the capture chambers 3, 5, 7 via the recirculation channel 19 to improve capture of the exosomes.

Following capture, the immunobeads are rinsed by the addition of PBS buffer into the sample input channel 15 and the exit of the rinsing water and sample fluid through the waste output channel 17.

After rinsing, detection reagents are added to the first chamber 3, second chamber 5 and third chamber 7 through their respective reagent input channels 9, 11 , 13. Figure 4b is a schematic representation of the detection of exosomes which occurs in each capture chamber 3, 5, 7 after the detection reagents are added. Figure 4b shows the addition of a PE-conjugated (Phycoerythrin-conjugated) antibody and the binding of the PE-conjugated antibody to the exosomes of the exosome capture complex formed in Figure 4a, to form an exosome detection complex. The formation of an exosome detection complex can then be detected to indicate a positive result for exosomes from a particular tumour type, in the sample.

The exosome detection complexes may be removed from the chip for further analysis, Alternatively, the TEVIPLEX chip may include further chambers for processing and characterisation of the exosomes. Figure 5 shows a schematic representation of a potential downstream applications, including a lysis chamber for lysing the exosomes and a downstream RNA, DNA or protein profiling chamber for analysing these components following lysis. There may also be a cfDNA capture and detection chamber.

An alternative arrangement of a TEVIPLEX chip is shown in Figure 5. Similar features to those of Figure 1 (described above) are provided with like reference numerals.

The chip includes a first recirculation channel 10, which permits recirculation of sample fluid through the first chamber 3. This arrangement improves capture of the EVs in the first capture chamber. The chip includes a second recirculation channel 12 which permits recirculation through the first and second capture chambers 3, 5. This arrangement improves capture of the EVs in the first and second capture chambers 3,5.

Example

Reaction process:

1. Beads introduced in each chamber & immobilised.

2. Chip is primed to remove air bubbles.

3. Sample (buffer diluted or otherwise) is introduced and passed through chambers sequentially capturing EVs.

4. Beads are washed to remove unbound

5. Detection antibody cocktail introduced

6. Unbound washed

7. Detection

Sample can be recirculated one or more times exclusively through chamber 1 only before allowing flow into chamber 2, 3, & 4. This can be achieved by adding inlets/outlet and valves between chambers 1 and 2. Circulation can occur through chambers 1 & 2 only, then 1 , 2 and 3 and finally 1 ,2,3 and 4.

Alternatively, a detection antibody cocktail can be mixed into the sample prior to capture. Then during sample flow-through, captured complexes may be detected in a real-time as they are momentarily immobilised in one chamber surface.

A potential arrangement for EV capture and detection is as follows.

Table 2

Chamber 1 detects the known characterisable cancer type EVs. Chambers 2 & 3 have more generic cancer markers, while cancer 4 has general EV markers to capture the remainder.

This approach uses a sequential, multiplexed capture, to capture and detect EVs in situ.

Examples of the invention are described in the following numbered clauses. 1. A method comprising: initiating flow of a sample fluid through at least one capture chamber in a device, in which one or more capture reagent is present in the or each capture chamber; and retaining the one or more capture reagent in the or each capture chamber as the sample fluid flows through the or each capture chamber, wherein: a) the device comprises a plurality of capture chambers and the one or more capture reagent in each capture chamber is retained in its respective capture chamber as the sample fluid flows through each capture chamber, wherein i) the one or more capture reagent in each capture chamber is different, and/or ii) the one or more capture reagent in each capture chamber is mobile; and/or b) the method comprises urging the one or more capture reagent in the or each capture chamber to reciprocate or oscillate independently of sample fluid flow, and/or c) the method comprises recirculating the sample fluid through the or each capture chamber.

2. A method according to clause 1 , wherein the device is a microfluidic device.

3. A method according to clause 1 or clause 2, comprising introducing the one or more capture reagent into the or each capture chamber.

4. A method according to any preceding clause, wherein the one or more capture reagent is in solution.

5. A method according to any preceding clause, wherein the one or more capture reagent is retained in the or each capture chamber by a semi-permeable physical barrier, optionally a filter.

6. A method according to any of clauses 1 to 4, wherein the one or more capture reagent comprises a magnetic component and is retained in the or each capture chamber, by a magnetic force.

7. A method according to clause 6, wherein the magnetic component is a magnetic bead.

8. A method according to clause 6 or clause 7, wherein the one or more capture reagent in each capture chamber is urged in the transverse direction relative to the direction of sample fluid flow, by a magnetic force, preferably an alternating magnetic force.

9. A method according to any preceding clause, wherein the device comprises at least three capture chambers.

10. A method according to any preceding clause, for capturing or isolating one or more analyte from a sample, preferably wherein the one or more analyte comprises an extracellular vesicle and/or cfDNA.

11. A method according to clause 10, wherein the one or more analyte comprises exosomes. 12. A method according to clause 10 or clause 11 , wherein the one or more capture reagent comprises a capture moiety that specifically binds the one or more analyte in the sample.

13. A method according to clause 12, wherein the capture moiety comprises an antibody and/or an oligonucleotide.

14. A method according to clause 12 or clause 13, wherein the one or more capture reagent comprises a magnetic bead coated with, or attached to, the moiety.

15. A method according to any of clauses 10 to 14, comprising introducing one or more detection reagent to the or each capture chamber, following capture of the one or more analyte.

16. A method according to clause 15, wherein the one or more detection reagent comprises a detection moiety that specifically binds to the one or more analyte.

