Simultaneous multiplex subtyping of breast cancer derived small extracellular vesicles

Abstract

The present disclosure relates to a lateral flow test device for isolating sEVs based on their expression of sEV specific proteins CD9, CD81 and CD63 and methods for detecting cancer sEVs in a biological sample obtained from a subject based on the expression of a cancer antigen by the sEVs. The methods disclosed herein also employ a Surface enhanced Raman spectroscopy (SERS) nanotag which is able to detect breast-cancer-specific sEVs comprising CD9, CD81 and CD63 thus providing for early diagnosis of breast cancer in a subject.

Description

[0001] Simultaneous Multiplex Subtyping of Breast Cancer Derived Small Extracellular Vesicles

[0002] Incorporation by reference

[0003] All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

[0004] Field

[0005] The present disclosure relates to a lateral flow test device for isolating sEVs based on their expression of sEV specific proteins CD9, CD81 and CD63 and methods for detecting cancer sEVs in a biological sample obtained from a subject based on the expression of a cancer antigen by the sEVs. The methods disclosed herein also employ a Surface enhanced Raman spectroscopy (SERS) nanotag which is able to detect breast-cancer-specific sEVs comprising CD9, CD81 and CD63 thus providing for early diagnosis of breast cancer in a subject.

[0006] Background

[0007] Early detection of breast cancer is crucial for improving survival rates. Patients diagnosed with stage I breast cancer have a significantly higher chance of survival, with a 5-year relative survival rate of nearly 100%. In contrast, those diagnosed at stage IV face a much lower survival rate of 31 -32% (Chhikara BS, et al., (2023) Chem Biol Lett. 10(1 ) :451 ). With breast cancer rates projected to increase, there is a need for a method that can detect breast cancer in the early stages ensuring improved patient outcomes.

[0008] Current diagnostic methods including medical imaging and tissue biopsies face significant challenges, as they often detect tumours only after full formation. Current medical imaging techniques including X-ray, computed tomography scan (CT-scan) and magnetic resonance imaging (MRI) can be expensive and time-consuming, reducing effectiveness at detecting breast cancers at early stages. On the other hand, tissue biopsies present heavy burdens on the healthcare system as they are costly, time-consuming, and highly invasive for patients. Thus, the demand for a technique that is non-invasive, rapid, and sensitive is becoming increasingly vital for improved patient outcomes.

[0009] Point-of-care testing (POCT) devices known as lateral flow immunoassays (LFIAs) are widely used today in applications such as pregnancy and COVID tests (Oliveira-Rodrfguez M, et al., (2017) Biosensors and Bioelectronics 87:38-45). They have proven to be sensitive, rapid and convenient for end-users due to the colorimetric nature of these tests allowing easy readouts that present either ‘yes’ or ‘no’. A major limitation to POCT for cancer diagnostics has been the lackof a suitable biomarker. Cancer cell-derived small extracellular vesicles (sEVs) have recently emerged as promising biomarkers because of their intrinsic role in tumor development, progression, and intercellular communication within cancerous tumor microenvironments. sEVs are lipid bilayer extracellular vesicles that are less than 200 nm in size, as denoted by the International Society for Extracellular Vesicles (ISEV) (Welsh JA, et al., (2024) J Extracell Vesicles 13(2):e12404). sEVs are readily available and abundant in bodily fluids allowing for easy sample acquisition.

[0010] However, sEVs are heterogenous in nature, demonstrating varying expression levels of common sEV markers, known as tetraspanins, such as CD9, CD81 and CD63. This makes accurate capture of sEVs difficult, with the common markers varying in different populations. Other proposed biosensors in this field use only one sEV tetraspanin for capture, limiting capabilities whereby some populations may not express or have limited expression of that given marker. sEVs also contain numerous cancer specific proteins on the lipid membrane surface, making them ideal for capture and detection in LFIA technologies. Given the heterogeneous nature of sEVs, molecular subtyping of cancer cell-derived sEVs allows for accurate capture while also providing comprehensive information on cancer proliferation, progression, and tumorigenesis at very early stages of cancer development. In addition, despite the potential of LFIA for cancer detection, there is no cancer-focussed LFIA using sEVs being used in clinical settings. This gap presents an opportunity for developing a new, rapid diagnostic tool that could improve early detection rates and patient outcomes.

[0011] There is an urgent need for detection methods that can identify cancer before tumours develop, to enable earlier treatment and monitoring.

[0012] Summary

[0013] To combat the issues faced in the field of cancer diagnostics with the use of sEVs, the inventors have developed a lateral flow immunoassay (LFIA) that utilises a colorimetric detection method and optionally further includes quantitative detection and analysis of sEVs. The assay allows the identification of breast cancer-specific sEVs as a proxy for early stage cancer detection before tumours develop to a size detectable by standard methods such as mammography. The sEVs are selected based on the presence of tetraspanins which are utilised for capture and subtyping of sEV populations. Cancer-specific sEVS are then selected based on their expression of a cancer antigen. Thus, the inventors have identified a particular combination of tetraspsins and cancer antigen that allows for the majority of the cancer-specific sEV population to be captured and ensure accurate diagnosis.

[0014] In one aspect, there is provided a lateral flow test device for optical detection of cell-derived small extracellular vesicles (sEVs) in a sample, the device comprising a porous matrixcomprising at least three distinct test locations on said porous matrix, each of the test locations comprising a reagent that binds to a marker present on the sEVs and wherein the reagents at the at least three test locations bind to different marker on the sEVs, and wherein the sample flows laterally along the test device and passes the test locations, and further wherein binding of the sEVs at the test locations is confirmed by an optical signal.

[0015] In one example, the makers present on the sEVs are tetraspanins. In one example, the markers are selected from the group consisting of CD63, CD81 , CD9, CD82 and CD151 . In a further example, the markers are CD63, CD81 and CD9.

[0016] In one example, the at least three test locations comprise a binding reagent immobilised on the surface of the test device which binds a tetraspanin biomarker on the sEVs. The binding reagents bind to, or specifically bind to different tetraspanin biomarkers. The binding reagent may be a full length antibody, or antibody fragment thereof, for example, a Fab fragment, a F(ab’)2 fragment, a Fd fragment, a Fv fragment, a dAb fragment, or a nanobody (VHH). In one example, the binding molecule is an aptamer or an i-body as described in WO 2005 / 118629. The binding reagents may be present in any order on the test device. In a particular example, the binding reagents comprise an anti-CD63, an anti-CD81 and an anti-CD9 binding reagent.

[0017] In one example, the sample comprises sEVs obtained from a subject with cancer. In one example, the sEVs comprise a size less than 200nm, more preferably a size of between 50nm to 80nm.

[0018] In one example, binding of the sEVs at the test locations is confirmed by detecting an optical signal present at one or more of, or all three of the test locations. In a particular example, the binding of the sEVs at the test locations is confirmed by contacting the bound sEVs with at least one nanotag that binds to a cancer antigen on the sEVs. In one example, the nanotag comprises a binding agent bound to an optically detectable nanoparticle. Preferably, for cancer detection, the binding agent is one which recognises a cancer antigen on the surface of the sEVs. The cancer antigen may be selected from the group consisting of one or more of EpCAM, PD-1 , PD-L1 , HER1 (EGFR), HER2, HER3, TROP2, MUC1 , PROCR, MET, CTLA4, CD44, CD47, CD133, E-cadherin, and N-cadherin. In a particular example the cancer antigen is EpCAM.

[0019] In one example, the sEVs are cancer-specific sEVs.

[0020] The nanoparticle may be selected from a core shell structure, a silica nanoparticle, a metal organic framework or a satellite structure. In one example, the core shell structure is a silver coated nanoparticle, a gold coated nanoparticle, or gold shell. The nanoparticle may be of any desired shape, for example nanorod, nanostar, nanoshell, nanotriange, nanocube or nanocage. Examples of other structures are described in Yaraki MT, et al., (2022) Nanoscale.

[0021] 14(41 ):15242-15268, the entire contents of which are incorporated by reference herein.In one example, the nanoparticle is a gold coated nanoparticle. Advantageously, gold nanoparticles absorb light at a wavelength of 530nm and this produces a pink colour that is visible to the naked eye.

[0022] The nanotag or SERS nanotag as used herein may comprise a binding agent that binds to, or specifically binds to a cancer antigen selected from the group consisting of one or more of EpCAM, PD-1 , PD-L1 , HER1 (EGFR), HER2, HER3, TROP2, MUC1 , PROCR, MET, CTLA4, CD44, CD47, CD133, E-cadherin, and N-cadherin. . In a further example, the binding agent binds to, or specifically binds to EpCAM.

[0023] In one example, the sample comprises sEVs obtained from a subject with cancer, more particularly breast cancer. The sample may be a liquid biological sample, for example, the sample may be selected from one or more of blood, urine or saliva. In one example, the sample is plasma or serum.

[0024] In some examples, the sample may be manipulated to increase the concentration of sEVs in the sample.

[0025] In one example, the nanotag comprises a binding agent selected from a full length antibody, an antibody fragment, an aptamer or i-body described herein bound to an optically detectable nanoparticle described herein. Binding of the nanoparticle and the binding agent may be by any means known in the art, including those described herein. In one example, the conjugation is a bioconjugation or passive conjugation as described herein. In one example, binding of the nanoparticle and binding agent is via a linker.

