Nanoparticles comprising a starch-iodine complex and their use for immunodetection

Abstract

The invention relates to nanoparticles comprising an iodine-starch complex in the core of the nanoparticles, or on their surface. The invention furthermore relates to a composition comprising a plurality of such nanoparticles, an immunodetection sensor comprising the nanoparticles and an immunodetection method using them for optical and / or electrochemical detection.

Description

[0001] Nanoparticles comprising a starch-iodine complex and their use for immunodetection

[0002] Technical field

[0003] The invention relates to nanoparticles comprising an iodine-starch complex in the core, or on their surface. The invention furthermore relates to a composition comprising a plurality of such nanoparticles, an immunodetection sensor comprising the nanoparticles and an immunodetection method using them for optical and / or electrochemical detection.

[0004] Background of the invention

[0005] COVID-19 pandemic has highlighted the need for simple, affordable, and easy-to-use point- of-care devices that can be used in situ for the patient without specialized personal. Furthermore, such devices are of great interest for environmental control or food safety applications. Lateral flow immunoassays (LFIAs) have become one of the dominant options along the years. They are based on immunochromatographic membranes where the sample and the antibodies diffuse, and nano-labels are used to create a visual signal in order to confirm the presence or the absence of the analyte. Different types of nano-labels can be used, gold nanoparticles (AuNPs) being the most common ones. Gold nanoparticles have great catalytic and conductive properties with simple and well-known synthesis procedures.

[0006] Immunochromatographic assays are based on using a nitrocellulose chromatographic membrane immobilized with a biological or biochemical receptors (antibody, antigen, aptamer or nucleic acid probes) on the test line (TL) and a secondary bioreceptor on the control line (CL). The sample is added to the sample pad and the analyte binds to the labelled biological or biochemical receptor on the conjugate pad. The sample then flows through the membrane by capillary action, where the target is captured by the TL receptor while the excess binds to the other receptor system on the CL. The result is interpreted by comparing the appearance of the two lines in a visual way with measurement of optical density signals as a semiquantitative method. However, this technique has obvious drawbacks such as low sensitivity or repeatability1, and the need to provide a quantitative result. Different alternatives have been investigated to improve the sensitivity of LFIAs, such as other types of nanoparticles to be used with other detection techniques with intrinsic better sensitivity such as fluorescence or magnetic nanoparticles, instead of optical density or reflectance analysis. But these require more complex and bulky readers. There remains thus a need for providing methods for enhancing the signal in LFIAs to overcome the above disadvantages and to allow the use of portable and small devices for the signal readout with ultra sensitivity.

[0007] Electrochemistry can be applied to the LFIAs because of the catalytic and conductive or electroactive properties of gold nanoparticles (AuNPs). This technique can improve the sensitivity of these tests in a cheap, simple, and rapid analysis by coupling the test line with an electrochemical cell. This involves applying or inserting screen-printed electrodes into the strip and using an electrochemical label, measuring a current signal or another electroanalytical property related to the label or the analyte. In this way, the POC device minimize matrix effects increasing the sensitivity.

[0008] Different labels can be used in electrochemical LFIAs (eLFIAs). There is research of eLFIAs based on enzymatic tracers2or quantum dots (QDs)3. However, gold nanoparticles which can be the most common label for visual analysis in lateral flow immunoassays, are rarely used for this purpose. In some of these cases they measure both the horseradish peroxidase (HRP) enzyme signal as an amplification probe4and dissolved Au(lll) ions by square wave voltammetry5, while in others they perform amperometric measurements of the analyte itself6.

[0009] Gold nanoparticles need to be conjugated to antibodies or other biological or chemical receptors. This process is frequently performed by passive adsorption taking advantage of the great affinity of sulfur atoms or thiol groups on the amino acid chains, for the gold surfaces. Then, once the biological or biochemical receptors are already adsorbed on the surface of the nanoparticle, the remaining free sites are blocked with proteins such as bovine serum albumin, caseine, or polymers such as polyethylene glycol7to avoid unspecific adsorptions.

[0010] In addition to the AuNPs, other types of nanoparticles have shown great potential in LFIA applications. On the one hand, colored latex microspheres (LM), also referred to as latex nanoparticles, offer comparable sensitivity to AuNPs and have the advantage of a wide range of colors, making them ideal for multiplexed assays for optical detection. Their polystyrene- based composition allows efficient adsorption of proteins via hydrophobic interactions, and their surfaces can be functionalized with acid or amine groups for robust bioconjugation11’12.

[0011] On the other hand, magnetic nanoparticles (MNPs), also have gained attention thanks to their superparamagnetic properties, which allow magnetic signal transduction for quantitative detection. Unlike optical detection, which is limited to the membrane surface, magnetic transduction measures particles throughout the depth of the membrane, improving the sensitivity of the assay. In addition, MNPs facilitates pre-concentration of the analyte by magnetic separation, a major advantage in complex biological matrices13’14.

[0012] Furthermore, biodegradable nanoparticles have attracted increasing interest in recent years for different purposes. Starch nanoparticles (SNPs) are natural, abundant, renewable and inexpensive to produce8. SNPs provide different physicochemical and biological properties compared to native starch. Among their advantages are higher solubility, reaction surface, absorptive capacity and biological penetration rate. The use of SNPs in various applications, such as the food industry, production of nanocomposite materials, for biomedical applications packaging, in cosmetics and as colloidal stabilizers has been described.

[0013] Iodine can be measured electrochemically through coulometry9, a technique where the potential of the working electrode is kept constant, allowing the analyte to exchange electrons across the electrode-solution interface. Various types of iodine nanoparticles and their use in different fields are disclosed in the prior art. Iodine nanoparticles and their use as X-ray reagents have been described for example in LIS2021 / 0069352 A1 , where the iodine nanoparticles are a reaction product of functionalized triiodo benzene, linking monomers, and biocompatible polymers. Nanoparticles formed from iodide and ions of an alkali metal or alkaline earth metal and their use for radiation therapy has also been described (WO2023 / 039415 A2). However, the use of such iodine-carrying nanoparticles in LFIAs has not been described to date.

[0014] A wide variety of targets can be detected using such nanoparticles and LFIAs containing such nanoparticles, such as for example extracellular vesicles (EVs). Over the past decade EVs have been recognized as essential elements in mediating intercellular communication that is fundamental to the maintenance of normal physiology and plays a critical role in a variety of pathological conditions. Ranging in size from nano- to micrometers and surrounded by a lipid membrane, these particles are secreted by virtually all cell types and are therefore present in almost all tissues and biological fluids15 16. Their ability to transport bioactive molecules highlights their importance in the regulation of cellular functions and their association with a wide range of diseases13. Therefore, EVs have been identified as promising non-invasive biomarkers in several diseases, such as cancer17 18and cardiovascular conditions19, as they reflect pathological changes through their quantity and specific content, opening new opportunities for early diagnosis and monitoring of disease progression.

[0015] Accurate detection of EVs is challenging because they are heterogeneous in size, origin and molecular composition. In addition, they are present in all body fluids along with a variety of other cellular and molecular components that can interfere with analytical results20. Techniques traditionally used to detect EVs include ultracentrifugation, which is considered the reference standard, microscopy, flow cytometry and enzyme-linked immunosorbent assay (ELISA)21. Although these approaches provide reliable results, they have important limitations, such as technical complexity, high cost and, in the case of immunoassays, low sensitivity, which makes it difficult to detect low concentrations of EVs in biological fluids, especially in the early stages of the disease.

[0016] To address these challenges, innovative methods for EV isolation and detection have recently been developed based on technologies such as microfluidics, biomolecular probes, nanomaterials, surface plasmons and magnetic technology22 23. These advanced strategies have brought closer the possibility of implementing liquid biopsies focused on the detection of EVs, with great potential to transform the early and non-invasive diagnosis of various diseases. However, many of these methods still face significant barriers to their routine use in the clinic, including low throughput and the need for signal amplification, which complicate their application in common diagnostic scenarios.

[0017] The inventors have set out to provide new nanoparticles that are suitable for use in LFIAs and eLFIAs that can overcome the disadvantages of the detection methods of the prior art, such as low sensitivity and repeatability, the requirement of complex and large readers when using fluorescence or magnetic nanoparticles instead of optical analysis to enhance the signal, and the fact that two detection methods can be used in the same system i.e. optical or electrochemical detection, or both together, depending on the LFIA chosen.

[0018] Summary of the Invention

[0019] The present invention therefore relates to a nanoparticle comprising an iodine-starch complex, preferably wherein the nanoparticle is suitable for immunodetection.

[0020] In one aspect of the nanoparticle of present invention the iodine-starch complex is located on the surface of the nanoparticle.

[0021] In one preferred embodiment of this aspect the nanoparticle is selected from a gold nanoparticle (AuNP), a magnetic nanoparticle and a latex nanoparticle.

[0022] In one embodiment the nanoparticle comprises antibodies, antibody fragments, proteins, peptides, nucleic acids, aptamers, or other biological or biochemical receptors conjugated to the surface.

[0023] In one embodiment the surface of the nanoparticle is entirely covered by the antibodies, antibody fragments, proteins, peptides, nucleic acids, aptamers, or other biological or biochemical receptors, and the iodine-starch complex. In one embodiment the nanoparticle has been blocked in a 0.1 % starch (w / v) starch-iodine solution.

[0024] In one embodiment the mean diameter of the AuNP without the iodine starch complex on the surface is between about 20 nm - 50 nm, preferably between about 30 nm - 40 nm.

[0025] In one embodiment the mean diameter of the AuNP with the iodine starch complex on the surface is between about 60 nm to 90 nm, preferably between about 70 nm to 80 nm.