17. A method according to clause 16, wherein the detection moiety comprises an antibody.

18. A method according to clause 16 or clause 17, wherein the one or more detection reagent comprises a fluorophore attached to the detection moiety.

19. A method according to any preceding clause, wherein there are a plurality of different capture chambers and a distinct capture reagent, or a distinct combination of capture reagents, is in each chamber.

20. A method according to any preceding clause, wherein a plurality of different capture reagents are introduced into the or each capture chamber.

21. A device, preferably a microfluidic device, for capturing one or more analytes from a fluid sample, the device comprising: at least one capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through the or each capture chamber; and a retainer for, in use, retaining a capture reagent in the or each capture chamber as the fluid sample flows through the or each capture chamber, wherein a) the device comprises a means for urging the capture reagent to reciprocate or oscillate, in use, independently of sample fluid flow; and/or b) the at least one capture chamber comprises a plurality of capture chambers, and i) a different capture reagent or a different combination of capture reagents, is in each capture chamber, and/or ii) there are no capture probes immobilised to the device; and/or c) the device comprises a channel for recirculating the sample through the or each capture chamber. 22. A device according to clause 21 , comprising a reagent inlet for introducing a capture reagent into the or each capture chamber.

23. A device according to clause 21 or clause 22, wherein the retainer comprises a magnet or a component that can exert a magnetic force. 24. A device according to any of clauses 21 to 23, wherein means for urging the capture reagent to reciprocate or oscillate, is a magnet or component that can exert a magnetic force.

25. A device according to clause 24, wherein the magnet or the component comprises a first part and a second part, and the first and second parts are positioned at opposing sides of the or each capture chamber, preferably wherein the magnetic force applied to the or each capture chamber, by the first and second parts, is variable.

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Claims

1. A method for capturing and optionally detecting extracellular vesicles, comprising: initiating flow of a sample fluid through a plurality of capture chambers in a device, in which a distinct combination of capture reagents is present in each capture chamber; and retaining the capture reagents in each capture chamber, as the sample fluid flows through each capture chamber.

2. A method according to claim 1 , wherein the device is a microfluidic device.

3. A method according to claim 1 or claim 2, comprising introducing the capture reagents into each capture chamber.

4. A method according to any preceding claim, wherein capture reagents are in solution.

5. A method according to any preceding claim, wherein the capture reagents are retained each capture chamber by a semi-permeable physical barrier, optionally a filter.

6. A method according to any of claims 1 to 4, wherein the capture reagents comprise a magnetic component and are retained in each capture chamber, by a magnetic force.

7. A method according to claim 6, wherein the magnetic component is a magnetic bead.

8. A method according to claim 6 or claim 7, wherein the capture reagents in each capture chamber are urged in the transverse direction, relative to the direction of sample fluid flow, by a magnetic force, preferably an alternating magnetic force.

9. A method according to any preceding claim, wherein the device comprises at least three capture chambers.

10. A method according to any preceding claim, wherein the capture reagents comprise a capture moiety that specifically binds the extracellular vesicles in the sample.

11. A method according to claim 10, wherein the capture moiety comprises an antibody, aptamer and/or an oligonucleotide.

12. A method according to claim 10 or claim 11 , wherein the one or more capture reagent comprises a magnetic bead coated with, or attached to, the moiety.

13. A method according to any preceding claim, comprising introducing one or more detection reagent to each capture chamber.

14. A method according to claim 13, wherein the one or more detection reagent is introduced following capture of the extracellular vesicles.

15. A method according to claim 13, wherein the one or more detection reagent is introduced to the capture chambers simultaneously with the sample.

16. A method according to claim 13, wherein the one or more detection reagent comprises a detection moiety that specifically binds to the extracellular vesicles.

17. A method according to claim 16, wherein the detection moiety comprises an antibody.

18. A method according to claim 16 or claim 17, wherein the one or more detection reagent comprises a fluorophore attached to the detection moiety.

19. A method according to any preceding claim, wherein the distinct capture reagents are distinguishable by fluorescence.

20. A method according to any preceding claim, wherein the capture chambers are arranged in series.

21. A device, preferably a microfluidic device, for capturing extracellular vesicles from a fluid sample, the device comprising: a plurality of capture chambers; a distinct combination of capture reagents in each capture chamber; a sample inlet arranged such that a sample introduced through the sample inlet can flow through each capture chamber; and a retainer for, in use, retaining a the capture reagents in each capture chamber as the fluid sample flows through each capture chamber.

22. A device according to claim 21 , comprising a reagent inlet for introducing the capture reagents into each capture chamber.

23. A device according to claim 21 or claim 22, wherein the retainer comprises a magnet or a component that can exert a magnetic force.

24. A device according to any of claims 21 to 23, which comprises a means for urging the capture reagents to reciprocate or oscillate, in use, independently of sample fluid flow, optionally wherein the means for urging is a magnet or component that can exert a magnetic force.

25. A device according to claim 24, wherein the magnet or the component comprises a first part and a second part, and the first and second parts are positioned at opposing sides of each capture chamber, preferably wherein the magnetic force applied to each capture chamber, by the first and second parts, is variable.

26. A device according to any of claims 21 to 25, comprising at least three capture chambers.

27. A device according to any of claims 21 to 26, wherein the capture chambers are arranged in series.

28. A method for diagnosing cancer comprising capturing and detecting extracellular vesicles from a sample as defined in any of claims 1 to 20.

29. Use of a device as defined in any of claims 21 to 27, for diagnosing cancer.