One-step competitive assays using surface-enhanced raman spectroscopy and functionalized nanoscale plasmonic surfaces

Abstract

The invention is notably directed to a method of performing a one-step competitive assay using surface-enhanced Raman spectroscopy and nanoscale plasmonic surfaces (e.g., of nanoparticles) functionalized with first molecules. The method first comprises bringing (S31 – S33) second molecules and third molecules in a liquid solution into contact with the nanoscale plasmonic surfaces. The first molecules and the second molecules form a dual set of complementary single strands of polynucleotides, whereby the second molecules can bind to the first molecules. The second molecules (e.g., cDNA) are labelled with Raman reporter molecules. The third molecules are analyte molecules that can bind either to the first molecules or to the second molecules, thereby competitively limiting the extent to which the second molecules can bind to the first molecules. Next, the method comprises measuring (S34, S35) a current Raman signal primarily caused by the Raman reporter molecules labelling the second molecules that effectively bind to the first molecules. No separation step is required. Finally, the method comprises determining a property of the third molecules by comparing the current measured Raman signal with one or more reference Raman signals. The invention is further directed to related systems.

Description

[0001] ONE-STEP COMPETITIVE ASSAYS USING SURFACE-ENHANCED RAMAN SPECTROSCOPY AND FUNCTIONALIZED NANOSCALE PLASMONIC SURFACES

[0002] TECHNICAL FIELD

[0003] The invention relates in general to methods of performing competitive assays using Surface-Enhanced Raman Spectroscopy (SERS), as well as related systems. In particular, it is directed to methods involving nanoscale plasmonic surfaces (e.g., nanopatterned surfaces, nanoparticles) functionalized with first molecules (e.g., aptamers), where second molecules (e.g., complementary DNA fragments) and third molecules (analyte molecules) are brought into contact with the nanoscale plasmonic surfaces. The analyte molecules can bind either to the first molecules or to the second molecules, which results in a competition limiting the extent to which the second molecules can bind to the first molecules.

[0004] BACKGROUND

[0005] Various spectroscopy techniques are known, which can be exploited for bioassay sensing. Of particular advantage are optical spectroscopies that can detect, identify, and quantify chemical species in liquid and soft media (e.g., absorption, fluorescence, Raman scattering). In particular, surface-enhanced Raman spectroscopy (SERS), also known as surface-enhanced Raman scattering, is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on plasmonic surfaces, such as rough metal surfaces, metallic nanostructures, or any metallic surface decorated with markers. The dramatic enhancement of the Raman scattering response of molecules in the proximity of metallic surfaces yields enhancement factors that can reach IO10or 1011, allowing up to the detection of single molecules in particular cases. The enhancement is mainly due to amplification of electromagnetic fields by the presence of metallic surfaces near the analyte molecules. This enhancement depends on the proximity, composition, and physical characteristics of the metallic surface and on the physico-chemical properties of the sample.

[0006] However, the detection of small molecules can be very challenging due to their size, because they cannot be simultaneously captured by a receptor and labelled with another molecule. This challenge can be addressed by competitive assays, where the presence of an analyte blocks the immobilization of the reporter molecule. Competitive assays typically require multiple reaction steps for removing the unbound reporter molecules. The quantification is carried out thereafter and is based on the loss of the signal from removed reporters. The requirement of multiple reactions steps has several drawbacks: It complicates assay miniaturization, requires external actuation, and increases the time to obtain results.

[0007] New approaches to competitive assays are therefore needed, which address the above challenges. Furthermore, such approaches should ideally not be limited to the detection of small molecules; they should allow larger molecules such as proteins and DNA fragments to be detected, too. The flexibility offered by such techniques would make it possible to implement complex diagnostic panels using the same core method. Moreover, such approaches should ideally allow multiplexing, for parallel detection of multiple analytes of different sizes, as multiplexing lowers costs and overall running times.

