Detection of biomarkers in a sample

Abstract

The present invention relates to a method for detecting a biomarker in a sample, comprising the steps of contacting a sample that potentially comprises a biomarker with optically responsive magnetic particles capable of binding the biomarker, wherein presence of a biomarker results in the generation of an optical response; concentrating the particles by means of magnetic force; resuspending the particles in a fluid; and detecting in the fluid whether an optical response is generated. The method is based on clustering or unclustering of the magnetic particles as a result of the presence of the biomarker and the optical response that is caused by clustering or unclustering. The magnetic particles are suitably coated with a noble metal.

Description

[0001] DETECTION OF BIOMARKERS IN A SAMPLE

[0002] The present invention relates to a method for detecting a biomarker in a sample. The invention further relates to a kit of parts for performing the method and to particles for use in the method.

[0003] Biomarkers are measurable indicators found in the body that can signal a wide range of physiological and pathological processes. Biomarkers are often disease-related reporter molecules and are used in medical research and healthcare to detect, diagnose, and monitor diseases.

[0004] Molecular biomarkers include DNA, RNA, peptides, proteins, and metabolites. They can indicate genetic mutations, protein levels, or metabolic changes associated with diseases. There are different types of markers, in particular endogenous and exogenous biomarkers. Endogenous biomarkers are produced by the body naturally. Exogenous biomarkers are markers that result for example from the interaction of administrated reagents with the body.

[0005] Biomarkers can help identify diseases at an early stage, often before symptoms appear. This early detection can significantly improve treatment outcomes and survival rates, especially for conditions like cancer. Furthermore, biomarkers can provide valuable information for diagnosing diseases and monitoring their progression. They may help in understanding how a disease is evolving and whether treatments are effective.

[0006] It is desirable for medical testing to be performed in a point-of-care (PoC) situation, i.e. near the site of patient care, rather than in a centralized laboratory. The goal of point-of-care testing and treatment is to provide immediate results and interventions, which can lead to faster decisionmaking and improved patient outcomes. PoC testing reduces the logistical and financial barriers associated with traditional testing, such as travel time and costs, thereby facilitating increased access and expanding the potential reach to a broader target population.

[0007] It is the object of the present invention to provide a novel method for the direct, point-of-care (PoC) detection and reporting of biomarkers, characterized by straightforward and easily interpretable signal generation, thus enhancing user accessibility and diagnostic efficiency.

[0008] In the research leading to the present invention, a combination in one system was developed, comprising a 2-step approach of first concentration enlargement of biomarkers present in a sample of body fluid or tissue and second, the detection or visualization of the biomarkers. The system was developed for nucleic acids including chemically modified nucleic acids, but can also be used for other biomolecules.

[0009] The invention thus relates to a method for detecting a biomarker in a sample, comprising the steps of:a) contacting a sample that potentially comprises a biomarker with optically responsive magnetic particles capable of binding the biomarker, wherein presence of a biomarker results in the generation of an optical response;

[0010] b) concentrating the particles by means of magnetic force;

[0011] c) resuspending the particles in a fluid; and

[0012] d) detecting in the fluid whether an optical response is generated.

[0013] Binding of the biomarker or analyte to the magnetic particle is either directly to a coating on the particle or to a receptor. The receptor may bind via hybridisation in the case of nucleic acids, such as DNA and RNA, or via ligand-receptor interaction, for example between a peptide or protein and its receptor or between an antigen and an antibody.

[0014] In a preferred embodiment, the binding of the biomarker to the magnetic particle is either specifically by ligand-receptor interaction or hybridisation, or non-specifically by electrostatic interactions. Non-specific binding in this context means that the interaction is not dependent on the structure or sequence of the biomarker but depends on a physical property of the biomarker, such as charge. There is some level of specificity, but it does not identify the biomarker per se, only a more general property thereof. For this type of interaction, the term ‘semi-specific’ will also be used in this application.

[0015] The use of specific ligand-receptor interaction in the method according to the invention is schematically shown in Figure 1. Figure 2 shows the variant based on non- or semi-specific binding of the biomarker to the magnetic particles by electrostatic interactions that is followed by the specific interaction in the next step, optionally after magnetic collection of the particles. In a preferred embodiment, non- or semi-specific binding in a first round of interaction between the particle and the biomarker is followed by specific binding in a second round of interaction.