[0026] The nanoparticle described herein is preferably one which has colorimetric properties. In one example, the nanoparticle is able to be viewed with the naked eye, for example, following contact by the sEVs with the at least three test reagents. The nanoparticle may be selected from a core shell structure, a silica nanoparticle, a metal organic framework or a satellite structure. In one example, the core shell structure is a silver coated nanoparticle, a gold coated nanoparticle, or gold shell. The nanoparticle may be of any desired shape, for example nanorod, nanostar, nanoshell, nanotriange, nanocube or nanocage. Examples of other structures are described in Yaraki MT, et al., (2022) Nanoscale. 14(41 ):15242-15268, the entire contents of which are incorporated by reference herein.

[0027] In one example, the nanoparticle is a gold nanoparticle having a size of about 50nm to about 80nm. In some examples, the nanoparticle has a UV-vis absorption peak of about 530nm. In one example, the nanoparticle has a zeta potential below -30mV.

[0028] In certain examples, a Raman reporter molecule may be bound to the nanoparticle. In some examples, the Raman reporter molecule is conjugated to the nanoparticle, for example using methods described herein. Raman reporter molecules are known in the art, examples suitable for use include 4-Mercaptobenzoic acid (4-MBA), 4-Nitrothiophenol (4-NTP), 4-Aminothiophenol (4-ATP), 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB), 2,3,5,6-Tetrafluoro-4-mercaptobenzoic acid (TFMBA), 4-Mercaptopyridine (2-Mpy), 2-Naphthalenethiol (2-NAT), 2,7-Mercapto-4-rnethylcoumarin (MMC) and Rhodamine X (ROX). In one example, the Raman reporter molecule is 4-MBA.

[0029] The porous matrix of the lateral flow test device may comprise nitrocellulose or similar material. The lateral flow device may also comprise an absorbent pad. In some examples, the lateral flow device may comprise a sample pad and / or conjugate pad.

[0030] In certain examples, the lateral flow test device comprises a control, for example a nonspecific binding agent that is able to capture the binding agent with the nanoparticle bound thereto.

[0031] In a second aspect, there is provided a complex comprising cancer sEVs bound to a nanotag, the nanotag comprising a binding agent that binds to a cancer antigen present on the sEVs and wherein the binding agent is bound to a nanoparticle having an optical property, and optionally wherein the nanoparticle further comprises a Raman reporter molecule bound thereto.

[0032] In one example, the binding agent is an antibody that binds to, or specifically binds to a cancer biomarker selected from the group consisting of one or more of EpCAM, PD-1 , PD-L1 , HER1 (EGFR), HER2, HER3, TROP2, MUC1 , PROCR, MET, CTLA4, CD44, CD47, CD133, E-cadherin and N-cadherin. In a further example, the binding agent binds to, or specifically binds to EpCAM. In some examples, the Raman reporter molecule is 4-MBA. In one example, different Raman reporter molecules may be used in combination and / or different nanoparticles may be used in combination.

[0033] The sEVs according to the second aspect, when bound to the binding agent may have a size range of from about 80 to about 10Onm, about 80 to 90nm, or about 85nm. In one example, the sEVs have a size range of up to 200nm.

[0034] In a third aspect, there is provided a method for optically detecting cancer cell-derived small extracellular vesicles (sEVs) in a sample, the method comprising:

[0035] (i) contacting the sample with the nanotag described herein;

[0036] (ii) applying the sample to the test device described herein, wherein the sample is applied to a site of the test device upstream of the test locations; and

[0037] (iii) detecting a colorimetric signal at the one or more test locations.

[0038] In one example, the colorimetric signal is detectable by eye, preferably naked eye. However, it will also be understood that the signal may be detectable by microscope. In one example, detecting a signal indicates that cancer-specific sEVs are present in the sample. In one example, detecting a signal indicates that cancer is present in the subject. In one example, the overall intensity of the three test locations are used to determine a positive result, wherein if one or more test lines show a signal, a positive result is indicated.In a fourth aspect, there is provided a method for subtyping cancer-specific sEVs in a sample, the method comprising:

[0039] (i) contacting the sample with the nanotag described herein;

[0040] (ii) applying the sample to the test device described herein, wherein the sample is applied to a site of the test device upstream of the test locations; and

[0041] (iii) detecting a colorimetric signal at the one or more test locations wherein the intensity of the signal at each of the test locations allows for sEV subtyping.

[0042] In one example, the sEV subtyping is based on the relative expression of CD63, CD81 and CD9.

[0043] In a fifth aspect, there is provided a method of detecting and / or diagnosing breast cancer in a subject, the method comprising:

[0044] (i) contacting the sample with the nanotag described herein;

[0045] (ii) applying the sample to the test device described herein, wherein the sample is applied to a site of the test device upstream of the test locations; and

[0046] (iii) detecting a colorimetric signal at one or more of the test locations corresponding to CD63, CD81 and CD9.

[0047] In some examples, the methods described herein may further comprise the use of Raman spectroscopy to detect and / or quantitate the signal detected at each of the test locations. In some examples, the method further comprises subsequently treating the subject with surgery and / or chemotherapy, and / or radiotherapy.

[0048] The skilled person will appreciate that based on the cancer to be detected, different tetraspanin binding reagents may be immobilised on the surface of the test device. These tetraspanins in combination with the choice of cancer antigen may be used to discriminate different types of cancers. Cancer detection can be improved by addition of binding agents to further cancer antigens.

[0049] In a sixth aspect, the disclosure provides a system for quantitatively detecting cancer cell-derived small extracellular vesicles (sEVs) in a sample, the system comprising:

[0050] (i) the test device described herein; and

[0051] (ii) a reader that comprises a light source and a photodetector to detect a detectable signal emitted by the Raman reporter molecule.

[0052] In one example, the system comprises the use of Raman spectroscopy.

[0053] In a seventh aspect, the disclosure provides a kit for quantitatively detecting cancer specific sEVs in a sample, the kit comprising:

[0054] (i) the test device described herein; and

[0055] (ii) instructions for using the test device to optically and / or quantitatively detect cancerspecific sEVs present in the sample.In some examples, the kit also comprises a liquid container comprising any suitable liquid and / or reagent. For example, the liquid container can comprise a binding agent (e.g. EpCAM antibody as exemplified herein) bound to a nanoparticle having a colorimetric property (e.g. gold nanoparticle), and optionally wherein the nanoparticle further comprises a Raman reporter molecule bound thereto.

[0056] The principles of the present test devices, kits, systems and methods can be applied, or can be adapted to apply, to the lateral flow test devices and assays known in the art. For example, the principles of the present test devices, kits, systems and methods can be applied, or can be adapted to apply, to the lateral flow test devices and assays described in one or more of the following patents and applications: U.S. Pat. Nos. 5,073,484, 5,654,162, 6,020,147, 4,695,554, 4,703,017, 4,743,560, 5,591 ,645, RE 38,430 E, 5,602,040, 5,633,871 , 5,656,503, 6,187,598, 6,228,660, 6,818,455, 7,109,042, 6,352,862, 7,238,537, 7,384,796, 7,407,813, 5,714,389, 5,989,921 , 6,485,982, 5,120,643, 5,578,577, 6,534,320, 4,956,302, RE 39,664 E, 5,252,496, 5,559,041 , 5,728,587, 6,027,943, 6,506,612, 6,541 ,277, 6,737,277, 7,175,992 B2, 7,691 ,595 B2, 6,770,487 B2, 7,247,500 B2, 7,662,643 B2, 5,712,170, 5,965,458, 7,371 ,582 B2, 7,476,549 B2, 7,633,620 B2, 7,815,853 B2, 6,267,722 B1 , 6,394,952 B1 , 6,867,051 B1 , 6,936,476 B1 , 7,270,970 B2, 7,239,394 B2, 7,315,378 B2, 7,317,532 B2, 7,616,315 B2, 7,521 ,259 B2, 7,521 ,260 B2, US 2005 / 0221504 A1 , US 2005 / 0221505 A1 , US 2006 / 0240541 A1 , US 2007 / 0143035 A1 , US 2007 / 0185679 A1 , US 2008 / 0028261 A1 , US 2009 / 0180925 A1 , US 2009 / 0180926 A1 , US 2009 / 0180927 A1 , US 2009 / 0180928 A1 , US 2009 / 0180929 A1 , US 2009 / 0214383 A1 , US 2009 / 0269858A1 , 6,777,198, US 2009 / 0311724 A1 , US 2009 / 0117006 A1 , 7,256,053, 6,916,666, 6,812,038, 5,710,005, 6,140,134, US 2010 / 0143941 A1 , 6,140,048, 6,756,202, 7,205,553, 7,679,745, US 2010 / 0165338 A1 , US 2010 / 0015611 A1 , 5,422,726, 5,596,414, 7,178,416, 7,784,678 B2, US 2010 / 094564 A1 , US 2010 / 0173423 A1 , US 2009 / 0157023 A1 , 7,785,899, 7,763,454 B2, US 2010 / 0239460 A1 , US 2010 / 0240149 A1 , 7,796,266 B2, 7,815,854 B2, US 2005 / 0244953 A1 , US 2007 / 0121113 A1 , US 2003 / 0119202 A1 , US 2010 / 0311181 A1 , 6,707,554 B1 , 6,194,222 B1 , 7,713,703, EP 0,149,168 A1 , EP 0,323,605 A1 , EP 0,250,137 A2, GB 1 ,526,708 and WO99 / 40438.

[0057] In the aspects described herein, the subject is a mammal, preferably a human.