[0008] SUMMARY

[0009] According to a first aspect, the invention is embodied as a method of performing a competitive assay using surface-enhanced Raman spectroscopy (SERS). The method is based on nanoscale plasmonic surfaces that are functionalized with first molecules, such as aptamers. The method first comprises bringing second molecules and third molecules in a liquid solution into contact with the nanoscale plasmonic surfaces.

[0010] The first molecules and the second molecules form a dual set of complementary single strands of polynucleotides, whereby the second molecules can bind to the first molecules. The second molecules are labelled with Raman reporter molecules, which preferably are molecules having a large Raman cross section, such as Nile blue, methylene blue, other chromophores, and / or fluorophore molecules. The third molecules are analyte molecules (e.g., small organic molecules, DNA fragments, proteins) that can bind either to the first molecules or to the second molecules. That is, either the third molecule can appreciably bind to the first molecules, in which case they do not appreciably bind the second molecules, or the third molecules can appreciably bind to the second molecules, in which case they do not appreciably bind to the first molecules. This constraint gives rise to a competition that limits the extent to which the second molecules can bind to the first molecules.

[0011] Next, the method comprises measuring a current Raman signal, i.e., a signal that is primarily caused by the Raman reporter molecules labelling the second molecules that effectively bind to the first molecules. Finally, the method comprises determining a property of the third molecules by comparing the current measured Raman signal with one or more reference Raman signals, which can be obtained in accordance with different calibration and / or normalization scenarios, as discussed herein in detail. The proposed method allows a one-step competitive assay to be performed, where the distance between Raman reporter molecules (which label the second molecules) and the plasmonic surfaces of the nanoscale elements can be controlled through the first molecules and the second molecules, which makes it possible to tune the assay sensitivity. By definition, the second and third molecules are in the liquid solution when contacting the nanoscale plasmonic surfaces. The fact that the third molecules (analyte) can bind to either the first molecules (only) or the second molecules (only) results in a competition, which limits (i.e., restricts) the extent to which the second molecules can bind to the first molecules on the plasmonic surfaces. Any molecule in excess remains in the liquid solution; the liquid solution remains in contact with the plasmonic surfaces when measuring the current Raman signal.

[0012] And this makes it possible to remove the usual intermediary steps. I.e., the present approach does not require any separation step (centrifugation, washing, etc.) to remove the unbound second molecules. The reason is that the latter do not produce a signal when they are in solution (i.e., away from the plasmonic surfaces), because the SERS signal intensity is highly dependent on the distance of the Raman reporter molecules to the plasmonic surfaces. Rather, the present approach makes it possible to mix every components and directly measure the resulting SERS signal, without requiring any intermediary step. So, the method is much preferably free of any separation or washing step. In that sense, the assay can be regarded as a one-step competitive assay. Moreover, the present approach is compatible with multiplexing and is not limited to small analyte molecules.

[0013] Different competition mechanisms can be contemplated. In a first class of embodiments, the third molecules can bind to the first molecules but cannot bind to the second molecules, whereby the second molecules and the third molecules compete to bind to the first molecules upon bringing the second molecules and the third molecules into contact with the nanoscale plasmonic surfaces. In a second class of embodiments, the third molecules can bind to the second molecules but not to the first molecules, whereby the second molecules can no longer bind to the first molecules once bounded by the third molecules.

[0014] In preferred embodiments, the dual set consists of, on the one hand, aptamers of singlestranded DNA, single-stranded RNA, or single-stranded XNA, and, on the other hand, molecule fragments of polynucleotides, wherein said fragments are complementary to the aptamers. Such a set enables the detection of small molecules, as well as larger molecules, such as proteins and DNA fragments. As a result, the proposed method is flexible and allows the implementation of complex diagnostic panels. Preferably, the aptamers are aptamers of single-stranded DNA, while the molecule fragments are complementary DNA fragments. A wide range of such molecules are commercially available. In embodiments, the nanoscale plasmonic surfaces are functionalized with aptamers such as evoked above, in which case the complementary fragments (in solution) are labelled with Raman reporter molecules. Conversely, the nanoscale plasmonic surfaces may be functionalized with polynucleotide fragments, while aptamers (in solution) are labelled with Raman reporter molecules.