[0016] In one embodiment, the magnetic particles are either coated with a transition metal, in particular a noble metal, such as gold, or are magnetic quantum dots. Clustering of both types of particles results in an optical response, such as a colour change.

[0017] The invention is based on the binding of the analyte to be detected to magnetic particles and easy detection or visualisation of this binding. A magnetic force is used to isolate these particles with the bound analyte from its original environment, such as a body fluid which besides the analyte may comprise a multitude of other components that may interfere with the detection of the analyte. The step of isolating the magnetic particles with the analyte bound thereto from its environment on the one hand leads to concentration of the analyte which becomes then more easily detectable and thus increases the sensitivity of the test. On the other hand, isolation of the magnetic particles with the bound analyte from its original environment leads to removal of interfering components. By resuspending the particles in a small volume of new, clean fluid, a test sample isobtained in which the analyte is concentrated and the environment is free from interfering molecules.

[0018] According to the invention one analyte can be detected in one test or multiple analytes can be detected in one test (multiplexing). When multiple analytes are detected in one test, the readout provides information on the presence or absence of these analytes, but it can not be determined which of the analytes to be detected are actually present. For a specific detection parallel tests with different receptors are needed.

[0019] The method of the invention can be used with one type of beads. Detection does not require another type of beads. The decoration of the beads used in an assay can be different though. In one embodiment, two beads decorated with different receptors binding the same analyte can be used to effect clustering. The core of these beads is still the same. In another embodiment, in a multiplex context, the thickness or type of the noble-metal coating, such as gold, can differ between beads, wherein one thickness or type represents one type of analyte and another thickness or type represents another

[0020] The detection indicates the presence or absence of the analyte. In one embodiment, the method of the invention leads to a yes or no response regarding the presence of the analyte to be detected in the sample. In another embodiment, a multiplex approach can be used in which multiple analytes are detected in the same pot. In this embodiment, the detection is also based on presence or absence, not on quantification. Alternatively, multiple analytes can be tested in parallel assays with different aliquots of the same sample.

[0021] The detection is done by generating an optical response which is representative for the presence of the analyte. The optical response is the result of clustering or unclustering of the magnetic particles with the analyte bound thereto.

[0022] The optical signal generated in the fluid is a change in the absorption and emission of electromagnetic radiation, in particular a colour change. Typically, the (colour) change is the result of either clustering of the particles in the fluid or the unclustering of particle aggregates, triggered by binding of the biomarker. Alternatively, the (colour) change can also be the result of clustering that follows direct binding of the biomarker to the coated particles. In this latter case, the analyte to be detected is not specifically bound to a receptor on the surface of a particle but can adhere to multiple particles thus acting as a kind of glue to keep particles together in a cluster. This cluster formation leads to the optical response. This type of clustering is typically used when the analyte is DNA that is chemically modified with a sulphur-bearing group.

[0023] The optical changes are either colour changes that are visible by the naked human eye, or changes in electromagnetic radiation that are invisible to the eye but can be observed by optical detectors. Optical detectors or photo detectors are sensors that convert electromagnetic radiation into electric signals that can be measured through an appropriate device. An example of an opticaldetector is the photodiode. The output electric signal thus obtained is proportional to the incident light or electromagnetic radiation. Optical detectors can be used to detect both visible and invisible electromagnetic radiation.

[0024] For the optical signal to occur, the biomarker must bind to the magnetic particles. For this, the particles are functionalized with a layer that interacts with the biomarker (also called analyte). This layer can for example consist of immobilized receptor molecules such as nucleic acid or derivatives thereof or peptide receptors that are able to specifically bind DNA, RNA or proteins and peptides. The specific binding is the result of hybridization of the nucleic acid biomarker with a complementary nucleic acid receptor molecule or ligand-receptor interaction in the case of peptides and proteins.

[0025] In one embodiment, the particles are suitably decorated with a first receptor that binds one part of the target analyte and a second receptor that binds the other part. For detecting nucleic acid analytes, the first receptor will hybridize with a first part of the nucleic acid, whereas the second receptor binds to another part of the nucleic acid analyte. This is also called “DNA handcuffing”. When the analyte to be detected is a protein or peptide, the first receptor may be an antibody binding one part of the protein or peptide and another antibody binding to another part. Peptides may also be bound to particles decorated with their natural receptor or an artificial receptor.