[0058] Figure 1 shows a schematic representation of the LFIA design with three test lines and a control line. Gold nanoparticles (AuNPs) are bound to the sEVs by the EpCAM antibodies which are then flowed through and captured by the three subtypes of sEV on the test lines via their express of tetraspanins proteins including CD63, CD81 and CD9. The control line contains goat antimouse IgG antibodies.Figure 2 shows characterisation of the AuNPs and cel line-derived sEVs. (A) Transmission electron microscopy (TEM) image of AuNPs. (B) Size distribution of AuNPs using Dynamic light scattering (DLS). (C) Peak shift using ultra-violet visible spectroscopy (UV-Vis) demonstrating a red shift when AuNPs are conjugated with EpCAM antibodies. (D) TEM image of MCF7 cell-derived sEV. (E) Size and concentration of MCF7 cell derived sEVs measured by Nano-Flow cytometry (NanoFCM). (F) Size and concentration of SKBR3 cell-derived sEVs by NanoFCM.

[0059] Figure 3 shows nanoparticle count obtained for sEVs in 0% and 1% Tween-20 running buffer.

[0060] Figure 4 shows three replicates of positive EpCAM expression of MCF7 and SKBR3 cell-derived sEVs (suspended in running buffer with conjugated AuNPs) and negative control (conjugated AuNPs and running buffer with no sEVs present).

[0061] Figure 5 shows (A) Three replicates of varying tetraspanin (CD63, CD81 and CD9) orders using MCF7 cell line-derived sEVs; and (B) Image J analysis of overall EpCAM expression per tetraspanin order.

[0062] Figure 6 shows expression of tetraspanins on MCF7 cell-derived sEVs characterized by NanoFCM. (A) Unstained sEVs as control to show the background signal, (B) DiO staining of sEVs to show the entire population of lipid nanoparticles captured; The expression level of CD81 (C), CD9 (D) and CD63 (E) on MCF7-derived sEVs.

[0063] Figure 7 shows size distributions (by NanoFCM) for four sEV samples isolated from plasma including breast cancer patients (BB0573, BB0695) and healthy (H0330, H9243).

[0064] Figure 8 shows healthy (H0330; H0930; H1030; H9243; H7530) and breast cancer patient samples (BB0606; BB0520; BB0695; BB0525; BB0573) from stages (0-IV) samples run using plasma directly.

[0065] Figure 9 shows SERS spectra of MCF7 (A) and SKBR3 (B) cell line-derived sEVs. Each spectra represents a line on the LFIA. The highlighted line indicates the characteristic peak of 4-MBA which was used for qualitative identification of SERS nanotag-sEV complexes captured on the test lines.Detailed Description

[0066] General

[0067] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0068] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0069] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0070] The steps, features, integers, compositions and / or therapeutic agents disclosed herein or indicated in the specification of this application individually or collectively, and any combinations of two or more of said steps or features.

[0071] Any example herein shall be taken to apply mutatis mutandisto any other example unless specifically stated otherwise.

[0072] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., immunology, protein chemistry, and biochemistry).

[0073] Unless otherwise indicated, any immunological techniques described herein are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991).

[0074] Selected Definitions

[0075] The term “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both means or for either meaning. Furthermore, a list or features including the phrase “and / or” between the second last and last feature means that any one or more of the listed features may be present in any combination.

[0076] Reference to the singular forms “a”, “an” and “the” is also understood to imply the inclusion of plural forms unless the context dictates otherwise.

[0077] The term “about”, as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0078] As used herein, “antibody” refers a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.” An “antibody” may be naturally occurring or manmade such as monoclonal antibodies produced by conventional hybridoma technology, various display methods, e.g., phage display, and / or a functional fragment thereof.

[0079] The term “epitope” refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually or often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0080] As used herein, the term “subject” is not limited to a specific species or sample type. For example, the term “subject” may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of mammalian or other species.As used herein, the term “sample” refers to anything which may contain sEVs for which the test device is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, or cerebral spinal fluid.

[0081] As used herein, a “binding reagent” or “binding agent” refers to any substance that binds to a target or a marker present on an sEV with desired affinity and / or specificity. Exemplary binding reagents can be an amino acid, a peptide, a protein, e.g., an antibody or receptor, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, e.g., DNA or RNA, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid, an aptamer and a complex thereof.

[0082] As used herein, the term “specifically binds” refers to the specificity of a binding reagent, e.g., an antibody or an aptamer, such that the binding reagent preferentially binds to a defined target or marker on an sEV. An binding reagent “specifically binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, a binding reagent that specifically binds to a target may bind to the target with at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, greater affinity as compared to binding to other substances; or with at least about twofold, at least about five-fold, at least about ten-fold or more of the affinity for binding to a target as compared to its binding to other substances. Recognition by a binding reagent of a target in the presence of other potential interfering substances is also one characteristic of specifically binding. Preferably, a binding reagent, e.g., an antibody or an aptamer, that is specific for or binds specifically to a target, avoids binding to a significant percentage of non-target substances, e.g., non-target substances present in a testing sample. In some embodiments, a binding reagent avoids binding greater than about 90% of non-target substances, although higher percentages are clearly contemplated and preferred. For example, a binding reagent can avoid binding about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99% and about 99.9% or more of non-target substances. In other embodiments, a binding reagent can avoid binding greater than about 10%, 20%, 30%, 40%, 50%, 60%, or 70%, or greater than about 75%, or greater than about 80%, or greater than about 85% of non-target substances.

[0083] The term “sample pad” as used herein refers to a material that absorbs and transports the sample, ensuring its even and controlled distribution to the conjugate pad.

[0084] The term “conjugate pad” as used herein refers to material that preserves and holds the reagents that bind the tetraspanin markers on the sEVs.The term “i-body” as used herein refers to a scaffold of a human protein comprising two binding loops that mimic the shape of shark antibodies. The loops are responsible for binding or interacting with a particular antigen.

[0085] As used herein the term “optically detect” means visible to the eye, preferably the naked eye.

[0086] As used herein, a “nanotag” or “SERS nanotag” refers to the complex of binding agent and nanoparticle (e.g. nanoparticle). In the examples, it is designated as AuNP@EpCAM. In some embodiments, the SERS nanotag also comprises a Raman reporter molecule which in the examples is referred to as AuNP@MBA@EpCAM.

[0087] The term “SERS” as used herein refers to surface-enhanced Raman spectroscopy. This technique amplifies the Raman scattering of molecules adsorbed on metal surfaces or nanostructures, allowing for the detection of low concentration analytes with high sensitivity.

[0088] The term “sEV” as used herein refers to small extracellular vesicles that have been isolated from a biological sample by selecting for binding to tetraspanin markers, in particular, the tetraspanins CD9, CD81 and CD63.

[0089] The term “cancer-specific sEV” refers to a subset of sEVs which have been selected based on their expression of one or more cancer antigens.

[0090] sEVs as a cancer biomarker

[0091] sEVs are denoted by the International Society for Extracellular Vesicles (ISEV) as being extracellular vesicles of less than 200 nm in size. They are released by the cells into extracellular space to then be circulated throughout the body and are detectable in all somatic fluids such as blood, saliva, urine, and cerebrospinal fluid. sEVs present as promising biomarkers for cancer diagnostics as they are readily available in bodily fluids, have multiple surface markers for capture and subtyping, and can share characteristics of their parent tumor cells. sEVs have a variety of markers which can be used for subtyping and detection. Within the EVs are nucleic acids, lipids, metabolites and proteins.

[0092] The biomarkers of sEVs vary greatly and mimic that of their parent cells. Examples include cancer specific proteins (e.g. HER2) to differentiate sEVs from normal and cancer cells, tetraspanins (CD9, CD81 , and CD63) which are common protein biomarkers among all sEVs, and miRNA (e.g. miR-21) derived from sEV cargo. While there are different assays for each biomarker, the most common one is the use of surface proteins as they can be readily captured with antibodies or aptamers. Single biomarkers have proven ineffective in accurately discriminating between cancer types, as many cancers share similar biomarkers. Due to the high heterogeneity of sEV populations, multiple biomarkers are often required to both capture sEVsand detect the presence of cancer, as each patient’s sEV samples can have varying expression levels.

[0093] Current isolation methods of sEVs include ultracentrifugation, acoustofluidics, solubility precipitation, size exclusion chromatography (SEC), immunoaffinity-based techniques using antibodies or aptamers, lipid self-assembly nanoprobes, charge dielectrophoresis, isolation kits and ion exchange chromatography. Among those, ultracentrifugation is considered the gold standard, however, it often results in low purity and prolonged processing times. SEC has become the preferred method as higher purities are able to be obtained.

[0094] As described herein, detection of cancer sEVs comprises the use of nanotags that bind to a cancer antigen on an sEV. The present methods have been exemplified using EpCAM, however it will be appreciated that other cancer antigens may be used, including EpCAM, PD-1 , PD-L1, HER1 (EGFR), HER2, HER3, TROP2, MUC1, PROCR, MET, CTLA4, CD44, CD47, CD133, E-cadherin, and N-cadherin.

[0095] Table 1 Exemplary cancer antigens

[0096]

[0097]

[0098] Nanoparticles for optical detection

[0099] Nanoparticles, particularly gold and silver nanoparticles are useful in optical detection due to their unique properties such as localised surface plasmon resonance (LSPR) enabling sensitive and specific detection of analytes (e.g. sEVs) in a sample. Any metal structure having resonance properties may be suitable for use in the device and methods described herein. Examples of nanoparticles that are useful for optical detection include gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), quantum dots (QDs), bimetallic nanoparticles and magnetic nanoparticles.

[0100] Nanoparticles comprising a core shell structure are described for example in Duffield C, et al., (2021) J Innov Opt Health Sci. 2021 ;14(04):2141007 and Fraire JC, et al., (2019) ACS Appl Mater Interfaces. 2019;11 (43):39424-39435.