[0026] The present invention furthermore relates to a method for preparing a nanoparticle as described in this aspect, the method comprising the steps of

[0027] (i) conjugating the nanoparticle with an antibody, antibody fragment, protein, peptide, nucleic acid, aptamer, or other biological or biochemical receptor; and

[0028] (ii) contacting the conjugated nanoparticle of step (i) with an iodine-starch solution, wherein step (i) is performed before step (ii).

[0029] In one embodiment of the method, the iodine-starch solution is a solution comprising between about 0.05 - 1% (w / v), preferably between about 0.05 - 0.5% (w / v) of starch, most preferred about 0.1% (w / v) of starch.

[0030] In one embodiment of the method of present invention the step (ii) is performed for at least 30 minutes, preferably for at least 45 minutes.

[0031] In one embodiment the step (ii) is performed at room temperature and at pH 7-9.

[0032] In another aspect the present invention relates to a nanoparticle having a core and a shell wherein the iodine-starch complex is located in the core of the nanoparticle.

[0033] In one embodiment the core of the nanoparticle comprises between about 1 % - 4% (w / v), preferably between about 1% - 2% (w / v) of the iodine-starch complex.

[0034] In one embodiment of this aspect the shell comprises at least one polyelectrolyte, at least one surface active agent and at least one stabilizer.

[0035] In one embodiment the polyelectrolyte is selected from the group consisting of polylactic-co- glycolic acid (PLGA), Polyethylene Glycol (PEG), polylactic acid (PLA), poly-e-caprolactone (POL), or combinations thereof. In one embodiment the surface-active agent is selected from the group consisting of phospholipids or non-ionic surfactants, or a combination thereof.

[0036] In one embodiment the phospholipid is phosphatidylcholine or phosphatidylserine or a combination thereof.

[0037] In one embodiment the non-ionic surfactant is Span 60 or Span 80, or combinations thereof.

[0038] In a preferred embodiment the nanoparticle has a core and a shell, and the iodine-starch complex is located in the core of the nanoparticle, wherein the shell comprises at least one polyelectrolyte, at least one surface active agent and at least one stabilizer, and wherein

[0039] (i) the polyelectrolyte is selected from the group consisting of polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), poly-e-caprolactone (PCL), or combinations thereof; and / or

[0040] (ii) the at least one surface-active is a phospholipid selected from phosphatidylcholine, phosphatidylserine, or a combination thereof, or a non-ionic surfactant selected from Span 60, Span 80, or a combination thereof.

[0041] In one embodiment the at least one stabiliser is selected from the group consisting of polyvinylalcohol, dodecanol, cholesterol, cholesteryl hemi succinate, or combinations thereof.

[0042] In one embodiment the mean diameter of the nanoparticle is between about 150 nm to about 250 nm, preferably between about 190 nm to about 210 nm.

[0043] In one embodiment the nanoparticle is conjugated with a protein, antibody, antibody fragment, peptide, nucleic acid, aptamer, or other biological or biochemical receptor.

[0044] In one embodiment the mean diameter of the nanoparticle conjugated with a protein or antibody is between about 200 nm to about 300 nm, preferably between about 250 nm to 280 nm.

[0045] In a third aspect the present invention relates to a composition comprising a plurality of nanoparticles as described in the above aspects.

[0046] In one embodiment of this aspect the composition comprises between 1 - 4% (w / v) of native quinoa starch nanoparticles previously stained with Lugol’s reagent (containing 5% (w / v) of iodine and 10% (w / v) of potassium iodide). In a preferred embodiment the nanoparticles have been stained with a volume ratio of nanoparticle to Lugol's reagent of 1 :2.

[0047] In a fourth aspect the present invention relates to an immunodetection sensor comprising the nanoparticle or the composition comprising a plurality of nanoparticles as described herein.

[0048] In a fifth aspect the present invention relates to an immunodetection method comprising the steps of

[0049] (i) mixing a sample with the nanoparticle(s) or the composition comprising a plurality of nanoparticles as described herein; and

[0050] (ii) detecting an electrochemical signal, an optical signal, or both.

[0051] In one embodiment of this aspect the signal is detected upon binding of the nanoparticle to an analyte selected from extracellular vesicles (EVs), bacteria, antibodies, proteins, metabolites, pollutants or allergens.

[0052] In a sixth aspect the present invention relates the use of the nanoparticle(s) or the plurality of nanoparticles, or the immunodetection sensor as described herein for the detection of an analyte, preferably wherein the analyte is selected from the group consisting of extracellular vesicles (EVs), bacteria, antibodies, proteins, metabolites, pollutants or allergens.

[0053] BRIEF DESCRIPTION OF THE FIGURES

[0054] Figure 1 : Hydrodynamic diameter distribution profile of SNPs stained with iodine.

[0055] Figure 2: TEM images taken at 100000x showing SNPs and agglomerates with a range of sizes from 50 nm up to 500 nm.

[0056] Figure 3: FTIR spectra of the SNPs before (dashed line) and after (solid line) staining them with iodine.

[0057] Figure 4: Scheme showing the synthesis and structure of the SNPs in PLGA-PVA / PC by emulsion / solvent evaporation method.

[0058] Figure 5: TEM micrograph of the lipid polymer hybrid nanoparticles encapsulating stained SNPs.

[0059] Figure 6: Hydrodynamic size distribution profiles of SNPs in PLGA-PVA / PC before (dotted line) and after (solid line) conjugation with a neutravidin concentration of 1 mg / mL. Figure 7: Photography (front view) and schematic illustration of the biotin-neutravidin affinity test (side view).

[0060] Figure 8: Coulometry performed at a fixed potential of + 0.75 V measuring signal at the test line in the CN95 membranes (dotted line for negative control and dashed line for positive TL) and FF120HP (long dash for negative control and solid line for positive TL).

[0061] Figure 9: A) Schematic illustration of the biotin-neutravidin affinity assay (side view) based on Au NPs as reporter labels blocked with iodine-doped quinoa starch. B) Scheme of Au NP blockage. Enlarged is the schematic of the starch-iodine complex, in which the l3’ ions are embedded in the amylose helix that forms starch together with amylopectin.

[0062] Figure 10: Hydrodynamic size distribution profiles of gold NPs before (dotted) and after conjugation with different blocking compounds: BSA (dashed) and starch-iodine (solid).

[0063] Figure 11 : Normalized absorbance UV-VIS spectra of colloidal gold (dotted) and the neutravidin conjugates blocked with BSA (dashed) and iodine-doped starch (solid). The maximum wavelength shifts towards red in the case of the conjugates.

[0064] Figure 12: Coulometry of lateral flow electrochemical assay: negative control (dotted), 0.15 mg / mL neutravidin (dashed), and 0.3 mg / mL neutravidin (solid) blocked with BSA 1 mg / mL (E = 0.5V, t = 100 s).

[0065] Figure 13: Coulometry of lateral flow electrochemical assay: negative control (dotted), 0.15 mg / mL neutravidin (dashed), and 0.3 mg / mL neutravidin (longdash) blocked with iodine-doped starch 1 mg / mL (E = 0.5V t = 90 s).

[0066] Figure 14: Representative TEM images of the bioconjugates of (A) Au NPs, where the scale bar corresponds to 20 nm, (B) MNPs, with a scale bar of 0.5 pm and (C) LM, whose scale bar corresponds to 0.5 pm.

[0067] Figure 15: Linear response for the developed gold-based eLFIA for quantification of EVs.

[0068] Figure 16: Linear response for the developed magnetic eLFIA for quantification of EVs.

[0069] Figure 17: Linear response for the developed latex-based eLFIA for quantification of EVs. Detailed description

[0070] The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention, and to the examples included therein.

[0071] As described in more detail above there is an unmet need for improved LFIAs with increased sensitivity, specificity, versatility and simplicity of use.

[0072] The inventors have set out to provide new nanoparticles that are suitable and beneficial for use in LFIAs that can overcome the disadvantages of the detection methods of the prior art, such as the low sensitivity and repeatability, the requirement of complex and large readers when using fluorescence or magnetic nanoparticles instead of optical analysis to enhance the signal and the fact that two detection methods can be used, i.e. only optical visualization only electrochemical detection depending on the LFIA chosen, or both types of detection on a single test.

[0073] The inventors have therefore developed nanoparticles that combine an enhanced signal, versatility in the method of signal detection depending on the needs and ease of use.

[0074] The present invention therefore relates to a nanoparticle wherein the nanoparticle comprises an iodine-starch complex.

[0075] The starch can be native quinoa starch, amaranth starch or maize starch, or can be derived from these or other starch types. In one embodiment the starch is selected from sources having longer chain amylose. In a preferred embodiment the starch is native quinoa starch.

[0076] As is described in more detail here below the inventors have found that the nanoparticles can be formed by encapsulating the iodine-starch complex in a micelle, also referred to as a shell herein. On the other hand, the iodine-starch complex can be doped onto the surface of a nanoparticle, such as for example a gold nanoparticle (AuNP), a magnetic nanoparticle or a latex nanoparticle.

[0077] NANOPARTICLES WITH ENCAPSULATED IODINE-STARCH COMPLEX

[0078] As described in more detail in examples 1 and 2 starch nanoparticles (SNPs) with longer chain amylose have been selected due to the strong blue color they obtain when iodine and the amylose form a complex and due to the stability of this complex. The iodine-stained SNPs were then encapsulated using a single emulsion / solvent method and hybrid nanoparticles, i.e., iodine-stained SNPs encapsulated in PLGA-PVA / PC were prepared.