[0015] In embodiments, the second molecules are labelled with the Raman reporter molecules in such a manner as to ensure, together with the first molecules that functionalize the nanoscale plasmonic surfaces, that an average distance between the Raman reporter molecules and said nanoscale plasmonic surfaces is between 0.1 nm and 20 nm, preferably between 0.1 nm and 10 nm, and more preferably between 0.1 and 5 nm. The interplay between the first and second molecules makes it possible to adjust the distance between the Raman reporter molecules. The smaller the average distance, the higher the Raman signal intensity.

[0016] In embodiments, the nanoscale plasmonic surfaces are surfaces of nanostructures or nanoparticles, and said nanostructures or nanoparticles comprise gold, which may possibly be passivated, oxidized, or coated, e.g., with silica or a polymer layer. This can be useful to mitigate contamination or prevent aggregation, although this also depends on the intended application. In particular, the nanoscale plasmonic surfaces can be surfaces of nanoparticles. In this case, the second molecules and the third molecules are brought into contact with the nanoscale plasmonic surfaces by mixing liquid solutions of the nanoparticles, the second molecules, and the third molecules, for the nanoparticles to form a colloidal dispersion in a resulting liquid solution. The functionalized nanoparticles can initially be provided as colloidal dispersion, which makes it easier to mix the various liquid solutions and provides greater control over the timing of the reactions. Also, this eliminates the need to have a device with nanostructures. Note, the term "colloidal dispersion" is to be understood in a broad sense, inasmuch as the nanoparticles may form colloids, sols, a dispersion, or a suspension.

[0017] A trade-off can be found between the average size of the nanoparticles (and therefore the average number of aptamers per nanoparticle) and the resulting Raman signal intensity. In that respect, the number of the aptamers that functionalize each of the nanoparticles will preferably be, on average, of between 100 to 10000.

[0018] In embodiments, bringing the second molecules and the third molecules into contact with the nanoparticles comprises pre-mixing liquid solutions of the nanoparticles and the third molecules to obtain a first liquid solution, prior to mixing a second solution of the second molecules with the first liquid solution. This favors initial interactions between the first molecules and the third molecules, hence giving the analyte a head start, something that can be useful to compensate for potential mismatches between the binding affinities. That said, one will generally try to match the binding affinities, to enable a fair competition and ease the subsequent quantification. In this respect, in embodiments, the binding affinity between the second molecules and the first molecules is equal to a binding affinity between the third molecules and the first molecules, subject to ± 10 % of an average binding affinity between the first molecules and each of the second molecules and the third molecules. The above definition assumes that the first molecules are molecules of a single type, the second molecules are molecules of a single type, and the third molecules are molecules of a single type. This, however, is not necessarily the case.

[0019] Indeed, in embodiments, the nanoscale plasmonic surfaces are functionalized with first molecules of multiple types, and the second molecules include single strands of complementary polynucleotides that can bind to each of the multiple types of the first molecules. The second molecules may notably include molecules of multiple types, where the multiple types of the second molecules can respectively bind to the multiple types of the first molecules. There are several reasons for wanting to do this, including to calibrate the assay, normalize the Raman signals, and / or to allow multiplexing.

[0020] In embodiments, the third molecules are molecules of a single type. Still, the multiple types of the first molecules have different binding affinities to the third molecules, which allows a broader dynamic range to be achieved.

[0021] In embodiments, the third molecules include analyte molecules of multiple types. The multiple types of the first molecules have respective binding affinities with the multiple types of the third molecules. The second molecules are molecules of multiple types, which are labelled with respective Raman reporter molecules. The multiple types of the second molecules have binding affinities with the multiple types of the first molecules that respectively match binding affinities of the multiple types of the third molecules with the multiple types of the first molecules, e.g., subject to ± 10% of the average binding affinities of the pairs considered. The method is performed as a multiplexed competitive assay for detecting properties of each of the multiple types of the third molecules, by measuring current Raman signals as primarily caused by the respective Raman reporter molecules labelling the second molecules that effectively bind to the first molecules.