[0101] Other types of nanoparticles include silica nanoparticles, for example as described in Su X, et al., (2023) ACS Appl Mater Interfaces. 15(31 ):37130-37142; nanoparticles comprising a metal organic framework as described in Qin X, et al., (2024) ACS Nano. 2024;18(22):14569-14582.; and satellite structures as described in Wang D, et al., (2022) Nanoscale 14(16):6212-6219 and Wang H, et al., (2025) Journal of Luminescence 277:120935.

[0102] A variety of shapes of nanostructures are contemplated herein for use with the device and methods described herein and may include nanospheres, nanorods, nanoshells, nanotriangles, nanocubes and nanocages as described in Yaraki MT, Tukova A, Wang Y. Emerging SERS biosensors for the analysis of cells and extracellular vesicles. Nanoscale.

[0103] 2022;14(41):15242-15268.Tetraspanins

[0104] The tetraspanins (TSPANs) are a family of proteins with four transmembrane domains (TM1 , TM2, TM3, and TM4), two extracellular loops (ECL1 and ECL2), and one intracellular loop (ICL). For a review see Zhou Z et al., (2023) Cell & Bioscience 13:59, the entire contends of which are herein incorporated by reference. In Homo sapiens, the TSPANs family has 33 members, namely TSPAN1-TSPAN33. The present invention is based on the use of three tetraspanin markers, namely CD9, CD81 and CD63 which distinguish a particular sEV subtype and which capture the majority of the EV population (Breitwieser K et al., (2022) Int J Mol Sci Aug 1 ;23(15):8544). More particularly, the combination of the three tetraspanins (CD63, CD9 and CD81) will capture more greater than 90%, greater than 95% or higher of the sEVs. Additional tetraspanins, CD82 and / or CD151 may be used in addition to CD9, CD81 and CD63. These markers are described in Table 2 below.

[0105] >

[0106]

[0107] 65 to 95% of amino acids are highly conserved among the TSPAN family members. The four transmembrane (TM) domains form a compact bundle in the membrane and facilitate interactions with other proteins. TM domains contain many polar residues that can stabilize TSPAN protein structure with the help of ECL2 disulfide crosslinks. ECL2 is essential to the functions of TSPANs since most of protein-protein interaction sites have been mapped to ECL2. ECL2 consists of a conserved domain and a variable domain. The conserved domain facilitates interactions between different TSPAN molecules, while the variable domain determines interactions with other non-TSPAN proteins. There are also some highly conserved motifs inECL2, such as CCG (Cys-Cys-Gly), PXSC (Phe-X-Ser-Cys) and EGC (Glu-Gly-Cys)]. These conserved motifs are basic structures for the interaction with other proteins.

[0108] TSPANs are expressed on the surface of most nucleated cells and play important roles in cell proliferation, differentiation, adhesion, migration, and cell-cell crosstalk. Although mainly located on cell membrane, TSPANs have no natural ligands.

[0109] Lateral flow immunoassays

[0110] In one aspect, the present disclosure provides a lateral flow test device for detecting (optically and optionally quantitatively) cancer cell-derived small extracellular vesicles (sEVs) in a sample. Preferably, the device comprises a porous matrix comprising at least three distinct test locations on said porous matrix, each of the test locations comprising a reagent that binds to a marker present on the sEVs. The reagents are preferably binding reagents that bind to, or specifically bind to tetraspanin markers present on the sEVs. A sample comprising sEV which is derived from a subject is applied to the test device such that the sample flows laterally along the test device and passes the test locations (ie. the tetraspanin binding agents). Binding of the sEVs to the test locations is visualised using a binding agent bound by a nanoparticle with optical properties. The presence of optical signal (i.e. a pink coloured line) given off by the nanoparticles (e.g. gold nanoparticle), indicates the presence of the cancer-specific sEVs in the sample.

[0111] In a preferred example, the test device is a dip stick.

[0112] The present disclosure is illustrated by reference to sEVs from breast cancer subjects. sEVs from both breast cancer cell lines and plasma from breast cancer patients were detected using the presently disclosed lateral flow device.

[0113] The test device is able to detect concentrations of sEVs within the range of about 106to 109nanoparticles / ml, preferably in the range of 107to 108nanoparticles / ml, more preferably about 108nanoparticles / ml. Advantageously, the inventors have found that the sEVs do not need to be concentrated prior to their application to the test device, allowing for a convenient and rapid result to be obtained. Further, the test device is able to provide a yes or no answer, akin to a Covid test. The overall intensity of the three test lines are used to determine the positive result. If one or more test lines shows a pink colour, a positive result is indicated. The control line must be visible in each test or else the assay is to be disregarded. The control line is read independent of the test lines.

[0114] Further, the device can be adapted to detect the presence of different cancers. For example, the nanotags can be designed with binders specific for various cancer antigens. The use of different nanotags binding to different cancer antigens can be used to increase the accuracy of cancer detection. Multiplexing using multiple nanotags recognising multiple cancer antigens are also contemplated by the present disclosure.Lateral flow immunoassays (LFIAs) are widely used paper-based diagnostic assays, commercially available in many forms, that leverage the capillary flow of porous membranes to transport a sample across the device and promote interaction with bioreceptors to detect target analytes. These assays are cost-effective and easy to use, making them suitable for point of care (POC) applications. A typical LFIA is made up of a sample pad, conjugate pad, nitrocellulose membrane (NC), and an absorbent pad which allows for a target analyte to flow through by capillary forces, interact with SERS nanotags and be captured on the test line to produce a signal.

[0115] The method of the present disclosure is modified from typical methods described above by conjugating the SERS nanotag (e.g. gold nanoparticle) to an antibody that binds sEVs in the sample. Thus, the present method does not rely on the analyte binding the nanoparticle, nor immobilising the nanoparticles to the solid substrate.

[0116] The label-based techniques described herein provides a targeted approach to sEV detection. The use of SERS nanotags to specifically label sEVs has improved specificity, added quantification capacities and reduced matrix effects. Within the field of label-based assays, LFIAs present a method that is rapid, cost-effective and easy for end-users.

[0117] The test device can comprise additional elements. In some examples, the test device can further comprise a sample application element upstream from and in fluid communication with the porous matrix. In other examples, the test device can further comprise a liquid absorption element downstream from and in fluid communication with the matrix.

[0118] In some examples, at least a portion of the matrix is supported by a solid backing. In other examples, half, more than half or all portion of the matrix is supported by a solid backing. The solid backing can be made of any suitable material, e.g., solid plastics.

[0119] The test device can further comprise a dried, labelled reagent (i.e. binding reagents that bind to tetraspanins on the sEVs).

[0120] The binding reagent / agents described herein can have any suitable binding affinity and / or specificity. In some examples, the binding reagents / agents (e.g. tetraspanins and EpCAM binding agents) bind, and preferably specifically bind, to sEVs in the sample. Preferably the affinity of the antibody may be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, preferred antibodies bind with affinities of at least about 107M‘1, and preferably between about 108M’1to about 109M‘1, about 109M’1to about 101°M-1, or about 101° M’1to about 1012M‘1. Affinity is calculated as Kd=koff / kon(koft is the dissociation rate constant, Konis the association rate constant and Kd is the equilibrium constant).

[0121] The test device can further comprise a housing that covers at least a portion of the test device, wherein the housing comprises a sample application port to allow sample applicationupstream from or to the test locations and an optic opening around the test locations to allow signal detection at the test locations. The optic opening can be achieved in any suitable way. For example, the optic opening can simply be an open space. Alternatively, the optic opening can be a transparent cover.

[0122] The present test device can further comprise a liquid container. The liquid container can comprise any suitable liquid and / or reagent. For example, the liquid container can comprise a binding agent (e.g. EpCAM antibody as exemplified herein) bound to a nanoparticle having a colorimetric property (e.g. gold nanoparticle), and optionally wherein the nanoparticle further comprises a Raman reporter molecule attached thereto. This mixture is combined with the sample (comprising sEVs) prior to application to the test device.

[0123] The present invention also provides for a kit for optically detecting sEVs in a sample, which kit comprises a test device as described above. In some examples, the kit can further comprise a liquid container as described above and instructions for using the test device to optically detect sEVs in the sample.

[0124] Enhancement of sensitivity of detection by SERS

[0125] Surface-enhanced Raman scattering (SERS) technology may optionally be used with the methods described herein to increase sensitivity of detection. The use of SERS may improve the detection limit allowing for detection of biomarkers which are in such a low concentration as to be undetectable by eye. Various label-based SERS multiplex assays have been developed in the field of SERS including bead assays, lateral flow immunoassays, microfluidic devices, and artificial intelligence (Al)-based label-free SERS chips, targeting multiple surface proteins to ensure comprehensive multiplex diagnostics.

[0126] Raman spectroscopy is an optical technique that provides insights into the vibrational and rotational properties of molecules (Lee W, et al., (2020) Journal of Raman Spectroscopy.

[0127] 2020;51 (2):293-300). However, since the Raman signals are inherently weak, enhancements are often required for practical applications. Surface-enhanced Raman scattering (SERS) is a powerful spectroscopic method that significantly amplifies Raman signals by utilizing molecules adsorbed on rough metal surfaces or nanostructures. SERS enhancement primarily occurs through two mechanisms: electromagnetic and chemical. Electromagnetic enhancement results from the resonance with surface plasmons, particularly when target molecules are adsorbed on nanoscale metallic surfaces such as gold (Au) or silver (Ag). Specific nanostructures, like gold nanostars (AuNS), can create "plasmonic hot spots" - regions of intense electromagnetic field enhancement due to localized surface plasmon resonance. Chemical enhancement involves charge transfer between the metal surface and attached molecules, often facilitated by Raman reporter molecules, and can increase signal intensity by a factor of 102to 104.Dyes such as NIR4f and NIR5e have been reported as ideal resonant Raman reporters for use with nanostars or nanospheres as they contribute to the signal intensity through molecular electronic resonances as demonstrated by Choi et al. (2023) Journal of Raman Spectroscopy.