[0079] In one embodiment of present invention the starch is selected among the sources with longer chain amylose. Starch derived from quinoa is preferred.

[0080] In one embodiment of the present invention the nanoparticle therefore has a core and a shell and the iodine-starch complex is located in the core of the nanoparticle. In one embodiment the core of the nanoparticle comprises between about 1% - 4% (w / v), preferably between about 1 % - 2% (w / v) of iodine-starch complex.

[0081] The shell of the nanoparticles can be further functionalized to bioconjugate it to the desired protein or antibody or other biological or biochemical receptor for detection. In addition, the shell allows to have more nanoparticles attached to a single antibody. This improves the sensitivity of the detection, versus to a single particle attached to a single unit of receptor.

[0082] The shell of the nanoparticles of present invention can comprise a polyelectrolyte that can bind the cargo, such as the starch iodine complex, surface active agents acting as main membrane components and stabilizers that provide the required stability to the micelles.

[0083] Encapsulation by Lipid-polymer hybrid nanoparticles (LPHNPs) has been previously disclosed10. Encapsulation by LPHNPs offers several advantages such as enhancing device sensitivity as well as the possibility to modify the properties of the surface to provide a better interaction with biological molecules.

[0084] In one embodiment of present invention the polyelectrolyte is selected from the group consisting of polylactic-co-glycolic acid (PLGA), Polyethylene Glycol (PEG), polylactic acid (PLA), poly-e-caprolactone (PCL), or combinations thereof.

[0085] Poly (lactic-co-glycolic acid) (PLGA) is described in the prior art as an alternative to other conventional nano colloids, such as nano emulsions or liposomes that allows the possibility to modify surface properties to provide stealthiness and / or better interaction with biological materials10. In a preferred embodiment the polyelectrolyte is therefore polylactic-co-glycolic acid (PLGA).

[0086] In one embodiment the surface-active agent is a phospholipid, such as for example phosphatidylcholine, or phosphatidylserine. In one embodiment the surface-active agent is a non-ionic surfactant, such as for example Span 60 or Span 80. Two or more surface-active agents can also be combined.

[0087] The preferred coating is phosphatidylcholine. Phosphatidylcholine (PC) is a neutral phospholipid that adsorbs and self-assembles onto the surface of the hydrophobic polymer through hydrophobic interactions with the goal of reducing the free energy of the system.

[0088] In one embodiment the at least one stabiliser is selected from the group consisting of polyvinyl alcohol, dodecanol, cholesterol, cholesteryl hemi succinate, or combinations thereof. Cholesteryl hemi succinate is preferred because it provides chemical groups for covalent binding to the detection antibody used in the immunoassay.

[0089] As described in Example 3 and shown in Figure 1 the diameter of the SNPs before iodine staining was about 54 to about 80 nm. The iodine-stained SNPs were shown to have a diameter of between about 400 nm nm to about 500 nm, meaning that the micelles with the encapsulated stained SNPs agglomerated. Aggregation is not wanted, as it impedes the flow through the membrane. However, since the analytical signal for detection is proportional to the number of nanoparticles used, it is necessary to use a plurality of nanoparticles. Therefore, a compromise between the degree of agglomeration (controlled) and maximum signal desired has to be achieved.

[0090] Furthermore, the size of the nanoparticles is important to allow the particle-antibody aggregates to flow through the pores of the membrane of a lateral flow immunoassay. The membranes are defined by their flow rate. Specifically suitable membranes for putting the nanoparticles of present invention into practice in a LFIA have a flow rate of between 75s / 4cm to 120s / 4cm. Especially suitable membranes are for example Sartorius CN95 (flow rate of 95s / 4cm), Cytiva FF120HP (flow rate of 120 s / 4cm), Millipore HiFlow Plus HFC07504 (flow rate of 75s / 4cm).

[0091] The nanoparticles of present invention comprise in one embodiment the mean diameter of the final core-shell nanoparticle of between about 150 nm to about 250 nm, preferably between about 190 nm to about210 nm. In the most preferred embodiment, the mean diameter is about 193 nm to about 200 nm. It is to be understood that this size is without conjugated antibody or protein to the nanoparticles. Once the protein or antibody is conjugated to the nanoparticle, the conjugated nanoparticle has a mean diameter of between about 200 nm to about 300 nm, preferably between about 250 nm to 280 nm. In the most preferred embodiment, the mean diameter is about 255 nm to about 271 nm. The particle morphology and the aggregation structure of the SNPs were determined by TEM and the agglomeration of the nanoparticles was confirmed with TEM images in which both individual nanoparticles and agglomerates of several different sizes were observed (Figure 2).

[0092] In one embodiment the present invention therefore relates to a composition comprising a plurality of nanoparticles as described herein, wherein the nanoparticles form agglomerates. Fourier transform infrared spectroscopy (FTIR) was used to determine the molecular structure of the iodine-stained SNPs and starch nanoparticles and to verify possible molecular structure alterations. The spectra of both the SNPs and the SNPs stained with iodine showed exactly the same bands, confirming that the complex amylose-iodine was created through secondary chemical interactions (Figure 3).

[0093] As described in Example 4, lipid-polymer hybrid nanoparticles encapsulating iodine-stained SNPs were synthesized by emulsion / solvent evaporation method. In figure 4, the scheme of the synthesis of the nanocapsule is depicted. The formulation consisted of an organic phase containing PLGA acting as a carrier entrapping the SNP transferred to this phase and cholesteryl hemi succinate to functionalize the hybrid nanoparticle. On the other hand, the aqueous phase contained phosphatidylcholine as the lipid layer coating and poly-vinyl alcohol, a non-ionic surfactant, as a stabilizer. The encapsulation was confirmed by TEM, shown in figure 5, where it can be seen that the final core-shell nanoparticles had a spherical shape. Whilst different shapes of nanoparticles have been used in lateral flow assays, such as for example nanorods or nanoflowers, the spherical shape provides the advantage of having a larger interfacial area being available. The spherical shape thus provides more surface for functionalization and detection.

[0094] Bioconjugation of lipid-polymer encapsulated iodine-stained SNPs was tested with Neutravidin (Example 5). The average hydrodynamic size of the SNPs was measured with Dynamic Light Scattering, (DLS, figure 6) before and after the conjugation, showing an increase of the size confirming a successful conjugation.

[0095] Finally, the use of the SNPs was tested in LFIAs based on the reaction between biotin and neutravidin as further detailed in Example 6. The color intensity of the test line was quantified by reflectance measurements by using a portable strip reader and the signal obtained was compared to another standard assay based also on Neutravidin / biotin LFIA but using 40 nm colloidal gold-neutravidin conjugates as a visual label. Only the neutravidin coated nanoparticles were retained on the biotin-BSA test line. A simplified schematic representation of the biotin-neutravidin interaction in LFIA is shown in Figure 7.

[0096] The dried test line was analyzed by reflectance measurements and the results of the reflectance measurements for gold and hybrid nanoparticles were compared in terms of intensity (mV), with the density of the latter being slightly higher for a similar concentration of neutravidin, making this label an advantageous alternative to the gold nanoparticles (see table 2 in Example 6).

[0097] To assess the suitability for electrochemical measurement of the signal, the iodine and triiodide present in the starch nanoparticles in the test line was measured through coulometry.

[0098] Results showed an increasing of charge in presence of nanoparticles in both types of membranes as it can be seen in Figure 8.

[0099] In summary, the inventors could show that the iodine-stained SNPs are suitable for visual and electrochemical detection and can thus provide for a sensitive, versatile and simple method for detection.

[0100] NANOPARTICLES WITH IODINE-STARCH COMPLEX ON SURFACE AND PREPARATION THEREOF

[0101] As described below and in the examples section in more detail the inventors have further developed a system based on the innovative functionalization of gold, latex and magnetic nanoparticles with iodine-doped starch. This approach not only improves the chemical stability of the assay but also enables the detection of electrochemical signals at ultra-low concentrations, overcoming the limitations of conventional optical formats. In particular, the use of magnetic nanoparticles introduces the potential for multiple signal transduction, combining magnetic, optical and electrochemical readouts, further extending the versatility of the platform, and also providing for the possibility for preconcentration. An LFIA comprising these advantageous nanoparticles is a promising new tool for clinical diagnostics and biomarker analysis.

[0102] In another aspect the present invention therefore relates to a nanoparticle suitable for use in immunodetection, wherein the iodine-starch complex is located on the surface of the nanoparticle. In one preferred embodiment of this aspect the nanoparticle is selected from a gold nanoparticle (AuNP), a magnetic nanoparticle and a latex nanoparticle.

[0103] As described in more detail in Example 7 gold particles (AuNPs) were bio-conjugated with neutravidin and subsequently iodine-stained starch molecules were attached to the surface of the AuNPs. The hydrodynamic size and the zeta-potential of the AuNP conjugates blocked with starch and BSA was determined by DLS measurements and were carried out to confirm bioconjugation by comparing the size of the hydrodynamic radius before and after the conjugation reaction (Fig. 10). As can be seen in table 3 the bare commercial AuNPs were smaller than both conjugates showing that the conjugation process was successful in both cases.

[0104] In one embodiment the mean diameter of the nanoparticle including the iodine-starch complex on the surface is between about 60 nm to about 90 nm, preferably about 70 nm to about 80 nm.

[0105] In one embodiment of the AuNPs of present invention the mean diameter of the nanoparticle is between about 20 nm - 50 nm, preferably between about 30 nm - 40 nm. In this size they provide for maximum intensity of their colour and can thus be visualized very well.