[0022] In embodiments, the second molecules of said multiple types are labelled with said respective Raman reporter molecules in such a manner as to ensure, together with the first molecules of the multiple types, distinct average distances between the Raman reporter molecules and said nanoscale plasmonic surfaces. This makes it possible to compensate for potential differences in the Raman signal intensities of the various Raman reporter molecules involved. In embodiments, the nanoparticles include multiple groups of nanoparticles, and nanoparticles in each of the groups are functionalized with a respective one of the multiple types of the first molecules. I.e., instead of functionalizing each nanoparticles with different types of the first molecules, different groups of nanoparticles are provided, which are functionalized with respective types of the first molecules. This makes it easier to set up the assay and also provides greater flexibility in the desired ratios of molecule types.

[0023] In embodiments, the method further comprises calibrating the assay by measuring said one or more reference Raman signals, as primarily caused by the Raman reporter molecules in absence, or in presence of known amounts, of the third molecules. The reference Raman signals can then be used to determine the property of interest.

[0024] In embodiments, the first molecules include two types of molecules. Each of the two types of first molecules is immobilized on at least some of the nanoscale plasmonic surfaces. In addition, each of the two types of first molecules has a binding affinity with the second molecules. However, only one of the two types of the first molecules has a binding affinity with the third molecules, while the other one of the two types of the first molecules has no binding affinity with the third molecules. For example, non-analyte-sensing DNA probes can be used for the purposes of internal calibration and / or assay signal normalization. The non-analyte-sensing DNA fragments can for instance be immobilized on the plasmonic surfaces, along with aptamers, and play the role of controls, which can serve for internal calibration. The hybridization of control molecules is not affected by the presence of analytes, and therefore the signal due to the corresponding reporter molecules can be used to calibrate the assay and / or normalize the Raman signals of interest.

[0025] According to another aspect, the invention is embodied as a system for performing a competitive assay using SERS, based on nanoscale plasmonic surfaces functionalized with first molecules. The system comprises a device, liquid supplies (which connect to the device), and a flow control system. The latter is operatively connected to the liquid supplies to bring second molecules and third molecules in a liquid solution into contact with the nanoscale plasmonic surfaces in the device. Consistently with the previous aspect of the invention, the first molecules and the second molecules form a dual set of complementary single strands of polynucleotides, whereby the second molecules can bind to the first molecules. The second molecules are labelled with Raman reporter molecules. The third molecules are analyte molecules that can bind either to the first molecules or to the second molecules, thereby competitively limiting the extent to which the second molecules can bind to the first molecules. The system further includes a Raman detector, which is configured in the system to measure a current Raman signal primarily caused by the Raman reporter molecules labelling the second molecules that effectively bind to the first molecules. Finally, the system includes a processing unit, which is configured to cause to determine a property of the third molecules by comparing the current measured Raman signal with one or more reference Raman signals. The device may notably include a set of one or more microfluidic chips, a microfluidic cartridge, and / or a microtiter plate.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] These and other objects, features and advantages of the present invention will become apparent from the following detailed description of illustrative embodiments thereof, which is to be read in connection with the accompanying drawings. The illustrations are for clarity in facilitating one skilled in the art in understanding the invention in conjunction with the detailed description. In the drawings:

[0028] FIG. 1 is a diagram schematically illustrating selected components of a system for performing a competitive assay, using surface-enhanced Raman spectroscopy (SERS) and functionalized nanoscale plasmonic surfaces, according to embodiments;

[0029] FIG. 2 is a diagram schematically illustrating selected components of a system similar to that of FIG. 1, where the system includes two connected microfluidic chips to gradually mix liquid solutions, as in embodiments;

[0030] FIG. 3 is a flowchart illustrating high-level steps of a method of calibrating and performing a competitive assay, with a view to determining a property of an analyte, according to embodiments;

[0031] FIG. 4 is a flowchart illustrating detailed steps of a method of performing a one-step, competitive assay, as in embodiments;