[0026] Since the analyte can thus bind to two particles and particles carry multiple receptors multiple particles can be linked to each other to form a cluster.

[0027] In one embodiment, the optical response is thus generated by binding of the biomarker to the particle and binding multiple particles together via multiple analyte molecules leading to a cluster of particles which clustering generates an optical response. The proximity of the magnetic particles or quantum dots leads to an observable shift in their absorption spectrum.

[0028] In another embodiment, the analyte is contacted with pre-clustered magnetic particles, and the optical response is generated by breaking the cluster formation. This unclustering may be the result of activating the collateral cleavage activity of a CRISPR-Cas enzyme by the target analyte. In a further embodiment for nucleic acid detection, unclustering is based on strand displacement, such as so-called “toehold-mediated” strand displacement. In this case, the particles are preclustered by hybridisation between complementary DNA or RNA strands. One of the strands has a loose end that is complementary to at least part of the analyte. When present in the reaction mixture, the analyte will bind to the loose end and displace the other strand thus disrupting the clustering of the two magnetic particles. In this embodiment, the target DNA thus binds to a piece of the DNA holding the cluster together and then zips off the rest because of its stronger binding by design.

[0029] The particles may be decorated with one type of receptor molecule with one specificity or with two, three, four or more different types of receptor molecule with each a separate specificity.When targeting multiple analytes, individual readout per analyte type is achieved by using multiple particle batches. In this case, each batch consists of various optically responsive particle types and is decorated with other receptor molecules and , to connect various colours with various biomarkers result in a differentiated readout (i.e. multiplexing!.

[0030] In a further embodiment, one particle batch can be functionalized with multiple receptor molecules on each particle and result in a yes / no (colour) change for the presence of any of the analyte molecules. This embodiment results in an undifferentiated readout. It only indicates that at least one of the analytes is present in the sample.

[0031] In a further embodiment, the method of the invention comprises adding a clustering agent to the reaction mixture. In a situation in which the clustering via the biomarker in the sample has already taken place a cluster is formed that comprises the biomarker. The clustering agent is then for facilitating the further clustering of the already clustered magnetic particles. When no biomarkers are present there is no prior clustering of the magnetic particles. The clustering agent will still cluster these particles. This additional clustering makes magnetic precipitation much easier, especially in case relatively small magnetic particles are used that may be more difficult to collect magnetically. De-clustering of these larger clusters can be achieved by adding a competitor molecule. The competitor molecule is capable of disrupting the larger clusters but not the clusters formed by the analyte to be detected. In case a biomarker had been bound by the magnetic particles these clusters remain intact and lead to an optical response. In case no biomarker was present the magnetic particles are still collected but de-clustering does not lead to an optical response.

[0032] Alternatively, or in addition, the particles are coated with a transition metal, and in particular a noble metal, more in particular selected from gold, silver, platinum, palladium, rhodium, ruthenium, osmium and iridium for binding the biomarker. In a preferred embodiment, the transition metal is a soft metal. In a more preferred embodiment, the soft metal is gold.

[0033] Thiols, dialkylsulfides, dialkyldisulfides and phosphorothioate are known to be chemisorbed with high affinity on gold. Therefore, DNA that is chemically modified with these sulphur-bearing groups can directly bind to a gold surface without the need for a receptor molecule. In a particular embodiment, the interaction between the particle and sulphur-containing biomarkers is thus directly with the surface of a gold layer.

[0034] Gold-coated particle clustering as a result of the presence of a sulphur-containing biomarker results in a detectable colour change. This binding is aspecific to the sequence, and multiplexing will be limited to the level of detecting the presence of any of the target biomarkers but not specifically which one. Thus, this type of assay can detect multiple biomarkers but cannot distinguish between them.

[0035] In a further embodiment, the sulphur-containing biomarker is phosphorothioate DNA. Phosphorothioate DNA is DNA in which a nonbridging oxygen is replaced by a sulphur atom.Binding of phosphorothioate DNA to the gold coated magnetic particle is non-sequence specific. The same applies to the other noble metals, like silver, platinum, palladium, etc.