[0106] After conjugation with a protein or antibody or antibody fragment (conjugated nanoparticles) they have a final diameter of between about 60 nm to about 90 nm, preferably about 70 nm to about 80 nm. In a preferred embodiment the mean diameter is between about 74 nm to 76 nm.

[0107] In one embodiment the mean diameter of the latex nanoparticle including the iodine-starch complex on the surface is between about 400 nm to about 520 nm, preferably about 450 nm to about 500 nm. After conjugation with a protein or antibody or antibody fragment the conjugated latex nanoparticles have a final diameter of between about 500 nm to about 580 nm, preferably about 510 nm to about 570 nm. In a preferred embodiment the mean diameter is between about 520 nm to 560 nm.

[0108] In one embodiment the mean diameter of the magnetic nanoparticles including the iodine- starch complex on the surface is between about 250 nm to about 320 nm, preferably about 280 nm to about 310 nm. After conjugation with a protein or antibody or antibody fragment the conjugated latex nanoparticles have a final diameter of between about 260 nm to about 360 nm, preferably about 280 nm to about 340 nm. In a preferred embodiment the mean diameter is between about 290 nm to 330 nm. The nanoparticles are first conjugated with the antibody, antibody fragment, protein, peptide, nucleic acid, aptamer, or other biological and biochemical receptor and then the iodine starch complex is added as blocking agent into the free spaces left on the surface of the nanoparticle between the conjugated entities. The inventors have found that by using these spaces between the conjugated entities the iodine starch provides for a double benefit. On the one hand the filling of the spaces reduces unspecific adsorption and on the other hand the particles at the test line can be better visualized.

[0109] In one embodiment the surface of the nanoparticle is entirely covered by the antibody, antibody fragment, protein, peptide, nucleic acid, aptamer, or other biological and biochemical receptor, and the iodine-starch complex thus not leaving any space for unspecific adsorption.

[0110] It is to be understood that “entirely covered” refers to a complete coverage of all available space on the surface of the nanoparticle after conjugation of the antibody, antibody fragment, protein, peptide, nucleic acid or aptamer, or other biological and biochemical receptor and the iodine-starch complex to the surface. In a preferred embodiment the surface of the nanoparticle is therefore at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, ideally 100% covered by the antibody, antibody fragment, protein, peptide, nucleic acid or aptamer, or other biological and biochemical receptor, and the iodine-starch complex.

[0111] In one embodiment of the nanoparticle of present invention the surface of the nanoparticles therefore further comprises antibodies, antibody fragments, proteins, peptides, nucleic acids, aptamers, or other biological and biochemical receptors conjugated to the surface.

[0112] The antibody fragments as referred to herein in all aspects of the invention include Fab fragments, scFv, single-domain antibody fragments like VH, VL or VHHs that bind to the same target as the entire antibody. These fragments can have the benefits of easier handling and reducing the size of the probes thus providing for the possibility of minimizing the size of the final sensors.

[0113] The other biological and biochemical receptors as referred to herein in all aspects of the invention can be selected from carbohydrates, lipids, fatty acids, glycolipids, sterols, monosaccharides, or small molecules, such as for example vitamins, hormones, antibiotics and metabolites. Furthermore, in one preferred embodiment bacteriophages can be conjugated to the nanoparticles of the invention. In one embodiment of present invention the plurality of nanoparticles comprises two different sets of nanoparticles, each set conjugated with a different antibody, antibody fragment, protein, peptide, nucleic acid or aptamer, or other biological and biochemical receptor that can either target different analytes or the same analyte. In a preferred embodiment the two different sets of nanoparticles target the same analyte.

[0114] In a preferred embodiment one set of nanoparticles is mixed with the sample in which the analyte is to be detected, and the other set of biological or biochemical receptor is located on the membrane. In this setup referred to as a “sandwich” one set of nanoparticles is used for detection of the analyte in the sample. The nanoparticles of present invention can be used for such a sandwich setup or other set ups involving competitive, inhibition or serum set ups commonly used in immunoassays. The conjugated nanoparticles can be added to the sample solution or immobilized at the conjugate pad in contact with the membrane.

[0115] Furthermore, the nanoparticles of present invention are suitable for the use in competitive inhibition assays in which only one type of bioreceptor is used.

[0116] In one preferred embodiment the antibodies used are antibodies that bind to extracellular vesicles, specifically to the membranes of the EVs. In one preferred embodiment two anti- tetraspanin antibodies: anti-CD9 for capture and anti-CD63 for detection are used. In this embodiment two different sets of bioreceptors are used: one is anti-CD9 antibody or antibody fragment and the other one an anti-CD63 antibody or antibody fragment, and only one of them (anti-CD63 in this case) was conjugated to the nanoparticles.

[0117] As further shown in the examples, the inventors verified both conjugation and agglomeration of the nanoparticles by measuring the ultraviolet-visible spectrum. The absorbance measurements of gold nanoparticles were employed to track the adsorption of proteins and antibodies on their surface. A strong absorbance peak between 460-560 nm was observed due to the surface plasmon resonance (SPR) of colloidal gold, and its maximum wavelength (Amax) varied depending on the size of the nanoparticles. The UV-Vis absorbance spectra of both the bare nanoparticles and the conjugates were measured between 350 nm and 800 nm (Fig. 11).

[0118] The maximum wavelength of AuNPs was 530 nm. A red-shift to a Amax of 532 nm and 534 nm was observed when the conjugation reaction was performed, blocking with BSA and starchiodine, respectively. This is an indication of a change in the refractive index of the nanoparticles due to the neutravidin layer on the surface of the nanoparticles. The inventors could therefore show that the conjugated gold nanoparticles were stable and could maintain the desired color, which also indicates that no aggregation occurred when blocked with starch. The developed nanoparticles are therefore suitable for the application in immunodetection as envisaged herein. lodine-doped starch-blocked gold nanoparticles were tested as labels in LFIAs based on the biotin-neutravidin affinity assay as described in Example 7 and electrochemical measurement was performed. Figure 12 shows the results of the coulometry for conjugates blocked with Bovine Serum Albumin (BSA). No significant trend in the measurements was observed as the analytical signal of the negative test is higher than the positive ones, which do not follow any trend either. However, in the case of the conjugates blocked with iodine-doped starch (Fig.13), a correlation between the electrochemical and visual signals was found. Both signals increase as the amount of conjugated neutravidin added increases. Therefore, with this procedure electrochemical measurements can be performed and used for quantification in LFIAs. This overcomes one of the actual limitations for simple and portable quantification of LFIAs.

[0119] The inventors have surprisingly found that when starch was used to modify the gold nanoparticle surface after the conjugation unspecific adsorption was not observed, indicating that starch is a very efficient blocking agent. When it is combined with iodine, it acts also as carrier for the electrochemical probe that generates the electroanalytical signal.

[0120] Finally, the inventors could show that also other nanoparticles, such as magnetic nanoparticles and latex nanoparticles are equally suitable. Specifically, and as described in more detail in Example 8, it was shown that the nanoparticles of present invention are highly sensitive for the accurate detection and quantification of analytes, such as extracellular vesicles (EVs). For this the inventors developed a novel biosensor using an electrochemical lateral flow immunoassay using nanoparticles as described herein, specifically gold nanoparticles, magnetic nanoparticles and latex particles were used. The biosensor demonstrated exceptional sensitivity, could detect ultra-low concentrations as low as 31 EVs / pL and quantifying a wide range of concentrations up to 10 000 EVs / pL through the selection of different nanoparticles. The incorporation of iodine-doped starch as a functional material not only stabilized the nanoparticles and minimized non-specific interactions, but also facilitated the integration of an electrochemical probe, streamlining the protocol and making the sensor straightforward for home use. The biosensor is a versatile and effective tool that provides for an improved method of extracellular vesicle detection. It is to be understood that this adaptability to different biomarkers of interest in EVs promises better and faster diagnosis and monitoring of various diseases. In summary, the inventors have found that iodine-stained starch polymers can be used as a label and / or blocking agent in immunoassays, specifically in combination with AuNPs, magnetic NPs or latex NPs. Some of the benefits of using such iodine-stained starch polymers labels or blocking agent is that they are renewable, biodegradable, biocompatible and cheap to produce11. The fact that the starch materials develop blue color through the interaction with iodine and that iodine can be measured electrochemically through coulometry opens a versatile detection system that can be used depending on its needs12 13.

[0121] In a fourth aspect the present invention relates to an immunodetection sensor comprising the nanoparticle suitable for immunodetection or the composition comprising a plurality of nanoparticles as described herein.

[0122] In a preferred embodiment said immunodetection sensor is a lateral flow immuno assay (LFIA) device, preferably an electrochemical lateral flow immuno assay (eLFIA) device.

[0123] In a fifth aspect the present invention relates to an immunodetection method comprising the steps of

[0124] (i) mixing a sample with the nanoparticle(s) or the composition comprising a plurality of nanoparticles as described herein; and

[0125] (ii) detecting an electrochemical signal, an optical signal, or both.

[0126] In this aspect the nanoparticles are conjugated nanoparticles. This means that the nanoparticles have been modified to contain an antibody, antibody fragment, protein, peptide, nucleic acid or aptamer, or other biological and biochemical receptors for detection bound to their surface which will detect the desired analyte in the sample.

[0127] Other analytes can be detected with the nanoparticles, the method and the immunodetection sensor or device. In one embodiment the analyte can be selected from the group consisting of extracellular vesicles, bacteria, antibodies, proteins, metabolites, pollutants or allergens.