[0128] 2023;54(9):929-939). Other suitable reporters that may be used in SERS assays for EV detection include 4-Mercaptobenzoic acid (4-MBA), 4-Nitrothiophenol (4-NTP), 4-Aminothiophenol (4-ATP), 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB), 2,3,5,6-Tetrafluoro-4-mercaptobenzoic acid (TFMBA), 4-Mercaptopyridine (2-Mpy), 2-Naphthalenethiol (2-NAT), 2, 7- Mercapto -4-methylcoumarin (MMC) and Rhodamine X (ROX). In SERS applications, it is essential to identify 'characteristic' peaks that correspond to specific biomarkers to ensure accurate diagnosis. This is typically done through the identification of the highest characteristic peak in the spectrum.

[0129] SERS has progressed into a rapidly developing field of research that allows for ultrasensitive biosensing, down to single molecule detection and imaging in a rapid and non-invasive manner. While SERS has many benefits, such as multiplexing capabilities and high sensitivity, it also does not have the typical issues often associated with fluorescence, such as photobleaching and overlapping emission spectra between fluorophores. SERS assays allow for real-time analysis for precision oncology and are highly sensitive due to increased signal generated by electromagnetic and chemical enhancements.

[0130] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.EXAMPLES

[0131] Reagents

[0132] All chemicals were purchased from Sigma-Aldrich, including Sodium tetraborate decahydrate (Na2B40y10H20; pH9), Boric acid (H3BO3; pH9), Bovine serum albumin (BSA), Tween-20, Surfactant 10g, Sucrose (C12H22O11), phosphate buffered solution (PBS), chloroauric acid (HAuCk), Sodium citrate (NasCeHsO?), Tris-base (TB; C4H11NO3), hydroquinone (CeHeC ), 4-mercaptobenzoic acid (4-MBA), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), NHydroxysuccinimide (sNHS), HSPEGCOOH, 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), Aqua Regia (HNO3+3 HCI) and Milli-Q water.

[0133] Antibodies

[0134] All antibodies were purchased from Abeam, except where noted. Antibodies used in the assay include EpCAM (323 / A3, # ab85987), CD9 (MEM-61 , # ab2215), CD81 (M38, # ab79559), CD63 (MEM-259; # ab8219) and Goat Anti-Mouse IgG (# a16092; purchased from Thermofisher scientific).

[0135] Lateral Flow assay components

[0136] All lateral flow assay components were purchased from MDI Advanced Microdevices (Haryana, India). The LFA is composed of a membrane (200CNPH-N-SS60-L2-P25;75x260 mm) and absorbent pad (AP045;27x260 mm).

[0137] Plasma Samples and ethical approval

[0138] Ethics approval was obtained from the Macquarie University Human Research Ethics Committees (approval no. 9230) and all studies followed the National Statement on Ethical Conduct in Human Research (2007) guidelines in accordance with the National Health and Medical Research Council Act 1992. All blood plasma samples were provided by St Vincent Hospital Biobank from a collaboration with general surgeon A / Prof Simon Tsao and were stored at -20 °C for use. Each sample was assigned a unique code either starting with ‘BB’ to indicate Biobank or ‘H’ to indicate Healthy.

[0139] Instruments

[0140] For the characterisation of the SERS nanotags, Jasco V-760 spectrometer (Jasco Corporation, Japan), Malvern Zetasizer Nano ZSP (Malvern Panalytical Ltd., United Kingdom) and JEOL JSM 71 OOF Field Emission Scanning Electron Microscope (JEOL Ltd., Japan; TEM) were utilised. For UV-Vis, samples were diluted by1 / a with milli-Q water before being placed in a UV-Vis cuvette (Cat# Z637092, Sigma Aldrich) for scanning at a wavelength of 400-700 nm.For dynamic light scattering (DLS) measurements, Zetasizer was used to determine the size and surface charge of the AuNPs. Each sample was run three times with a minimum of 11 runs, using a UV-Vis cuvette was used with a 633 nm laser at 25°C to determine the size. The surface charge was determined with1 / 4 diluted in milli-Q water using folded capillary cells (DTS1070; Malvern Panalytical).

[0141] Transmission electron microscopy (TEM) was used to image the SERS nanotags and the sEVs. The instrument was used at 100 kV voltage and 60,000x magnification. For the SERS nanotags characterisation by TEM, a copper grid was prepared by dropping 10 pL onto the grid, allowing to process for 2 min before drying with filter paper. This was repeated three times to ensure adequate concentration on the grid. To prepare the sEV samples for TEM, 5 pL of the sample was placed on the grid and left for 3 minutes (min) before removing excess liquid using a filter paper. Uranyl acetate (5 pL; 2 wt. %) was subsequently added on the grid and left for 3 mins before being removed with filter paper. The grids were left to dry overnight.

[0142] The LFIAs were constructed using a dispenser (KinBio XYZ dispenser HM3035, China) and Lateral flow assay guillotine (Automatic Strip Cutter ZQ2002, China). The LFIAs were cut to 2.5 mm and each line was dispensed at 0.6 uL / cm and 80 mm / s for both test (1 mg / mL) and control lines (1 mg / mL). The Sierra IM-52 Portable Raman Microscope (Snowy Range Instruments, the United States of America; 785 nm laser; 100 mV power) was used to obtain the Raman spectra from the LFIAs. For validation purposes a scanning electron microscope (SEM: JEOL 6480LA; 15.0kV voltage; 4300x, 23,000x or 37,000x magnification) was used. The LFIA was prepared for SEM by cutting it into small pieces (1 mm x 1 mm) and coating in gold nanoparticle (10 nm) spray coating. A flatbed scanner (Epson Perfection V39; Officeworks; Sydney, NSW AUS), was used for imaging the LFIAs. For characterisation of the sEV samples, NanoFCM flow cytometry (Tokyo; Japan) was utilised. Each sample was run three times diluted 1 in 10 in filtered 0.1 mM PBS. The samples were run using a 488 nm continuous wave (CW) laser at a power of 6 / 50 mW with 1 .0 Kpa pressure.

[0143] Synthesis of 50 nm spherical gold nanoparticles (AuNP)

[0144] Gold nanoparticles (AuNPs) were synthesized using the seeded growth method. Briefly, chloroauric acid (HAuCk; 0.5 mL; 25 mM) was added to the boiling milli-Q water (48 mL) under stirring conditions (650 rpm) for 10 min. To synthesize the gold seed, sodium citrate (1 % w / v; 1 .5 mL; 34 mM) was then added rapidly and refluxed for a further 30 min. The reaction was then cooled to room temperature before being used as the gold seeds for the growth of 50 nm AuNPs. All glassware was cleaned using aqua regia for the following steps. Milli-Q water (45.5 mL) was boiled under stirring conditions (650 rpm) before adding Tris-buffer (2 mL; 100 mM) and incubating for 5 min. The as prepared AuNP seeds (2 mL) was then added with extra chloroauricacid (HAuCk; 0.5 mL; 25 mM) successively. The reaction was refluxed for 30 min under continuous stirring before allowing to cool and performing characterisation.

[0145] Conjugation with Raman reporter molecules

[0146] Conjugation of AuNP with 4-MBA

[0147] For conjugation with AuNPs, 4-MBA (10 pL; 1 mM in ethanol) was added to 1 mL of AuNPs. The sample was then incubated under shaking conditions (Rotator; mode 45; rpm 60) for 2 hours (h) before being centrifuged (5500 rpm; 8.5 min) and resuspended in milli-Q water (1 mL).

[0148] Bioconjugation methods

[0149] Covalent bioconjugation via DTSSP

[0150] DTSSP (1 mg / mL; 4 pL) was first incubated with EpCAM antibody (0.5 mg / mL in PBS; 4 pL) under shaking conditions (600 rpm; Room temperature) for 30 min. The mixture was then added to AuNP (1 mL) with or without @MBA and incubated for another 30 min (600 rpm; room temperature) to form AuNP@EpCAM or AuNP@MBA@EpCAM (linked with DTSSP). The AuNP@EpCAM or AuNP@MBA@EpCAM was then centrifuged (5500 rpm; 8.5 min) and resuspended in storage buffer (300 pL; 0.05% BSA, 10% sucrose and Borate buffer 10 mM).

[0151] Passive bioconjugation

[0152] Borate buffer (100 mM; 30 uL) was added to AuNP to change the solution pH to be 9 for passive bioconjugation. EpCAM antibodies (0.5 mg / mL; 3.3 pL) were then added and incubated under shaking conditions for 30 min (600 rpm; room temperature). The AuNP@EpCAM or AuNP@MBA@EpCAM was then centrifuged (5500 rpm; 8.5 min; room temperature) and resuspended in a storage buffer (10% sucrose, 0.5% BSA and 10 mM borate buffer).

[0153] Cell line-derived sEV Isolation

[0154] Breast cancer cell lines MCF7 and SKBR3 were used for the proof-of-concept experiments and optimisation. The cell culture and isolation of the sEVs was conducted by a laboratory technician. Briefly, cell culture was performed using aspirated culture medium (RPMI+10% FBS and 1 / 100 unit of Penicillin / Streptomycin) combined with PBS and trypsin (warmed to 37°C). The cells were washed with PBS (5 mL) before being combined with trypsin (1 mL). The cells were then incubated at 37°C for 5 minutes for the cells to detach from the flask. The cells were then centrifuged (1200 rpm) for 5 minutes before resuspending in media (5 mL) as described in Zhang W, et al., (2020) ACS Sens.5(3):764-771 .Extraction of cell line and plasma sEVs was performed by ultracentrifugation following the standard sEV isolation protocol outlined in Ngo et Al. (2023) Analyst. 148(13):3074-3086.