[0036] The method of the invention is also very useful in the detection of DNA barcodes that are used in the PATROL platform (Zhong Q, Tan EKW, Martin-Alonso C, Parisi T, Hao L, Kirkpatrick JD, Fadel T, Fleming HE, Jacks T, Bhatia SN. Inhalable point-of-care urinary diagnostic platform. Sci Adv. 2024 Jan 5;10(l):eadj9591. doi: 10.1126 / sciadv.adj9591. Epub 2024 Jan 5. PMID: 38181080; PMCID: PMC10776015). The PATROL platform formulates a set of DNA-barcoded peptides into an inhalable format. Each peptide is specifically cleaved by a particular protease that is representative for a particular form of cancer. Upon cleavage, the associated DNA-barcode is released and excreted via the urine. The DNA-barcode detected in a urine sample is thus diagnostic for the cancer that is present in the patient. The method of the invention is particularly useful to detect these DNA barcodes. In this embodiment, the barcode is the biomarker to be detected.

[0037] The particles are also magnetic, which allows the pull-down of particles with bound analytes from (sometimes large) liquid biopsy volumes. Reconstitution of the particles that are separated from the sample in a clean, controlled and much smaller volume, effectively results in analyte concentration increase and purification.

[0038] The receptor molecules with which the magnetic particles are decorated can be nucleic acids, nucleic acid derivatives, peptides, proteins.

[0039] In a further embodiment, the magnetic particles are decorated with a receptor molecule capable of binding the biomarker and subsequently coated with a charge coating. The charge coating is capable of binding analytes with an opposite charge. In the case of a nucleic acid biomarker for example, a positively charged outer coating, such as Poly-L-Lysine or Q-Dextran, will non-specifically capture all negatively charged nucleic acid molecules (like ssDNA, dsDNA,RNA or synthetic derivatives thereof, such as phosphorothioate DNA ) in the sample. After separating the particles from the sample by means of a magnetic force, the charge coating can be removed, for example enzymatically, to release the nucleic acid target molecules, which subsequently bind specifically to the receptors on the particles, if there is a specific match between target and surface receptor.

[0040] The method as claimed thus comprises the further step of releasing the charge coating between steps c) and d). Such two-layer coating enables the stepwise capture of the biomarker using electrostatic interactions between the DNA / RNA and the positive charge, followed by triggered charge removal such as enzymatic degradation of the charge coating. This is schematically shown in Figure 2. In this embodiment, the sample concentration and binding of the analyte to be detected are integrated in one particle. In this two-layer approach, the outer layer is degradable by means of a chosen trigger, such as an enzyme. After degrading this coating theactual receptor is exposed to bind the analyte. This simple two-step process can be performed without the need for laboratory equipment or specialised skills. Multiple receptors on still the same particle allows for multiplexing.

[0041] This two-step method enables detection of analytes in samples with a low analyte concentration or a complex composition. Only a few malignancies lead to a high analyte concentration. With this method other malignancies can also be detected. Liquid biopsies may be too complex to allow direct analyte detection without prior sample preparation. The method of the invention can be used in both situations.

[0042] The advantage of the additional charge coating is that all targets will bind to all beads, requiring a total of less beads in the (large) sample to achieve target binding at a similar rate. If multiple particle batches without the charge coating are used to allow multiplexing, each batch will need to be used at a similarly high concentration as the double layer ensemble batch to achieve similar binding rate of the specific target, requiring the total combined bead loading to be much higher. In addition, the denaturation of dsDNA targets in the case of large (urine) samples is impractical, complicating the detection of double stranded DNA (dsDNA) in this situation. By addition of the charge coating, all DNA is strongly concentrated before the specific hybridization to the receptors on the particles. This makes denaturation of dsDNA much more feasible.

[0043] An additional advantage of the additional charge coating is that the specific binding step can be performed in a clean and user-determined matrix rather than the sample matrix. This greatly benefits achieving a specific and robust assay.

[0044] In a further embodiment, the particles are quantum dots (QD). Quantum dot clustering results in colour change. Using magnetic QDs facilitates magnetic precipitation. In order to detect and indicate each multiplex biomarker, various colours QDs can be combined in one-pot and the outcome colour can indicate the specific biomarker(s) present in the sample.

[0045] In one embodiment, the surface charge on the particle is in the form of a strong ion exchange coating, such as a coating bearing quaternary ammonium ions, to facilitate binding of short DNA or DNA-derivative oligonucleotides even in high-salt media such as urine.