[0128] In a sixth aspect the present invention relates the use of the nanoparticles or the plurality of nanoparticles, or the immunodetection sensor as described herein for the detection of an analyte, preferably wherein the analyte is selected from the group consisting of extracellular vesicles (EVs), bacteria, antibodies, proteins, metabolites, pollutants or allergens. EXAMPLES

[0129] Example 1 : SNPs (Starch nanoparticles) staining with iodine

[0130] Native starch from both quinoa and amaranth were dissolved separately with a concentration of 20 mg / mL in milliQ water. Then, a solution of Lugol's reagent (5% I2, 10% KI) was added to native starch solution at a volume ratio of 1 :2 followed by a reaction time of 30 min at stirring conditions of 24 rpm. Nanoparticles obtained were precipitated with absolute ethanol and centrifuged at 9500 rpm for 5 minutes. Afterwards, two washes with milliQ water and ethanol were performed to finally resuspend the nanoparticles in milliQ water.

[0131] Different trends were observed after adding the Lugol's solution to the colorless starch solutions of both quinoa and amaranth. A reddish like solution was observed in case of amaranth starch, whilst, in case of quinoa starch, the solution instantly became dark blue. This can be explained based on the foundations of the starch reaction with the triiodide ion (10). Starch is composed by two glucose polymers: amylopectin, which is the major one (around 75%) and amylose (around 25%). The molecular iodine and the triiodide ion react to amylose through secondary chemical interactions (rearranging with dipole moments parallel to the axis of the amylose helix). Depending on the chain length of the amylose, in the case of short chains, the amylose-iodine complex can have a purple-brown color, which would be the case of the amaranth solution, as both SNPs from amaranth and quinoa are reported to have a similar percentage of amylose (20.90% and 20.95% respectively) (11). It is known that only the blue complex between the triiodide and the iodine with the amylose is stable whereas the complex of the iodine species with the amylopectin (red) is less stable. Hence, quinoa SNPs were selected due to their strong blue color, making them suitable for this assay.

[0132] Example 2: Synthesis of hybrid nanoparticles encapsulating SNPs-lodine

[0133] Iodine-stained SNPs were encapsulated using a single emulsion / solvent method. An aqueous phase was prepared containing 10 mg phosphatidylcholine, as the sole membrane component, and 1% polyvinyl alcohol (w / v) in milliQ water, whilst an organic phase, consisting of a 12.5% (v / v) solution of methanol in chloroform, containing 30 mg of poly (lactic-co-glycolic acid) (PLGA) and 1% (w / v) cholesteryl hemisuccinate of the total membrane compounds (PC).

[0134] The preparation of the hybrid nanoparticles (SNPs-iodine in PLGA-PVA / PC) was done following the next protocol: 2 mL of organic phase, containing 1 mL of dispersed SNPs-iodine, were emulsified in 6 mL of the aqueous phase, obtaining an oil-in-water (O / W) emulsion under continuous sonication at 55% amplitude for 2 min in an ice bath. The emulsion was then placed on a mechanical stirrer (witeg Labortechnik GmbH, Germany) with a magnet to evaporate the organic solvent and form the particles overnight. Hybrid nanoparticles obtained were purified by size exclusion chromatography (SEC) with a solution of SepharoseTM CL-4B in milli-Q water 67.5% (v / v).

[0135] Example 3: Characterization of the SNPs-l and LPHNPs

[0136] 3.1. Particle size distribution

[0137] Hydrodynamic size and homogeneity of the SNPs-lodine particles were obtained by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, UK). Samples were measured with the 173° backscatter detector in low volume disposable cuvettes (Malvern Instruments Ltd., Malvern, UK). The average diameter was 434 ± 34 nm with three different populations of nanoparticles (Figure 1). Size of nanoparticles before staining them was 68 ± 13 nm, suggesting an agglomeration of the nanoparticles. A great polydispersity was also observed as a PDI value of 1.00 was obtained.

[0138] 3.2. Particle morphology

[0139] Particle morphology, and the aggregation structure of the SNPs was determined by TEM. An aliquot of an aqueous suspension of the samples was placed on a transparent carbon sheet with copper grid support and analyzed with MET JECL-2000 EXH TEM (Saint-Herblain, France). The agglomeration of the nanoparticles could be confirmed with TEM images (Figure 2) in which both individual nanoparticles and agglomerates of several different sizes were observed.

[0140] 3.3. Molecular structure

[0141] Fourier transform infrared spectroscopy (FTIR) was used to determine the molecular structure of the iodine-stained SNPs and starch nanoparticles and to verify possible molecular structure alterations. FTIR spectra were recorded in a Fourier transform infrared spectrophotometer (Varian 620-IR, Thermo Fisher Scientific Inc., U.S.A.) at room temperature. Dried powder samples of approximately 1 mg were measured directly and spectra were recorded between 650-4000 cm-1(medium infrared band).

[0142] The spectra of both the SNPs and the SNPs stained with iodine showed exactly the same bands, lacking any halogen bonding band, therefore confirming that the complex amyloseiodine was created through secondary chemical interactions (Figure 3). In both cases, the hydroxyl groups appeared at a wavelength around 3300 cm-1. At a wavelength of 2900 cm-1, the characteristic band corresponding to C-H stretching vibration of the glucose unit12 was observed. The peak around 1600 cm-1 may correspond to the bending vibration of the H-O-H group of starch-bound water (13).

[0143] Example 4: Synthesis and characterization of the lipid-polymer hybrid nanoparticles encapsulating SNPs-iodine

[0144] Then lipid-polymer hybrid nanoparticles encapsulating iodine-stained SNPs were synthesized by emulsion / solvent evaporation method. In figure 4, the scheme of the synthesis of the nanocapsule is depicted. The formulation consisted of an organic phase containing PLGA acting as a carrier entrapping the SNP transferred to this phase and cholesteryl hemisuccinate to functionalize the hybrid nanoparticle. On the other hand, the aqueous phase contained phosphatidylcholine as the lipid layer coating and poly-vinyl alcohol, a non-ionic surfactant, as a stabilizer.

[0145] The encapsulation was checked by TEM, shown in figure 5, where it can be seen that the nanoparticles had a spherical shape.

[0146] Example 5: Bioconjuqation of lipid-polymer SNPs-lodine

[0147] A fresh solution 0.097 M of EDC and 0.261 M of NHS in MES buffer pH 5.5 was prepared. 100 pL of this solution was added to 1 mL of the lipid-polymer SNPs-l. In order to activate the carboxylic groups of the nanocapsule, it was allowed to react for 15 minutes under shaking. Then, it was centrifuged at 5000 x g for 4 minutes and washed with 200 pL of MES buffer, followed by an addition of 200 pl of a 0.5 mg / mL Neutravidin solution in MES pH 7.4 and left shaking for 3 h. After that, another shaking was done. After this time, another centrifugation at 5000 x g for 4 minutes was done followed by another washing step with MES buffer.

[0148] After the activation of carboxylic groups, a blocking step of un unreacted carboxylic groups unbound with neutravidin was done. 100 pL solution of CH3O-PEG-NH2 (5mg / mL) was added followed by a reaction time of 20 min. Finally, the solution was centrifugated under the same conditions as above and after washing, resuspend in 100 pL of 10 mM phosphate buffer pH 7.4.

[0149] The average hydrodynamic size was measured with DLS (figure 6) before and after the conjugation, showing an increase of the size confirming a successful conjugation (table 1) (14). Table 1. Hydrodynamic diameter of SNPs in PLGA-PVA / PC before and after conjugation with neutravidin.

[0150] Example 6: Lateral flow immunoassays

[0151] 6.1. Preparation of the strips

[0152] The LFIA was based on a dipstick format. The test strips consist of a sample pad, a nitrocellulose membrane, an absorbent pad, and a backing plastic card. First, the 25 mm-wide- nitrocellulose membranes (CN95, Sartorius, Germany, FF120HP, Cytiva, Germany) was attached to a backing plastic card to obtain a robust system. Then, a test line of biotin-BSA was immobilized across the membrane by the IsoFlow dispenser (Imagene Technology, Lebanon, NH, USA) at a rate of 0.100 pL / mm with CN95 membranes and at 0.060 pL / mm with FF120HP membranes. After this, the membrane was dried for 20 min at 37 °C. Finally, the sample pad and the absorbent pad were stuck onto the backing card overlapping between them of 2 mm. The complete card was cut into 5 mm wide strips for the subsequent individual assays.

[0153] 6.2. Lateral flow immunoassays assays (LFIA)

[0154] The use of starch nanoparticles as label in LFIAs were tested based in the reaction between the vitamin biotin and the protein neutravidin.

[0155] 10 pL of the lipid polymer SNP-l-neutravidin conjugates were transferred into Eppendorf tubes with running buffer up to a final volume of 100 pL. The strips were added vertically and allowed to run for 15 min. After that, color intensity of the test line was quantified by reflectance measurements by using a portable strip reader ESE-Quant LR3 lateral flow system (Qiagen Inc., Hilden, Germany).

[0156] The signal obtained was then compared to another standard assay based also in Neutravidin / biotin LFIA but using 40 nm colloidal gold-neutravidin conjugates as a visual label.

[0157] Only the neutravidin coated nanoparticles were retained on the biotin-BSA test line. A simplified schematic representation of the biotin-neutravidin interaction in LFIA is shown in Figure 7. The dried test line was analyzed by reflectance measurements (table 2). The results of the reflectance measurements for gold and hybrid nanoparticles were compared in terms of intensity (mV), with the density of the latter being slightly higher for a similar concentration of neutravidin, making this label a possible competitor for the gold nanoparticles.

[0158] Table 2. Comparison of the intensity profiles measured for the gold nanoparticles and for the stained SNPs in the PLGA-PVA / PC nanoparticles.