[0032] FIGS. 5A - 11 are diagrams schematically illustrating nanoscale plasmonic surfaces functionalized with first molecules (single strands of polynucleotides), which interact with second molecules (complementary single strands of polynucleotides, labelled with Raman reporter molecules) and third molecules (i.e., analyte molecules), where the third molecules that can bind either to the first molecules or to the second molecules. This gives rise to a competition that limits the extent to which the second molecules can bind to the first molecules immobilized on the plasmonic surfaces, according to embodiments. Each series of diagrams (e.g., FIGS. 5A and 5B) is accompanied by separately numbered legend (e.g., FIG. 5C is the legend of FIGS. 5A and 5B); and

[0033] FIGS. 12A to 14B show experimental Raman signals obtained in experiences carried out by the inventors for the purpose of demonstrating the viability of the present approach.

[0034] The accompanying drawings show simplified representations of devices or parts thereof, as involved in embodiments. Technical features depicted in the drawings are not necessarily to scale. Similar or functionally similar elements in the figures have been allocated the same numeral references, unless otherwise indicated.

[0035] Systems and methods embodying the present invention will now be described, by way of non-limiting examples.

[0036] DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0037] Referring to the flowcharts of FIGS. 3 and 4, a first aspect of the invention concerns a method of performing a competitive assay. This method and its variants are collectively referred to as the "present methods" in this documents. The present methods can notably be performed using systems 1 such as depicted in FIGS. 1 and 2. Such systems concern another aspect of the invention, which is described later in detail. All references Sn refer to methods steps of the flowcharts of FIGS. 3 and 4, while numeral references (and occasionally letters) pertain to devices, components, and other concepts, as involved in embodiments of the present invention.

[0038] The method relies on surface-enhanced Raman spectroscopy (SERS) and requires nanoscale plasmonic surfaces, also referred to as "plasmonic surfaces" herein, for short. The plasmonic surfaces can be surfaces of nanoparticles P, such as nanorods, or nanopatterned structures, such as flat elements. In all cases, the plasmonic surfaces are surfaces of nanoscale elements that have structures in the nanoscale range and can be used for SERS. Such elements are preferably made of metal, although this is not a strict requirement. For instance, graphene can be contemplated, too. The nanoscale elements can contain metal, whether in bulk or surface. Nanoparticles may for instance be passivated, or coated, e.g., with silica. The metal surface can be purposely oxidized, if necessary, or coated with an organic polymer. Nanoscale plasmonic surfaces are known per se.

[0039] The present method essentially includes three steps, which aim at: (i) bringing different molecules into contact, for them to interact; (ii) measuring one or more Raman signals; and (iii) determining a property of the analyte molecules. Three categories of molecules are involved, which are referred to as "first molecules", "second molecules", and "third molecules", or as 1stmolecules, 2ndmolecules, and 3rdmolecules, in this document.

[0040] In detail, the 1stmolecules 100 - 110 functionalize nanoscale plasmonic surfaces P, Pl - P3, see FIGS. 5A - 11. That is, the molecules 100 - 110 are immobilized on the plasmonic surfaces, as known per se. As also illustrated in FIGS. 5A - 11, the 2ndmolecules 200 - 210 are labelled with Raman reporter molecules 250 - 260. The 1stmolecules 100 - 110 and the 2ndmolecules 200 - 210 form a dual set of complementary single strands of polynucleotides. As a result, the 2ndmolecules can bind to the 1stmolecules. The 3rdmolecules 300 - 307 are analyte molecules. The goal of the assay is to determine a property (e.g., a mass or a number concentration, or an amount) of the analyte molecules.

[0041] The method comprises a step of bringing the 2ndmolecules and the 3rdmolecules into contact with the nanoscale plasmonic surfaces, thanks to a liquid solution, see steps S31 - S33 in FIGS. 3 and 4. When they come into contact with the plasmonic surfaces, the 2ndand 3rdmolecules are in the liquid solution. The plasmonic surfaces may for instance be surfaces of nanoparticles that form part (e.g., as a colloidal dispersion) of this liquid solution, too. In variants, the plasmonic surfaces are surfaces of nanostructures, which are patterned on a surface (e.g., of a microchannel or a microplate well). In that case, the nanostructures are wetted by the liquid solution containing the 2ndand 3rdmolecules, which gives rise to the desired interactions.