[0155] Lateral Flow Assay Preparation and Optimisation

[0156] Lateral Flow assay preparation and procedure

[0157] The LFIAwas prepared using a nitrocellulose membrane with an absorbent pad attached. The assay has three test lines with three common sEV tetraspanins (CD63, CD81 and CD9) and a control line. The lines were dispensed using an LFIA dispenser (0.6 pL / cm; 80 mm / s; tetraspanin antibody 1 mg / mL and goat anti-mouse IgG 1 .5 mg / mL). The lines were made to be 4 mm apart. The membranes were then dried in a desiccator for 2h at room temperature. The assays were then cut to 2.5 mm wide strips using a LFIA guillotine and subsequently used for experiments. The standard procedure for the LFIA assay uses cell-derived sEVs, running buffer (10 pL; 1% Tween-20, 10% Sucrose, 0.5% BSA, 10 mM borate buffer) and nanoparticles (AuNP@EpCAM or AuNP@MBA@EpCAM; 10 pL). The nanoparticles were incubated with sEVs (SKBR3 or MCF7 or patient plasma sample derived; 5 pL; 109nanoparticles / mL) before the LFIA was inserted and allowed to proceed for 10 min. The nanoparticle-EV complex then flows through and binds to the common sEV tetraspanins on each of the test lines. The excess is absorbed by the absorbent pad. For plasma samples, the same standard procedure was used except the sEVs were substituted with half diluted plasma (5 pL; diluted in 10 mM borate buffer).

[0158] Fixing tetraspanin antibodies to the lateral flow membrane

[0159] The LFIA membrane is comprised of nitrocellulose which has a negative charge on its surface (i.e. -NO2 groups). When proteins (e.g. tetraspanin antibodies) are dispensed onto the membrane they bind via electrostatic interactions and thus become immobilised.

[0160] The assay has three test lines with three common sEV tetraspanins (CD63, CD81 and CD9) and a control line. The lines were dispensed using an LFIA dispenser (0.6 pL / cm; 80 mm / s; tetraspanin antibody 1 mg / mL and goat anti-mouse IgG 1 .5 mg / mL). The lines were made to be 4 mm apart. The membranes were then dried in a desiccator for 2h at room temperature.

[0161] Optimisation

[0162] Optimisation studies were conducted by testing incubation conditions (4°C, 22°C and 37°C), incubation times (0 min, 15 min, 30 min, 1 hr, 2 hr) and bioconjugation technique (covalent vs passive).Data

[0163] Image J analysis was conducted on the scanned images of the assays to find the overall intensity of the bands indicating EpCAM expression. The bands indicate 1 ) the presence of sEVs due to binding to tetraspanin markers, and 2) the EpCAM expression due to binding to the AuNPs. This was done through the ‘gels’ feature and the overall intensity was taken as the sums of the area under the curves for each test line. The summed intensity of three replicates was then used for statistical analysis. The data was tested for Gaussian distribution using the Shapiro-Wilk test. The non-parametric Kruskal-Wallis test and Dunn multiple comparison test was then used to determine if the means were significantly different using the obtained p-values.

[0164] Example 1 Lateral flow immunoassay design

[0165] To address the challenge of using sEVs in cancer diagnosis, the inventors have produced a multi-line lateral flow immunoassay (LFIA) which architecture is shown in Figure 1A. The innovative part of the LFIA design is the multiplexed detection and EV subtyping capabilities.

[0166] The assay serves two purposes 1) subtyping cancer-derived sEVs based on their tetraspanin expression (CD63, CD81 and CD9); and 2) detecting EpCAM on sEVs using the overall colorimetric intensity of the three test lines. The control line contains an IgG protein for general capture of the conjugated god nanoparticles (AuNPs). For cancer-specific detection, the cancer biomarker EpCAM is used on the conjugated AuNPs.

[0167] A test sample is incubated with antibody conjugated AuNPs to form a complex. The complex flows through the lateral flow membrane to be captured by the test lines of tetraspanin antibodies CD9, CD63 and CD81 forming a visible pink line indicating a positive result. If there are no sEVs present in the sample, no interaction occurs between the conjugated AuNPs and the test lines. The intensity of the three lines combined contribute to the determination of whether EpCAM+ sEVs are present in the test sample. The test lines can be observed colorimetrically to obtain a yes or a no response which is visible by naked eye.

[0168] In other embodiments, the LFIA comprises an architecture shown in Figure 1 B. In this embodiment, The assay serves two purposes: 1) detecting EpCAM on sEVs using the overall intensity of the three tetraspanin bands, and 2) subtyping cancer derived sEVs based on their tetraspanin expression (CD63, CD81 and CD9) through both colorimetric and SERS measurements. This LFIA design includes sEVs being initially incubated with the SERS nanotags to form a sEV-SERS nanotag complex. The complex then flows through the membrane to be captured by the tetraspanin antibodies (CD9, CD63 and CD81), forming a pink line. The pink colour comes from the AuNPs in the SERS nanotag design. Free SERS nanotags are captured by the control line which features a goat-anti mouse IgG protein. The goat anti-mouse IgG protein allows for the capture of the EpCAM antibody on the SERS nanotag as it is mouse anti-human.If there are no sEVs, no interaction occurs between the SERS nanotags and the test lines. Each test line is scanned using SERS or by eye (colorimetrically) and the overall intensity correlated to the amount of EpCAM expressing sEVs captured.

[0169] Example 2 Conjugation of gold nanoparticles and EpCAM antibody

[0170] Conjugation of EpCAM antibody and gold nanoparticles were characterised by transmission electron microscopy (TEM), dynamic light scattering (DLS) and ultra-violet visible spectroscopy (UV-VIS) as depicted in Figure 2 (A to C).

[0171] AuNPs were synthesised using the seeded growth as compared to the Turkovich method due to the improved reproducibility and homogeneity across the sample (Turkevich J, et al., (1951 ) Discuss Faraday Soc. 11 :55). AuNPs with a diameter of 50 nm were selected for several reasons. Firstly, 50 nm AuNPs demonstrate excellent plasmonic properties for SERS enhancements due to the strong electric field enhancements they induce. They also provide suitable a surface area for the adsorption of Raman-active molecules, further enhancing SERS sensitivity. Secondly, they exhibit favourable absorbance at -530 nm, resulting in a visible pink colour that is advantageous for colorimetric detection. However, other size nanoparticles ranging between 40nm to 10Onm, or between 40nm to 50nm also demonstrate a strong pink colour and thus are also suitable for use in the methods described herein.

[0172] As shown in the TEM image (Figure 2A), AuNPs were found to have a size around 55 + 2nm with a spherical shape. This was further confirmed by DLS, whereby the nanoparticles were found to have an average hydrodynamic size of 56 + 0.4nm. A slight discrepancy in size as compared to that obtained by TEM is due to DLS measuring the hydrodynamic size, which is slightly larger than the size of the nanoparticles by TEM as a consequence of the electric double layer of the nanoparticles (Figure 2B).

[0173] This size was selected as it has desirable optical properties (visible pink colour) while also being large enough to ensure adequate binding of multiple antibodies. AuNPs were seen to increase in size to 83.5 ± 1 .3 nm when they were successfully conjugated by antibodies. AuNPs were also found to be ideal for colorimetric applications with a UV-Vis absorption peak at 533 nm demonstrating a strong pink colour detectable by eye (Figure 2C).

[0174] Additionally, AuNPs are negatively charged as evidenced by the zeta potential of -34.53 ± 21 .27 mV. The zeta potential of below -30 mV indicates that the nanoparticles demonstrate a high

[0175] degree of stability, meaning they are less prone to aggregation.Example 3 SERS nanotaq synthesis and bioconiuqation

[0176] SERS nanotags were prepared following two steps. The as-synthesised AuNPs were first conjugated with Raman reporter molecule, 4-MBA, before being functionalised by passive conjugation with EpCAM antibodies.

[0177] 4-MBA was chosen as Raman reporter molecule because it has two distinct peaks (1078 cm-1 and 1590 cm-1) and there is no fluorescent broadening at a 785 nm laser excitation wavelength. The thiol group (-SH) on the 4-MBA binds covalently to AuNPs, forming a strong Au-S bond. The conjugated AuNPs@MBA were characterised using SERS spectroscopy, where the main characteristic peak was observed at 1078 cm-1 with a sufficient intensity of 10,980 a.u for further biosensing applications. This is important to ensure the maximum detection sensitivity of LFIAs. A hydrodynamic size of 58.75 ± 0.72 nm using DLS further confirms the conjugation with 4-MBA as the size was seen to increase when compared to AuNPs alone.

[0178] The as-synthesised AuNP@MBA were then passively bioconjugated with EpCAM antibodies for functionalisation. Passive bioconjugation involves carefully modifying the surface charge of the AuNPs by adjusting the pH to 8. This ensures that the AuNPs are negatively charged, and proteins positively charged, allowing for electrostatic interactions to occur. While the antibodies are able to successfully adsorb onto the surface of the AuNP@MBA, it is difficult to control the orientation of the antibodies, which may potentially affect binding with the sEVs. However, passive bioconjugation has been routinely used and demonstrates lower non-specific binding as compared to other techniques. Furthermore, while the passive binding are weaker than other covalent bioconjugation methods available, the benefit of using this method includes simplification, reduced costs and time.