[0159] 6.3. Electrochemical measurement

[0160] The iodine and triiodide present in the starch nanoparticles in the test line was measured through coulometry. Nitrocellulose membranes with different surfactant treatments were used. After dried, test line was cut with a size of 4 mm x 4 mm, long enough to cover the working electrode. 2 pl of H2SO40.5 M were added to the strip and overlayed to the SPCE. The auxiliary and reference electrode were not in contact with the membrane so another addition of 2 pl for each electrode was done to cover the entire electrochemical cell. Measurements were done by coulometry by applying a potential of 0.75 V for 900 s.

[0161] Next step was the measuring of hybrid nanoparticles in the test line after a lateral flow assay. Both types of membranes previously mentioned were tested. Results showed an increasing of charge in presence of nanoparticles in both types of membranes as it can be seen in Figure 8.

[0162] Example 7: Electrochemical LFIA based on gold nanoparticles blocked with iodine-doped starch

[0163] 7.1 Iodine staining of starch

[0164] A solution of native guinoa starch (reportedly with a 20.95% of amylose and a 79.05% of amylopectin 14) in milliQ water was heated at 80°C for 30 min. Then, 2 mL of a solution of I2 5% and KI 10% were added, followed by the addition of 7 mL of absolute ethanol to precipitate the stained starch. The sample was then centrifuged for 4 min at 15880 x g. Finally, the supernatant was discarded, and the pellet was resuspended in milliQ water. 7.2 Bioconjuqation of Au NPs

[0165] A solution of 1.5 mL of Au NPs with a 40 nm size was mixed with 100 pL of a neutravidin solution at different concentrations (0.15 and 0.3 mg / mL). After 1 h of reaction, 100 pL of a blocking solution containing native quinoa starch 0.1 % stained with iodine were added. The mixture was incubated for 45 minutes with constant agitation. Then the nanoparticles were centrifuged for 20 min at 9184 x g and, after discarding the supernatant, they were resuspended in 2 mM phosphate stabilizing buffer at pH 7.4 with a concentration of 1 % BSA and 10% sucrose. For comparison purposes, the same procedure was followed using 0.1% BSA as blocking agent.

[0166] 7.3 Characterization of nanoparticle conjugates

[0167] 7.3.1. Particle size distribution and potential

[0168] The hydrodynamic size and homogeneity of the Au NPs and conjugates were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, UK). Samples were measured using the 173° backscatter detector in low volume disposable cuvettes (Malvern Instruments Ltd., Malvern, UK). The zeta potential was also measured using this technique in single use folded capillary cells (DTS1070).

[0169] The hydrodynamic size and the zeta-potential of the AuNP conjugates blocked with starch and BSA was determined by DLS measurements were carried out to confirm bioconjugation by comparing the size of the hydrodynamic radius before and after the conjugation reaction (Fig. 10). Table 3 summarizes the results obtained for each condition.

[0170] Table 3: DLS measurements ( average, Pdl and ^-potential) of the bare Au NPs and the conjugates blocked with BSA and iodine-doped starch.

[0171] The difference in size among the bare commercial nanoparticles and both conjugates proves that the conjugation process was successful in both cases. The main differences found between the conditions were found in the case of Pdl, analysis although the conjugates with the different blocks have similar sizes, in the case of the AuNPs blocked with starch-iodine the particles are slightly more polydisperse. This corresponds with what was observed in the potential measurements.

[0172] 7.3.2. UV-Vis

[0173] The ultraviolet-visible spectrum was also measured to verify both conjugation and agglomeration of the nanoparticles. For this purpose, the absorption spectra of both the nanoparticles and the conjugates were measured on a Cary 60 UV-Vis spectrophotometer from Agilent Technologies (Palo Alto, CA, USA).

[0174] The absorbance measurements of gold nanoparticles were employed to track the adsorption of proteins and antibodies on their surface. A strong absorbance peak between 460-560 nm was observed due to the surface plasmon resonance (SPR) of colloidal gold, and its maximum wavelength (Amax) varied depending on the size of the nanoparticles. The UV-Vis absorbance spectra of both the bare nanoparticles and the conjugates were measured between 350 nm and 800 nm (Fig. 11).

[0175] The maximum wavelength of AuNPs was 530 nm. A red-shift to a Amax of 532 nm and 534 nm was observed when the conjugation reaction was performed, blocking with BSA and starchiodine, respectively. This is an indication of a change in the refractive index of the nanoparticles due to the neutravidin layer on the surface of the nanoparticles.

[0176] 7.4 Preparation of the strips

[0177] The LFIA was carried out based on a dipstick format. The test strips consisted of a sample pad, a nitrocellulose membrane (CN95, Sartorius, Germany), an absorbent pad, and a plastic backing card. First, the 25 mm wide nitrocellulose membranes were attached to a plastic backing card to obtain a robust system. A test line of biotin-BSA was then immobilized across the membrane using an IsoFlow dispenser (Imagene Technology, Lebanon, NH, USA) at a rate of 0.100 pL / mm. The membrane was then dried at 37°C for 30 minutes. Finally, the sample and absorbent pads were adhered to the backing card with an overlap of 2 mm. The complete card was cut into 5 mm wide strips for the subsequent individual assays.

[0178] 7.5 Lateral flow immunoassay with electrochemical measurement

[0179] The iodine-doped starch-blocked nanoparticles were tested as labels in LFIAs based on the biotin-neutravidin affinity assay. The assay buffer was prepared by adding 10 pL of the gold nanoparticles conjugated (either the iodine-doped starch- or the BSA-blocked) to microcentrifuge tubes with running buffer, containing BSA, Tween 20 and NaCI up to a final volume of 100 pL. The strips were added vertically and allowed to run for 15 minutes. At the end of the test, the test line (TL) was cut and put in an Eppendorf tube with 300 pL of PBST (0.01 M + 1% Tween-20) for 2 min to clean it from unspecific adsorptions. Then it was dried with paper and prepared to the screen-printed platform. Next step was the addition of 3pL of sulfuric acid 0.5 M to the membrane as an inert electrolyte. Then, it was coupled to the electrode and two additions of 2 pl of sulfuric acid were done to the auxiliary and reference electrode respectively. Finally, the electrochemical measurement was done by coulometry by applying a potential of +0.5 V for 100 s.

[0180] Only the neutravidin-coated nanoparticles were retained on the biotin test line. The test line was then left to dry, cut, to a stipulated size to cover the working electrode (4 mm), and handled as described above. Figure 12 shows the results of the coulometry for conjugates blocked with BSA. No significant trend in the measurements was observed as the analytical signal of the negative test is higher than the positive ones, which do not follow any trend either.

[0181] However, in the case of the conjugates blocked with iodine-doped starch (Fig.13), a correlation between the concentration of neutravidin and the charge was obtained suggesting that by simply changing the blocking of the conjugates, electrochemical measurements can be performed, obtaining an electrochemical quantification procedure by using a standard LFIA based on Au NPs and overcoming one of its main limitations.

[0182] Example 8: Development of a highly sensitive eLFIA for the accurate detection and quantification of EVs

[0183] 8.1. Materials

[0184] A solution of 40 nm gold nanoparticles (AuNPs) was obtained from BBI Solutions (Cardiff, UK). Estapor® polystyrene microspheres were also obtained from Merck (Darmstadt, Germany), some with a size of 0.5 pm with colored latex (K1-050) and others with a size of 0.3 pm with magnetic dye (53.4%, dry weight) (M1-030 / 40).

[0185] Iodine, reagent ACS (99.8%), was obtained from Fisher Scientific. The native quinoa starch used in this study was generously provided by Lund University (Lund, Sweden) and was characterized in previous studies.

[0186] The mouse anti-CD63 monoclonal antibody was obtained from Immunostep (Salamanca, Spain). For the components of the test strip, the membrane (Hi-Flow™ Plus HF180) and glass fiber sample pads (GFCP001000) were purchased from Millipore (Darmstadt, Germany), the carrier cards (KN-V1080) were provided by Kenoshatapes (Amstelveen, Netherlands) and the absorbent pads were purchased from Whatman (Piscataway, USA).

[0187] Additional reagents such as anti-mouse IgG, N-hydroxysuccinimide (NHS), 1-ethyl-3-[3- dimethylaminopropyl]-carbodiimide hydrochloride (EDC), bovine serum albumin (BSA), MES, TRIS, sodium phosphate monobasic and sodium phosphate dibasic were purchased from Sigma-Aldrich (Darmstadt, Germany). HEPES was supplied by Fisher Scientific. These reagents were used to prepare phosphate buffer (PB), MES, TRIS and HEPES buffer at 10 mM concentration and pH 7.4, followed by filtration for later use.

[0188] Instrumentation included an IsoFlow reagent dispensing system from Imagene Technology (USA) for dispensing detection lines. Strips were cut using a Fellowes Gamma guillotine (Spain).

[0189] Electrochemical measurements were performed using screen-printed electrode cards (DRP- 110) from Metrohm Dropsens (Spain) with carbon ink for the working and auxiliary electrodes and a silver pseudoreference electrode. The electrodes were connected using a DSC connector (DRP-DSC) from the same manufacturer. A pStat-l 400s potentiostat controlled by Dropview 8400 software was used for the measurements.