[0046] In one embodiment, the detection of the optical response is phone-camera aided. Using a phone-camera for detection the response minimizes observer-bias and facilitates read-out for visually impaired people, in particular colour-blind people. Alternatively, the change is observed with a spectroscope or another device that can detect changes in visible and invisible electromagnetic radiation.

[0047] The method of the invention may further comprise a step for amplifying the optical response generated in a positive feed-back loop, in particular by means of a cascade reaction, or by means of an autocatalytic process, or by means of a collateral cleavage effect.Sensitivity increase is then obtained by presentation of the receptor molecules in such a manner that binding of the biomarker results in a positive feedback loop. This can subsequently enable (un)clustering of particles or results in the generation of a fluorescence signal making use of the Forster Resonance Energy Transfer (FRET) effect to generate or quench fluorescence.

[0048] Increasing the amount of clustering can be achieved by using the positive feedback loop to effectively generate or liberate more target molecules which result in the clustering. Liberation of molecules refers for instance to a conformation change DNA ‘hairpins’ in the presence of a target molecule, enabling further hybridization binding. Increasing the amount of unclustering can be achieved in the same manner if the unclustering is achieved by means of a toehold switch.

[0049] Increasing the amount of unclustering can be additionally achieved by using the collateral cleavage effect of CRISPR-Cas enzymes activated by the target molecule.

[0050] The feedback loop can be generated by a cascade reaction where the binding of one biomarker changes the receptor molecule in such a way that neighboring receptor molecules also change. This causes one target to change multiple receptors which thus can result in increased clustering. An example of such feedback loop is the hybridization chain reaction (HCR; Evanko, D. Hybridization chain reaction. Nat Methods 1, 186 (2004)).

[0051] In another embodiment, increase of the sensitivity of the assay is achieved by an autocatalytic process where the binding of the biomarker releases an agent that also bind to the receptor molecule. In this way, one biomarker will generate multiple agents that each interact with the bead-bound receptors and therefor increase clustering. An example is the technology described in “RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides” (Y uki Goto and Hiroaki Suga; Accounts of Chemical Research 54 (18), 3604-3617 (2021)) where the target DNA gets translated multiple times into various peptides allowing signal amplification and multiplexing.

[0052] In one embodiment, the optical response is detected by means of a particle-based lateral flow assay. These assays are typically more sensitive than colour change from gold particle clustering. Multiple test-spots can facilitate the detection and identification of each multiplex biomarker.

[0053] The optical response can also be the result of using pre-clustered particles (like gold or QD) that will be released and change in colour as an effect of collateral cleavage activity of CRISPR-Cas 12, activated in presence of the biomarker. In this case, the particles are clustered by design at the start of the assay. Once the CRISPR-Casl2 enzyme is activated by the target DNA molecule, it will start to ‘collaterally’ cleave all present DNA including the DNA that is used to hold together the pre-clustered beads. CRISPR-Casl3 can do the same with RNA targets. The unclustering will result in the optical signal. The collateral cleavage effect can increase overall sensitivity. In order to detect and indicate each multiplex biomarker, the sample would need to be split in as many aliquots. This can be combined with autocatalytic sensitivity increase by using asystem such as “CRISPR-Cas-only amplification network (CONAN) (K. Shi, S. Xie, R. Tian, S. Wang, Q. Lu, D. Gao, C. Lei, H. Zhu and Z. Nie, Set. Adv., 2021, 7, eabc7802). In this platform, recognition of the DNA biomarker activates transducer 2, which comprises Casl2a and a dsDNA probe, and the resulting Casl2a trans-cleavage activity liberates a caged crRNA that can then target transducer 2, creating a positive feedback circuit that generates an exponentially increasing fluorescent signal.

[0054] The invention further relates to a kit of parts for performing the method, comprising optically responsive magnetic particles coated with a noble metal and / or decorated with one or more receptor molecules and means for detecting an optical signal generated by the particles after binding of a biomarker.

[0055] Also part of the invention are particles for use in the method, comprising optically responsive magnetic particles coated with a noble metal and / or decorated with one or more receptor molecules.

[0056] In a further embodiment, the optically responsive magnetic particles may be provided with a double layer, an inner layer comprising the receptor molecules and an outer layer comprising a charge coating. These particles may also be present in the kit.

[0057] The present invention will be further elucidated in the examples that follow and that are given for illustration purposes only and are not intended to limit the invention in any way.