[0179] The functionalisation was confirmed by UV-Vis and DLS characterisation. UV-Vis absorption spectra highlighted the red-shift (3 nm) observed once the antibodies were successfully bioconjugated which occurs due to changes in the refractive index on the surface of the AuNPs. This was further confirmed with DLS whereby an increase in hydrodynamic size to 83.53 ± 1.29 nm was observed (Table 3). The zeta potential of -31.20 ± 16.40 mV for the AuNP@MBA@EpCAM indicated a high degree of stability for the conjugated SERS nanotags as the more negative the zeta potential, the greater the stability. The polydispersity index (Pdl) for the AuNPs, AuNP@MBA and Au NP@MBA@ EpCAM were all seen to be less than 0.4, indicating that the samples have an ideal size distribution for the DLS technique. No TEM images were taken of the AuNP@MBA or AuNP@MBA@EpCAM as the Raman reporter molecules and antibodies are too small to be seen. Therefore, SERS nanotags were found to be successfully synthesised for further biosensing applications.Table 3 Hydrodynamic size and zeta potential of AuNP, AuNP@MBA and AuNP@MBA@EpCAM

[0180]

[0181] Example 4 Cell line-derived sEV characterisation

[0182] Human breast cancer derived sEVs from cell lines MCF7 and SKBR3 (a Her2 overexpressing line) were characterised by TEM and NanoFCM to confirm their morphology, size and concentration (Figure 2D-F). The size distribution was found to be broad with a range from 50 - 200 nm. They were found to have an average size of 84.2 ± 4.7 nm and 85.0 ± 4.2 nm for MCF7 and SKBR3, respectively. As each cell-line is less than 200 nm, the size falls within the naming guidelines for sEVs defined by ISEV (Welsh JA, et al., (2024) J Extracell Vesicles.

[0183] 2024;13(2):e12404). A concentration of (6.1 ± 1 .2) x 109nanoparticles / mL was obtained for MCF7 sEVs and (2.1 ± 1 .7) x 109nanoparticles / mL for SKBR3 sEVs. These concentrations were used for subsequent assay preparation. As the running buffer used in the LFIA can be harsh for sEVs due to the use of surfactants (Tween-20), nanoparticle counts from NanoFCM were obtained to ensure ideal stability. The sEVs were found to be sufficiently stable over a two hour incubation period in the running buffer (Figure 3). A running buffer containing 1% Tween-20 was found to be ideal for running conditions and has sufficient nanoparticle numbers for colorimetric applications.

[0184] Example 5 LFIA using cell line derived sEVs.

[0185] Optimisation

[0186] Optimisation of the LFIA was a key factor to ensure optimal binding and point-of-care running conditions. Various conditions including temperature, bioconjugation methods, sEV stability and incubation time were tested. The ideal conditions chosen were analysed with respect to their colorimetric visual appearance allowing for the darkest intensity to be observed, best running conditions for point-of-care testing, reproducibility, stability and accuracy (low nonspecific binding).

[0187] To ensure optimal binding of sEVs to SERS nanotags, temperatures at 4°C, 22°C and 37°C were examined during the incubation of sEVs and SERS nanotags. An incubation time of 2 hours between the SERS nanotag and sEVs was chosen. Room temperature (22°C) produced the darkest visible bands. This was confirmed by image J analysis. Room temperature is alsoideal for point-of-care biosensing applications whereby cooling and heating would induce extra costs for the required equipment.

[0188] As bioconjugation of antibody to SERS nanotags is a crucial component to the functionality of the LFIA, different bioconjugation techniques were tested and analysed based on their colorimetric intensity and degree of non-specific binding. Using a linker such as DTSSP is a common bioconjugation technique that ensures a high degree of stability due to strong covalent bonding. DTSSP has a disulphide bond in the middle of the molecule and two NHS-ester functional groups at either end. The NHS-ester functional groups bind to the amino groups on the proteins before the disulphide bonds are broken to form a covalent Au-S bond with AuNPs. To test each bioconjugation method, MCF7 cell line-derived sEVs at a concentration of (6.1 ± 1.2) x 109nanoparticles / mL (5 pL) were used as the positive sample. Hence, for the positive samples, SERS nanotags, sEVs and running buffer were incubated together and run using the LFIA. The negative control was the SERS nanotags and running buffer alone with no sEVs. Passive bioconjugation, where MBA is electrostatically bonded to AuNPs was found to be more suitable than DTSSP as less non-specific binding was observed.

[0189] Different incubation times were tested (15 min, 30 min, 1 hr and 2 hrs). While the assays showed clear bands from 15-minute incubation for CD9 and CD63 test lines, the CD81 test line did not become evident until the 1 -hour incubation time point. At 2-hours, the CD81 line became clearer and ideal for colorimetric applications, making it the ideal incubation time for cell line derived sEVs. Hence, a 2-hour incubation time was determined to be optimal for cell line-derived sEVs.

[0190] Assay characterisation

[0191] As proof of concept, the LFIA was evaluated using sEVs derived from two breast cancer cell lines: MCF7 and SKBR3, due to their high EpCAM expression. Figure 4 illustrates triplicate assays for sEVs from each cell line, along with a negative control consisting of conjugated AuNPs and running buffer without sEVs. This negative control serves to detect any potential nonspecific binding between conjugated AuNPs and test line antibodies. The MCF7 and SKBR3 LFIAs exhibited three distinct test line bands, confirming positive EpCAM expression. Furthermore, the assays successfully subtyped the sEVs populations based on their tetraspanin expression (CD9, CD81 , and CD63). Among these, CD9 demonstrated the highest expression in the breast cancer cell line derived sEVs, followed by CD63 as the second brightest line. CD81 was faintly visible, approaching the colorimetrically visible limit of detection.

[0192] The negative control LFIA replicates showed only the control line, indicating the absence of non-specific binding between the conjugated AuNPs and test line antibodies, thus validating the assay's specificity. In conclusion, the LFIA successfully demonstrated colorimetric detectionof EpCAM expression in two breast cancer cell lines by a ‘yes’ or ‘no’ colorimetric response, while also providing insights into the relative expression levels of common sEV tetraspanins.

[0193] Due to the sequential filling nature of LFIAs from the first line to the last, it is crucial to ensure an abundance of sEV-conjugated AuNPs complexes capable of binding to each test line. This abundance is essential for minimizing result variations and guaranteeing accurate detection and subtyping capabilities.

[0194] To verify the use of sufficient sEV-conjugated AuNPs complexes, the inventors implemented comprehensive testing of each of the possible tetraspanin permutations. Multi-line LFIA designs were created, each featuring a different order of tetraspanins (CD9, CD63, and CD81 ) test line. The tetraspanin antibodies were used at a concentration of 0.5 mg / mL with MCF7 cell line-derived sEVs at a concentration of (6.1 ± 1 .2) x 109nanoparticles / mL. This concentration of capture antibodies was determined to ensure consistent overall EpCAM expression. Three replicates were produced for each condition and tetraspanin order (Figure 5A). The inventors examined two key aspects for each tetraspanin order: the overall expression level and the relative colorimetric intensity of the subtyped population. These analyses were conducted using Imaged software for colorimetric assessment, ensuring consistency in result output across different LFIA designs (Figure 5B).

[0195] The LFIA permutations illustrated in Figure 5A demonstrated consistent colorimetric results for overall EpCAM expression, irrespective of the order of the test lines. This consistency is evident in the combined intensity of all three bands across different permutations. Image J analysis further corroborated these findings, as shown in Figure 5B, revealing no statistically significant difference in band intensities among the various permutations (p = 0.076; Kruskal-Wallis test). This p-value, being greater than the significance threshold of 0.05, supports the conclusion that the overall intensity of EpCAM expression remains consistent regardless of the order of the test lines on the LFIA. Furthermore, a general trend can be observed in the relative expression of each tetraspanin test line, as further validated by NanoFCM characterisation of the sEV populations.

[0196] NanoFCM data showed that the expression level of CD9 has higher expression (6.2%) than CD63 (2.2%) and CD81 (0.9%) (Figure 6). This trend was also evident regardless of tetraspanin order on the LFIA, whereby CD9 has the highest visual expression in every combination, with CD81 and CD63 being similar. Therefore, the LFIA was demonstrated to be able to successfully detect the presence of EpCAM regardless of tetraspanin order as well to successfully subtype the captured sEV population. To conclude, the inventors show that the sEV-conjugated AuNPs complexes are in excess, allowing for the overall intensity to remain consistent regardless of tetraspanin order. Furthermore, the assay can successfully subtype sEVs based on their tetraspanins expression.To ensure the clinical applicability of the proposed multi-line LFIA assays, the inventors also tested the assay with a cohort of patient samples. To confirm the presence of sEVs in human plasma, sEVs were isolated from plasma using ultracentrifugation and characterized by NanoFCM for their size and concentration (Figure 7). Due to the heterogenous nature of sEVs, the broad spectrum of sizes was observed. Although the sizes vary, they would not impact the functionality of the LFIA design which is designed to target any sEV with 40 - 200 nm in size.

[0197] The concentrations were found to range from 108-109nanoparticles / mL, making them ideal for subsequent LFIA testing. The concentration of sEVs at 108nanoparticles / mL were used in the LFIA design to ensure there was an abundance of sEVs to bind with the AuNPs for initial testing purposes.

[0198] Sample pre-processing still remains a large issue in translating new device designs to clinic, as it induces high costs, requires speciality knowledge, and extensive time. Hence, to limit sample preprocessing, plasma was directly used (i.e. without sample pre-processing) in the present study with the optimised LFIA design. To ensure the plasma samples had sufficient sEVs in the adequate size range for LFIA biosensor, characterisation was conducted using TEM and NanoFCM to identify the size, shape, concentration and surface expression of plasma derived sEVs.