[0190] 8.2. Methods a) Preparation of starch-iodine complex

[0191] A solution of native quinoa starch was prepared at 1 mgVmL (with a composition of 20.95% amylose and 79.05% amylopectin, as reported25) in Milli-Q water and heated at 80°C for 30 min. Lugol's reagent (5% I2 and 10% KI solution) was then added and the mixture stirred. Absolute ethanol was added to precipitate the colored starch. The sample was centrifuged at 15,880 x g for 4 minutes, the supernatant was discarded, and the pellet was resuspended in 10 mM phosphate buffer pH 7.4 for subsequent use with Au NPs or in TRIS buffer pH 8 for use with LM and MNPs, adjusting to the original volume of the solution. b) Bioconjuqation of AuNPs

[0192] A 1 .5 mL solution of 40 nm commercial AuNPs was gently combined with 100 pL of 0.15 mg / mL anti-CD63 solution, which served as the detection antibody. After a 1-hour reaction, 100 pL of a blocking solution containing 0.1% iodine-doped quinoa starch was added. The mixture was incubated for 45 minutes with constant agitation. The nanoparticles were then centrifuged at 9184 x g for 20 minutes, the supernatant was discarded, and the pellet was resuspended in 250 pL of 2 mM phosphate buffer (pH 7.4) containing 1 % BSA and 10% sucrose for stabilization. c) Bioconjugation of LM and MNPs

[0193] Microspheres (100 pL at 10% w / v) were washed three times with 50 mM MES buffer pH 6.0 and centrifuged (14,000 rpm, 7 min for MNPs; 12,000 rpm, 5 min for LM). After resuspension in MES buffer, activation was performed by adding 24 pL of 200 mM EDC and 240 pL of 200 mM sulfo-NHS, followed by mixing for 30 min at room temperature. The microspheres were then washed three times in MES buffer and resuspended in 700 pL of the same buffer. Anti- CD63 (300 pL at 2 mg / mL) were added to achieve a coating concentration of 40 mg / g, and the suspension was incubated on a rotary disc for 2.5 hours. The suspension was quenched with 30 pL ethanolamine and incubated for 30 minutes. The microspheres were washed and resuspended in blocking buffer (50 mM TRIS, pH 8.0, 0.5% iodine-doped starch), mixed for 2 h and washed twice with 50 mM TRIS, pH 8.0, 0.5% casein. Finally, the microspheres were adjusted to 1 % w / v and stored at 4°C until further use. d) Characterization of bioconjugates

[0194] Transmission electron microscopy (TEM) was used to study particle morphology, size and aggregation. A small volume of the aqueous nanoparticle suspension was deposited on a carbon-coated copper grid and analysed using a JECL-2000 EXH TEM (Saint-Herblain, France). The average particle size of the bioconjugates of LM and MNPs was then determined by random measurements employing Imaged software.

[0195] The hydrodynamic size and ^-potential of the Au NPs and their bioconjugates were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively, using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd, Malvern, UK).

[0196] Ultraviolet-visible (UV-Vis) spectra were also recorded to assess the conjugation and agglomeration of the AuNPs. Absorbance measurements of the aqueous suspensions, including both bare particles and their conjugates, were performed between 350 and 800 nm using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). e) Preparation of the strips

[0197] The LFIA was based on a dipstick format. The test strips consist of a sample pad, a nitrocellulose membrane, an absorbent pad, and a backing plastic card. First, the 25 mm-wide- nitrocellulose membrane was attached to a backing plastic card to obtain a robust system. Then, a test line of anti-CD9 and a control line of anti-IgG were immobilized across the membrane by the IsoFlow dispenser at a rate of 0.100 pL / mm. After this, the membrane was dried for 20 min at 37 °C. Finally, the sample pad and the absorbent pad were stuck onto the backing card overlapping between them of 2 mm. The complete card was cut into 5 mm wide strips for the subsequent individual assays.

[0198] 8.3. Lateral flow immunoassay with electrochemical measurement

[0199] The iodine-doped starch-blocked nanoparticles were tested as labels in LFIAs. The assay buffer was prepared by adding either 10 pL of the bioconjugated Au NPs or 5 pL in the case of LM or M NPs to microcentrifuge tubes with 5 pL of sample with a known concentration of EVs, and running buffer, containing 1 % BSA, 0.05% Tween 20 and 150 mM NaCI in HEPES 10 mM pH 7.4 up to a final volume of 100 pL. The strips were added vertically and allowed to run for 20 minutes.

[0200] At the end of the test, the test line (TL) was cut and put in an Eppendorf tube with 300 pL of PBST (0.01 M + 1% Tween-20) for 2 min to clean it from unspecific adsorptions. Then it was dried with paper and prepared to the screen-printed platform. Next step was the addition of 3pL of sulfuric acid 0.5 M to the membrane as an inert electrolyte. Then, it was coupled to the electrode and two additions of 2 pl of sulfuric acid were done to the auxiliary and reference electrode respectively. Finally, the electrochemical measurement was done by coulometry by applying a potential of +0.5 V (Au NPs), + 0.65 V (LM), + 0.7 V (MNPs) for 90 s.

[0201] 8.4. Results and discussion

[0202] Principle of the immunoassay

[0203] The immunoassay used in this study uses a sandwich configuration with two anti-tetraspanin antibodies: anti-CD9 for capture and anti-CD63 for detection. These antibodies were selected based on previous findings by the research group2627. Both proteins are commonly found in the membranes of extracellular vesicles (EVs) and are therefore considered general biomarkers for EV detection.

[0204] Human plasma EV samples were diluted in a buffer to achieve the desired concentration range for constructing a calibration curve for the method developed with each kind of NPs (0-10 000 EV / pL). For the dipstick assay, the strip was placed in a microcentrifuge tube containing the NPs, the EV sample and the running buffer, which consisted of 1% BSA, 0.05% Tween20, 150 mM NaCI in HEPES buffer at pH 7.4. As the assay progressed, NPs-[EV CD63+] complexes moved along the strip by capillary action and were captured at the test line by recognition of CD9+ EVs by the anti-CD9 antibody immobilized there. Excess complexes were retained on the control line functionalized with antimouse IgG antibodies.

[0205] Assessment of starch as a stabilizer and characterization of conjugates

[0206] Bare NPs were functionalized with EV-specific antibodies either by direct adsorption for AuNPs, thanks to the strong affinity between thiol groups in antibodies and the gold surface, or by carbodiimide chemistry for carboxyl functionalized Estapor® microspheres.

[0207] In both approaches, starch — a renewable, biodegradable, biocompatible, and cost-effective natural polymer — served as a blocking agent and functional material to reduce unspecific interactions. This choice was made due to the specific ability of starch to form a stable complex with polyiodides, which offers potential as an electrochemical probe. The resulting particles integrated blocking and electrochemical sensing functionalities.

[0208] Transmission electron microscopy (TEM) confirmed the spherical morphology of the conjugates (Figure 14) and provided size measurements (Table 4) of the bioconjugates for the cases of LM and MNPs, as DLS measurements could not be performed.

[0209] Table 4: Mean sizes of the polystyrene microspheres before (according to their certificate of analysis) and after bioconjugation with anti-CD63.

[0210] These measurements indicated successful bioconjugation, with an increase in size observed after the conjugation process. TEM results further demonstrated the uniform distribution of the particles without aggregation.

[0211] The hydrodynamic size and potential of the AuNP conjugates were also determined by DLS and ELS, respectively, shown in table 5. Table 5: DLS and ELS measurements ( average, Pdl and potential) of the bare Au NPs and the conjugates blocked with BSA and iodine-doped starch.

[0212] The ultraviolet-visible spectrum was also measured to verify both conjugation and agglomeration of the Au NPs. The absorbance measurements of Au NPs were employed to track the adsorption of proteins and antibodies on their surface. A strong absorbance peak between 460-560 nm was observed due to the localized surface plasmon resonance (LSPR) of colloidal gold, and its maximum wavelength (Amax) varied depending on the size of the nanoparticles.

[0213] The maximum wavelength of AuNPs was 530 nm. A red-shift to a Amax of 533 nm was observed when the conjugation reaction was performed, blocking with iodine-doped starch. This is an indication of a change in the refractive index of the nanoparticles due to the neutravidin layer on the surface of the nanoparticles.

[0214] - Electrochemical detection of the NPs

[0215] AuNPs are standard for LFIA, but their use in electrochemical quantification requires dissolution with hazardous chemicals or enzymatic digestion, limiting their practicality for home use. The proposed iodine-doped starch-blocked AuNPs integrate an electrochemical probe directly into the LFIA design, eliminating the need for external reagents, simplifying the procedure and reducing potential interferences. However, the electrochemistry of gold and iodine presents challenges due to overlapping oxidation-reduction reactions within similar potential windows. Gold forms complexes such as Aul2' and Au , while iodine undergoes reactions such as triiodide formation and reduction, making it difficult to distinguish between the processes. In addition, iodine stabilizes oxidized gold states, further influencing the redox behavior of the system

[0024] .

[0216] Using commercially available screen-printed electrodes, AuNPs showed a distinct redox peak at +0.45 V when a cyclic voltammetry (CV), confirming this electrochemical activity. CVs were also performed on both polystyrene Estapor® microspheres to characterize the redox behavior of iodine in each system. For the colored latex microspheres, the redox peak was identified at +0.65 V, while the magnetic microspheres showed a peak at +0.7 V. These variations highlight the influence of nanoparticle composition on the stabilization and electrochemical behavior of iodine species within each configuration.

[0217] - Analytical performance of the gold-based eLFIA for EVs quantification

[0218] A linear response was obtained for the concentration range 0 to 270 EV / pL. This linear response (Figure 15) was fitted to the equation:

[0219] Q (pC) = 0.056 [Concentration (EV / pL)] + 4.8 (Eq. 1) with a linear regression coefficient of 0.9904. This gives an LOD (following the criterium of 3SD / slope, being SD the standard deviation of the blank) of 31 EV / pL, which is equivalent to 1.86 fM. It is also important to note that this method was highly reproducible, with an RSD of 6.52%.