[0058] EXAMPLES EXAMPLE 1

[0059] Differentiated target binding

[0060] In this example, the particles are coated with a DNA receptor layer that directly and specifically bind the ssDNA target from the sample as schematically shown in Figure 1 and specified in that figure as “differentiated”.

[0061] Nanoparticles ranging from 15 to 100 nm in diameter with a magnetic core and gold shell ranging in thickness between 3 to 40 nm thickness were synthesized according to literature procedures (M. Mehdipour et al., J. Mater. Chem. C, 2021, 9, 1034-1043 or

[0062] M. Mehdipour et al., Part. Part. Syst. Charact. 2022, 39, 2200051) or alternatively are commercially available, for example from the company CD Bioparticles (htps: / / www.cd-bioparticles.com / ) or nanoComposix (https: / / nanocomposix.com / ).

[0063] For each target DNA, a set of two receptor functionalized particles are produced. One particle batch binds one half of the target DNA molecule specifically. The other particle batch binds the other half of that same DNA molecule specifically. As a result, the DNA target can bind one particle from each batch together, so-called “DNA handcuffing”.These particles are functionalized with DNA probes using one of the following methods.

[0064] Method 1

[0065] Magnetic gold nanoparticles (MAuNPs) (from CD Bioparticles or nanoComposix) are transferred to PBS buffer, pH 7.4. An excess of thiolated DNA probes is added to a final concentration of IpM in the nanoparticle solution. The reaction mixture is mixed by vortexing and incubated at room temperature for at least Ih. NaCl solution is added gradually to the mixture to reach a final concentration of 0.3 M. The mixture is incubated at room temperature overnight. The MAuNPs are pelleted using a magnet and the supernatant containing unbound DNA is removed and replaced with PBS. The washing with PBS is repeated 3 more times . The DNA-conjugated nanoparticles are stored at 4°C.

[0066] Method 2

[0067] Poly-L-Lysine (PLL) with 5% of the lysine residues functionalized with Strained Alkyne groups (PLL-SA) such as dibenzocyclooctyne (DBCO) or bicyclononyne (BCN) are synthesized according to literature protocol (Langmuir 2020, 36, 16, 4272-4279

[0068] (htps : ZZdoi . orgZ 10 , 1021 Zacs . langmuir . OcOO 144)) or purchased from specialized vendor such as Susos (susos.com).

[0069] The MAuNPs are transferred to PBS buffer, pH 7.4. An excess PLL-SA is added to a final concentration of 1 mgZmL in the nanoparticle solution. The mixture is incubated for 1 h on rollershaker.

[0070] The MAuNPs are magnetically pelleted and excess PLL-SA is replaced with PBS. The washing is repeated 3 more times. Excess azide functional DNA probes are added to a final concentration of IpM in the nanoparticle solution. The mixture is incubated at room temperature for 2 h. The MAuNPs are pelleted using a magnet and the supernatant containing unbound DNA removed and replaced with PBS. The washing is repeated 3 more times. The DNA-conjugated nanoparticles are stored at 4°C.

[0071] MAuNPs display different optical properties by tuning the size of the particle from 15 to lOOnm and thickness of the gold layer in the range of 3 to 40 nm. For each target biomarker, a different combination of two particles is selected and decorated with DNA probes as specified above. The various nanoparticle batches are mixed to allow multiplexing in one-pot.

[0072] The nanoparticle mixture is added to the sample, e.g. urine. The sample can contain no, one or multiple single stranded DNA (ssDNA) reporters.

[0073] After incubation for Ih the beads are magnetically collected and the supernatant removed. The beads are resuspended in lOOpL of 4xSSC buffer at pH 7.4 and allowed to further interact for 15 min. This concentrated formulation will allow further development of the “DNA handcuffing”process but is also needed for effective optical readout since DNA hybridization is strongly saltdependent and works well under for instance 4xSSC buffer at pH 7.4.

[0074] The final color is compared to a separate particle batch of the same original batch that had not been in contact with the sample for reference.

[0075] The signal can be read out using a phone camera and deconvoluted to determine which nanoparticles are clustered and thus which biomarkers were present in the original sample. The diagnostic result can be sent digitally to a healthcare professional.