[0199] The LFIA was tested with 5 breast cancer patient samples at stages varying from (0-IV) and 5 healthy plasma samples. All samples were women of a post-menopausal age. 3 pL of halfdiluted plasma was directly incubated with the nanoparticles before being run with the assay.

[0200] When tested with the designed LFIA, breast cancer patients at each stage were found to have clear positive test lines (Figure 8), with the darkest visible lines being from stage IV and faint but visible lines at stage 0. The healthy individuals were found to be significantly fainter in expression of EpCAM as compared to the breast cancer patients, which correlates to the low-level expression of EpCAM that can be observed in healthy people (Schmelzer E, et al., (2008) Front Biosci. 2008;13:3096-3100). As EpCAM is a cancer specific biomarker, it was able to successfully detect high amounts in patient plasma as compared to healthy ones (Abhange K, et al., (2021) Bioactive Materials. 2021 ;6(11):3705-3743). This trend is expected, with overexpression of EpCAM being reported to indicate the presence of cancer (Osta WA, et al., (2004) Cancer Research. 2004;64(16):5818-5824). Therefore, the LFIA was able to successfully subtype cancer sEVs, and provide a colorimetric ‘yes’ or ‘no’ response, indicating the potential for use as a clinical aid in the detection and subtyping of cancer derived sEVs.

[0201] Example 6 SERS-based quantitative detection

[0202] SERS based- LFIA design is key for quantitative detection with high sensitivity. The purpose of using SERS detection is to lower the detection limit further to any SERS nanotag-sEVs complexes captured without necessarily being visible by eye. The Raman characteristic peak at 1080 cm-1 of 4-MBA on the SERS nanotag can be used to qualitatively determine the presence of sEVs and quantitatively determine the concentration of sEVs on each test line.

[0203] As can be seen in Figure 9, the intensities for MCF7 and SKBR3 observed by eye correlated with the SERS spectra obtained. The control line was seen to have the highest intensity for both cell line derived sEVs, alongside CD9 which had the second highest. For SKBR3, CD63 was found to have a higher Raman intensity than CD81 , however for MCF7 cell line-derived sEVs, the Raman spectra was very similar between CD81 and CD63, with CD81 being slightly higher. This is due to the point-based Raman system that was used for measurements whereby ten points were taken along each test line and averaged.

[0204] To summarise, the ongoing prevalence of breast cancer and the lack of suitable rapid test for diagnostics have highlighted the critical need for a rapid, sensitive, and accurate device. This study implemented a multi-line LFIA design with two primary objectives: to detect overall EpCAM expression and to subtype the population of sEVs based on their tetraspanins expression for clinical application. The proposed novel multi-line LFIA design for detection and subtyping of cancer sEVs presents a promising clinical aid for breast cancer diagnosis. The assay successfully detected EpCAM using the overall intensity of the three test lines (p=0.076) and subtyped sEV tetraspanins regardless of order. A significant advantage for clinical translation is the LFIA's ability to use plasma directly, potentially reducing the need for sample pre-processing.

[0205] The LFIA shows promising potential as a clinical aid for comprehensive sEV analysis and cancer detection, offering a powerful and promising tool in early breast cancer diagnostics.

Claims

CLAIMS:

1. A lateral flow test device for optical detection of cell-derived small extracellular vesicles (sEVs) in a sample, the device comprising a porous matrix comprising at least three distinct test locations on said porous matrix, each of the test locations comprising a reagent that binds to a marker present on the sEVs and wherein the reagents at the at least three test locations bind to different markers on the sEVs, and wherein the sample flows laterally along the test device and passes the test locations, and further wherein binding of the sEVs at the test locations is confirmed by an optical signal.

2. The device of claim 1 , wherein the markers present on the sEVs are tetraspanins.

3. The device of claim 1 or 2, wherein binding of the sEVs at the test locations is confirmed by contacting the bound sEVs with at least one nanotag that binds to a cancer antigen on the sEVs.

4. The device of claim 3, wherein cancer-specific sEVs are detected.

5. The device of claim 1 or 2, wherein the markers present on the sEVs are selected from the group consisting of CD63, CD81 , CD9, CD82 and CD151.

6. The device of claim 5, wherein the markers are CD63, CD81 and CD9.

7. The device of any one of claims 1 to 6, wherein the at least three test locations comprise a binding reagent immobilised on the surface of the test device which binds a tetraspanin biomarker on the sEVs.

8. The device of claim 7, wherein the binding reagents comprise an anti-CD63, an anti-CD81 and an anti-CD9 binding reagent.

9. The device of any one of claims 1 to 8, wherein the sEVs are obtained from a subject with cancer, preferably breast cancer.

10. The device of claim 3, wherein the nanotag comprises a binding agent bound to an optically detectable nanoparticle.11 . The device of claim 10, wherein the binding molecule binds to a cancer antigen on the surface of the sEVs.

12. The device of claim 11 , wherein the cancer antigen is selected from the group consisting of one or more of EpCAM, PD-1 , PD-L1 , HER1 (EGFR), HER2, HER3, TROP2, MUC1 , PROCR, MET, CTLA4, CD44, CD47, CD133, E-cadherin, and N-cadherin.

13. The device of claim 12, wherein the cancer antigen is EpCAM.

14. The device of claim 10, wherein the binding agent is selected from a full length antibody, an antibody fragment, an aptamer or an i-body.

15. The device of any one of claims 1 to 14, wherein the nanoparticle is selected from a core shell structure, a silica nanoparticle, a metal organic framework or a satellite structure.

16. The device of claim 15, wherein the core shell structure is a silver coated nanoparticle, a gold coated nanoparticle, or gold shell.

17. The device of claim 15 or 16, wherein the nanoparticle is a nanosphere, nanorod, nanostar, nanoshell, nanotriange, nanocube or nanocage.

18. The device of claim 17, wherein the nanoparticle is a gold coated nanosphere.

19. The device of any one of claims 1 to 18, wherein the sample comprises sEVs obtained from a subject with cancer.

20. The device of any one of claims 1 to 19, wherein the sample is blood, urine or saliva.21 . The device of any one of claims 9 to 20, wherein a Raman reporter molecule is bound to the nanoparticle.

22. The device of claim 21 , wherein the Raman reporter molecule is selected from the group consisting of 4-Mercaptobenzoic acid (4-MBA), 4-Nitrothiophenol (4-NTP), 4-Aminothiophenol (4-ATP), 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB), 2,3,5,6-Tetrafluoro-4-mercaptobenzoic acid (TFMBA), 4-Mercaptopyridine (2-Mpy), 2-Naphthalenethiol (2-NAT), 2,7-Mercapto-4-methylcoumarin (MMC) and Rhodamine X (ROX).

23. The device of claim 22, wherein the Raman reporter molecule is 4-MBA.

24. A complex comprising cancer sEVs bound to a nanotag, the nanotag comprising a binding agent that binds to a cancer antigen present on the sEVs and wherein the binding agent is bound to a nanoparticle having an optical property, and optionally wherein the nanoparticle further comprises a Raman reporter molecule bound thereto.

25. A method for optically detecting cancer cell-derived small extracellular vesicles (sEVs) in a sample, the method comprising:(i) contacting the sample with a nanotag comprising a binding agent bound to an optically detectable nanoparticle;(ii) applying the sample to the test device of any one of claims 1 to 23, wherein the sample is applied to a site of the test device upstream of the test locations; and(iii) detecting a colorimetric signal at the one or more test locations.

26. A method for subtyping cancer-specific sEVs in a sample, the method comprising:(i) contacting the sample with a nanotag comprising a binding agent bound to an optically detectable nanoparticle;(ii) applying the sample to the test device of any one of claims 1 to 23, wherein the sample is applied to a site of the test device upstream of the test locations; and(iii) detecting a colorimetric signal at the one or more test locations wherein the intensity of the signal at each of the test locations allows for sEV subtyping.

27. A method of detecting and / or diagnosing breast cancer in a subject, the method comprising:(i) contacting the sample with a nanotag comprising a binding agent bound to an optically detectable nanoparticle;(ii) applying the sample to the test device of any one of claims 1 to 23, wherein the sample is applied to a site of the test device upstream of the test locations; and(iii) detecting a colorimetric signal at one or more of the test locations corresponding to CD63, CD81 and CD9 reagents.

28. The method of claim 27, further comprising subsequently treating the subject with surgery and / or chemotherapy, and / or radiotherapy.

29. The method of any one of claims 25 to 28, further comprising the use of Raman spectroscopy to detect and / or to quantitate the signal detected at each of the test locations.

30. The method of claim 27 to 29, wherein detecting a signal indicates that cancer is present in the subject.31 . A system for quantitatively detecting cancer cell-derived small extracellular vesicles (sEVs) in a sample, the system comprising:(i) the test device of any one of claims 1 to 23; and(ii) a reader that comprises a light source and a photodetector to detect a detectable signal emitted by the Raman reporter molecule.

32. The system of claim 31 , wherein the system comprises the use of Raman spectroscopy.

33. A kit for quantitatively detecting cancer specific sEVs in a sample, the kit comprising:(i) the test device of any one of claims 1 to 23; and(ii) instructions for using the test device to optically and / or quantitatively detect cancerspecific sEVs present in the sample.

34. The kit according of claim 33, further comprising a container comprising a binding agent that binds to a cancer biomarker bound to a nanoparticle having a colorimetric property and optionally wherein the nanoparticle further comprises a Raman reporter molecule bound thereto.

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