[0220] - Discussion on the performance of the biosensor

[0221] In this study, the detection of EVs isolated from human plasma is carried out using different types of NPs, all functionalized during their bioconjugation process with iodine-doped quinoa starch.

[0222] Table 6 shows the calibration ranges, detection limits and reproducibility obtained for each type of NP.

[0223] Table 6: Detection ranges, LCDs and reproducibility achieved for each NP.

[0224] These data highlight the remarkable versatility of the biosensor. By using the most common nanoparticles in visual lateral flow assays produced by multinational companies, and thanks to its wide linear detection range (from 0 to 10,000 EV / pL), the biosensor allows the selection of the appropriate type of nanoparticle according to the expected concentration of EVs in the samples to be quantified.

[0225] The innovative nature of eLFIA offers numerous advantages, including high sensitivity and specificity, which are essential for the accurate detection of EVs in complex biological samples.

[0226] The sensitivity of these methods is excellent, with the use of Au NPs and MNPs providing LODs that, to the authors' knowledge, have not been achieved before. Recently, a study using fluorescent Eu NPs achieved a LOD of 240 EV / pL

[0025] , while in our study Au NPs detected down to 31 EV / pL and MNPs down to 157 EV / pL with a linear range of 0 to 270 and 3500 to 5000 EV / pL, respectively.

[0227] The specificity of the immunoassay is ensured by the sandwich format, which uses two different antibodies to detect EVs, thus avoiding cross-reactivity and achieving a more accurate quantification of the target EVs.

[0228] The electrochemical readout of the eLFIA allows rapid analysis and quantification, making it a valuable tool for clinical diagnostics. The integration of electrochemical detection with a lateral flow design represents a significant advance in the field, providing a simple and efficient means of analyzing EVs and potentially other biomarkers.

[0229] Conclusions

[0230] This study presented a novel biosensor using an electrochemical lateral flow immunoassay for the detection and quantification of extracellular vesicles. The device demonstrated exceptional sensitivity, capable of detecting ultra-low concentrations as low as 31 EVs / pL and quantifying a wide range of concentrations up to 10 000 EVs / pL through the selection of different nanoparticles. The incorporation of iodine-doped starch as a functional material not only stabilized the nanoparticles and minimized non-specific interactions, but also facilitated the integration of an electrochemical probe, streamlining the protocol and making the sensor straightforward for home use.

[0231] The results of this research establish the biosensor as a versatile and effective tool that can complement, and in some cases, improve traditional methods of extracellular vesicle detection. Its adaptability to different biomarkers of interest in EVs promises better and faster diagnosis and monitoring of various diseases. Further ITEMS of the invention

[0232] The present invention furthermore relates to the following items:

[0233] 1. A nanoparticle for immunodetection based on lateral flow assay, wherein the nanoparticle comprises an iodine-starch complex.

[0234] 2. The nanoparticle of item 1 , wherein the nanoparticle has a core and a shell, and the iodine-starch complex is located in the core of the nanoparticle.

[0235] 3. The nanoparticle of items 1 or 2, wherein the core of the nanoparticle comprises between about 1 % - 4% (w / v), preferably between about 1 % - 2% (w / v) of the iodine- starch complex.

[0236] 4. The nanoparticle of items 2 or 3, wherein the shell comprises at least one polyelectrolyte, at least one surface active agent and at least one stabilizer.

[0237] 5. The nanoparticle of item 4, wherein

[0238] (i) the polyelectrolyte is selected from the group consisting of polylactic-co- glycolic acid (PLGA), polylactic acid (PLA), poly-e-caprolactone (PCL), or combinations thereof; and / or

[0239] (ii) the at least one surface-active is selected from phospholipids or non-ionic surfactants, or a combination thereof.

[0240] 6. The nanoparticle of item 5, wherein

[0241] (i) the phospholipid is selected from phosphatidylcholine, phosphatidylserine, or a combination thereof; and / or

[0242] (ii) the non-ionic surfactant is selected from Span 60, Span 80, or a combination thereof.

[0243] 7. The nanoparticle of any one of items 4 to 6, wherein the at least one stabilizer is selected from the group consisting of polyvinyl alcohol, dodecanol, cholesterol, cholesteryl hemi succinate, or combinations thereof.

[0244] 8. The nanoparticle of any one of items 1 to 7, wherein the mean diameter of the nanoparticle is between about 150 nm to about 250 nm, preferably between about 190 nm to about 210 nm. The nanoparticle of item 1 , wherein the iodine-starch complex is located on the surface of the nanoparticle. The nanoparticle of item 9, wherein the nanoparticle is a gold nanoparticle (AuNP). The nanoparticle of items 9 or 10, wherein the nanoparticle has been blocked in a 0.1 % starch (w / v) starch-iodine solution. The nanoparticle of any one of items 9 to 11 , wherein the mean diameter of the nanoparticle including the iodine-starch complex on the surface is between about 60 nm to about 90 nm, preferably about 70 nm to about 80 nm. A composition comprising a plurality of nanoparticles of any one of the preceding claims. An immunodetection sensor comprising the nanoparticle for immunodetection based on lateral flow assay or the composition comprising a plurality of nanoparticles according to any one of the preceding claims. An immunodetection method comprising the steps of

[0245] (i) mixing a sample with the nanoparticle(s) according to any one of items 1 to 12 or the composition comprising a plurality of nanoparticles according to item 13, wherein the nanoparticle(s) have been conjugated to the antibody or protein for detection of an analyte in a sample; and

[0246] (ii) detecting an electrochemical signal, an optical signal, or both.

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Claims

CLAIMS1 . A nanoparticle comprising an iodine-starch complex, preferably wherein the nanoparticle is suitable for immunodetection.

2. The nanoparticle of claim 1 , wherein the iodine-starch complex is located on the surface of the nanoparticle.

3. The nanoparticle of claim 2, wherein the nanoparticle is selected from a gold nanoparticle (AuNP), a magnetic nanoparticle and a latex nanoparticle.

4. The nanoparticle of any one of claims 1 to 3, wherein the surface of the nanoparticle comprises antibodies, antibody fragments, proteins, peptides, aptamers, nucleic acids or other biological or biochemical receptors conjugated to the surface.

5. The nanoparticle of any one of the preceding claims, wherein the surface of the nanoparticle is entirely covered by the antibodies, antibody fragments, proteins, peptides, aptamers or nucleic acid probes or other biological or biochemical receptors, and the iodine-starch complex.

6. A method for preparing a nanoparticle according to anyone of claims 1 to 5, the method comprising the steps of(i) conjugating the nanoparticle with an antibody, antibody fragment, protein, peptide, aptamer, nucleic acid or another biological or biochemical receptor, and(ii) contacting the conjugated nanoparticle of step (i) with a starch-iodine solution, wherein step (i) is performed before step (ii).

7. The method of claim 6, wherein the iodine-starch solution is a solution comprising between about 0.05 - 1% (w / v), preferably between about 0.05 - 0.5% (w / v) of starch, most preferred about 0.1 % (w / v) of starch.

8. The method of claims 6 or 7, wherein the step (ii) is performed for at least 30 minutes, preferably for at least 45 minutes.

9. The method of claims 7 or 8, wherein the step (ii) is performed at room temperature and at pH 7-9.

10. The nanoparticle of claim 1 , wherein the nanoparticle has a core and a shell, and the iodine-starch complex is located in the core of the nanoparticle, wherein the shell comprises at least one polyelectrolyte, at least one surface active agent and at least one stabilizer, and wherein(i) the polyelectrolyte is selected from the group consisting of polylactic-co- glycolic acid (PLGA), polylactic acid (PLA), poly-e-caprolactone (PCL), or combinations thereof; and / or(ii) the at least one surface-active is a phospholipid selected from phosphatidylcholine, phosphatidylserine, or a combination thereof, or a nonionic surfactant selected from Span 60, Span 80, or a combination thereof.1 1 . The nanoparticle of claim 10, wherein the core of the nanoparticle comprises between about 1% - 4% (w / v), preferably between about 1 % - 2% (w / v) of the iodine-starch complex.

12. The nanoparticle of claims 10 or 11 , wherein the at least one stabilizer is selected from the group consisting of polyvinyl alcohol, dodecanol, cholesterol, cholesteryl hemi succinate, or combinations thereof.

13. The nanoparticle of any one of claims 10 to 12, wherein the mean diameter of the nanoparticle is between about 150 nm to about 250 nm, preferably between about 190 nm to about 210 nm.

14. A composition comprising a plurality of nanoparticles of any one of the preceding claims.

15. An immunodetection sensor comprising the nanoparticle according to any one of claims 1 to 5 and 10 to 13, or the composition comprising a plurality of nanoparticles according to claim 14.

16. An immunodetection method comprising the steps of(i) mixing a sample with the nanoparticle according to any one of claims 1 to 5 and 10 to 13 or the composition comprising a plurality of nanoparticles according to claim 14; and(ii) detecting an electrochemical signal, an optical signal, or both.

17. The immunodetection method of claim 16, wherein the analyte is selected from the group consisting of extracellular vesicles (EVs), bacteria, antibodies, proteins, metabolites, pollutants, allergens.

18. Use of the nanoparticle of any one of claims 1 to 5 and 10 to 13 or the composition comprising a plurality of nanoparticles according to claim 14 for the immunodetection of an analyte, preferably wherein the analyte is selected from the group consisting of extracellular vesicles (EVs), bacteria, antibodies, proteins, metabolites, pollutants, allergens.

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