[0076] EXAMPLE 2

[0077] Particles with charge coating

[0078] In this example, the particles are coated with 2 layers. An inner receptor layer and an outer PLL layer. All DNA is first captured aspecifically by the PLL layer after which the PLL layer is cleaved enzymatically to expose the inner layer, after which the DNA biomarkers bind specifically to the DNA receptors in the inner layer.

[0079] MAuNPs are functionalized with DNA probes according to method 1 of example 1, so using the thiolated DNA method only. DNA-fiinctionalized MAuNPs are then incubated with Img / mL of PLL in PBS for 2h on a roller-shaker. Excess PLL is washed 3x using magnetic precipitation of particles.

[0080] This 2-layer MAuNPs is then added to the sample, e.g. urine. The sample can contain no, one or multiple ssDNA reporters or double stranded DNA (dsDNA) targets. All ssDNA or dsDNA now binds to the outer PLL coating by charge-interaction.

[0081] The MAuNPs are pelleted magnetically and washed 3x with PBS. IOOUL of 2.5mg / mL trypsin solution in HEPES saline buffer at pH 7.4 is added to the cleaned bead pellet and incubated for Ih at room temperature on a roller shaker. During this process, the outer PLL layer is enzymatically broken down and the DNA bound thereto released.

[0082] Now there are 2 options depending on the presence of ssDNA or dsDNA targets. ssDNA may bind in a sequence-specific manner to the inner DNA receptor layer, resulting in MAuNPs clustering and subsequential color change in the presence of the specific biomarker(s).

[0083] For dsDNA targets the now small sample volume is heated to 95 °C to denature the dsDNA after which the sample is cooled down and the now ssDNA target bind in a sequence- specific manner to the inner DNA receptor layer, resulting in MAuNPs clustering and subsequential color change.

[0084] The final color is compared to a separate batch of the same particles that have not been in contact with the sample for reference. The signal can be read out using a phone camera and deconvoluted to determine which nanoparticles are clustered and thus which biomarkers werepresent in the original sample. The diagnostic result can be sent digitally to a healthcare professional.

[0085] EXAMPLE 3

[0086] Method with clustered particles

[0087] In this example, particles are pre-clustered in small clusters and the un-clustering will give the optical signal. Particles are clustered by making 2 batches or MAuNPs. One with DNA probe “A” and one with DNA probe “B”. A third and free dissolved synthetic oligo complementary to both “A” and “B” is used to ‘handcuff the particles together. This third oligo “C” additionally has a short single stranded section after binding to “A” and “B” that is susceptible to the collateral cleavage activity of CRISPR-Casl2 proteins, which will result in un-clustering and consequential color change.

[0088] MAuNPs are functionalized with the DNA probes according to method 1 of example 1, so using the thiolated DNA method only. However, only 2 DNA receptors are used now (“A” and “B”) rather than one set of two probes for each specific target biomarker. So, a final total of 2 batches of functionalized MAuNPs is created, independent of the amount to different targets.

[0089] The MAuNPs batch with receptor “A” is incubated with luM of oligo “C” and allowed to hybridize for Ih. The MAuNPs are pelleted magnetically and washed 3x with PBS. Now, under vigorous stirring, the MAuNPs batch with receptor “B” is very slowly titrated to these particles to a final 1:1 molar ratio of the “A” and “B” particles. The now clustered particles are coated with the 2ndlayer (PLL) as described in Example 2.

[0090] DNA from the sample is captured, concentrated and released as in Example 2. CRISPR-Casl2 loading with the RNA guide gives a Ribonucleoprotein (RNP) Complex specific for the dsDNA target complementary to the RNA guide. RNP complexes can be commercially purchased. For each target sequence, a complementary RNA guide for CRISPR-Casl2 is be designed to allow multiplexing.

Claims

CLAIMS1. Method for detecting a biomarker in a sample, comprising the steps of:a) contacting a sample that potentially comprises a biomarker with optically responsive magnetic particles capable of binding the biomarker, wherein presence of a biomarker results in the generation of an optical response;b) concentrating the particles by means of magnetic force;c) resuspending the particles in a fluid; andd) detecting in the fluid whether an optical response is generated.

2. Method as claimed in claim 1, wherein the optical response is generated by binding of the biomarker to the particles, which binding leads to clustering of the particles resulting in the optical response.

3. Method as claimed in claim 1, wherein the particles are pre-clustered and the optical response is generated by presence of the biomarker, which leads to unclustering of the particles resulting in the optical response.

4. Method as claimed in claim 3, wherein unclustering is the result of activating the collateral cleavage activity of a CRISPR-Cas enzyme by the presence of the biomarker.

5. Method as claimed in any one of the claims 1 to 4, wherein the optical response is the result of clustering or unclustering of magnetic particles that are coated with a transition metal, in particular a noble metal selected from gold, silver, platinum, palladium, rhodium, ruthenium, osmium and iridium.

6. Method as claimed in claim 5, wherein the noble metal is gold.

7. Method as claimed in claim 5 or 6, wherein the biomarker binds to the transition metal coating on multiple particles to form a cluster of magnetic particles.

8. Method as claimed in claim 7, wherein the particles are coated with gold for binding a biomarker comprising a sulphur-bearing group, such as phosphorothioate DNA.

9. Method as claimed in claim 7 or 8, wherein binding to the transition metal coating is non-specific.

10. Method as claimed in any one of the claims 1 to 3, wherein the optical response is the result of clustering or unclustering of magnetic quantum dot particles.

11. Method as claimed in any one of the claims 1 to 10, wherein the particles are decorated with a receptor molecule capable of binding the biomarker.

12. Method as claimed in claim 11, wherein clustering is the result of biomarkers binding to at least two receptors on separate particles.

13. Method as claimed in claim 11 or 12, wherein binding of the biomarker to the receptor is specific.

14. Method as claimed in claim 12 or 13, wherein the at least two receptors bind different parts of the biomarker.

15. Method as claimed in any one of the claims 11-14, wherein the receptor molecule is selected from nucleic acids, nucleic acid derivatives, peptides, proteins.

16. Method as claimed in claim 15, wherein the receptor molecule is selected from ssDNA, dsDNA, RNA.

17. Method as claimed in any one of the claims 1 to 16, wherein the optical response is a colour change resulting from clustering of the particles or unclustering of the particles.

18. Method as claimed in claim 17, wherein the colour change is visible for the naked eye or in the invisible spectrum.

19. Method as claimed in any one of the claims 1 to 6 and 10 to 18, wherein the optically responsive magnetic particles are decorated with a first layer of receptor molecules capable of binding the biomarker and coated with a second layer of a charge coating for binding oppositely charged biomarkers.

20. Method for detecting a biomarker in a sample, comprising the steps of:a) contacting a sample that potentially comprises a biomarker with the optically responsive magnetic particles as defined in claim 19 and allowing binding of oppositely charged biomarkers to the charge coating;b) concentrating the particles by means of magnetic force;c) resuspending the particles in a fluid;d) removing the charge coating while releasing the bound biomarkers;e) allowing the released biomarkers to bind to the receptors in the first layer and cluster the magnetic particles to generate an optical response;andf) detecting in the fluid whether an optical response is generated.

21. Method as claimed in any one of the claims 1 to 20, wherein the particles are decorated with one type of receptor molecule with specificity for one biomarker or wherein the particles are decorated with two or more different types of receptor molecule with each a different specificity for one biomarker.

22. Method as claimed in claim 21, wherein a type of receptor molecules with specificity for one biomarker comprise at least two different receptor molecules, each binding to a different part of the biomarker.

23. Method as claimed in any one of the claims 1 to 22, further comprising a step for amplifying the optical response generated in a positive feed-back loop.

24. Method as claimed in claim 23, wherein the optical response is amplified by means of a cascade reaction such as the hybridization chain reaction (HCR).

25. Method as claimed in claim 23, wherein the optical response is amplified by means of an autocatalytic process.

26. Method as claimed in claim 23, wherein the optical response is amplified by means of a collateral cleavage effect.

27. Method as claimed in any one of the claims 1 to 26, wherein the optical response is detected by means of a particle-based lateral flow assay, a spectroscope, or a smartphone, optionally after deconvolution of the signal.

28. Kit of parts for performing the method as claimed in any one of the claims 1 to 27, comprising optically responsive magnetic particles coated with a noble metal and / or decorated with one or more receptor molecules and means for detecting an optical signal generated by the particles after binding of a biomarker.

29. Particles for use in the method as claimed in any one of the claims 1 to 27, comprising optically responsive magnetic particles coated with a noble metal or magnetic quantum dots, which particles or dots are optionally decorated with one or more receptor molecules.

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