[0042] In this respect, the 1st, 2nd, and 3rdmolecules are assumed to be such that the 3rdmolecules can bind either to the 1stmolecules or to the 2ndmolecules. That is, the propensity of the 3rdmolecules to bind to one of the two categories of molecules (the 1stand 2ndmolecules) is negligible in comparison to its the propensity to bind to the other one of the two categories. As a result, either the 3rdmolecule can appreciably bind to the 1stmolecules (in which case they do not appreciably bind the 2ndmolecules), or the 3rdmolecules can appreciably bind to the 2ndmolecules, in which case they do not appreciably bind to the 1stmolecules. In other words, the 3rdmolecules have a negligible binding affinity with only one of the two categories of molecules. This constraint results in a competition that limits the extent to which the 2ndmolecules can bind to the 1stmolecules. This point is further discussed later in detail and exemplified.

[0043] So, the 1stmolecules refer to those molecules that functionalize the nanoscale plasmonic surfaces. These molecules may be in the liquid solution (e.g., nanoparticles), or not (e.g., nanopatterned surfaces). The 2ndmolecules refer to complementary molecules in the liquid solution, which can bind to the 1stmolecules. And the 3rdmolecules are analyte molecules in the liquid solution, which can bind to one or the other category of molecules, such that the 3rdmolecules compete with the 2ndmolecules, in one way or another.

[0044] Next, the method comprises measuring S34, S35 a current Raman signal, i.e., a signal that is primarily caused by those Raman reporter molecules that label the 2ndmolecules that effectively bind to the 1stmolecules immobilized on the plasmonic surfaces. Note, this implies that the liquid solution is still in contact with the nanoscale plasmonic surfaces at the time of the measurement. Finally, the method a step of determining S40 a property of the 3rdmolecules, i.e., the quantification. This is achieved by comparing the current measured Raman signal with one or more reference Raman signals. Such reference signals can be obtained in accordance with different calibration and / normalization scenarios, as discussed later in detail.

[0045] The proposed method allows a one-step competitive assay to be performed, where the distance between Raman reporter molecules (which label the 2ndmolecules) and the plasmonic surfaces of the nanoscale elements can be controlled through the 1stand 2ndmolecules. The 3rdmolecules (i.e., the analyte molecules) compete with the 2ndmolecules, because of one of the following two scenarios:

[0046] (i) The 3rdmolecules 300 - 305 can only bind to the 1stmolecules 100 - 105, as illustrated in FIGS. 5A - 9D. And when the 3rdmolecules 300 - 305 bind to the 1stmolecules 100 - 105, the 2ndmolecules 200 - 205 (which carry the Raman reporter molecules 250 - 255) can no longer bind to the 1stmolecules 100 - 105 on the plasmonic surfaces. In other words, the 3rdmolecules can bind to the 1stmolecules but cannot bind to the 2ndmolecules, such that the 2ndmolecules and the 3rdmolecules compete to bind to the 1stmolecules when coming into contact with the plasmonic surfaces;

[0047] (ii) The 3rdmolecules 307 can only bind to the 2ndmolecules 207 (which carry the Raman reporter molecules 257). When they do so, the 2ndmolecules 207 can no longer bind to the 1stmolecules 107. In other words, the 3rdmolecules can bind to the 2ndmolecules but not to the 1stmolecules, and the 2ndmolecules can no longer bind to the 1stmolecules once bounded by the 3rdmolecules.

[0048] Each of the above scenarios results in competitively limiting the extent to which the 2ndmolecules can bind to the 1stmolecules. And in each case, the molecules in excess (whether the 2ndor 3rdmolecules) remain in the liquid solution, away from the 1stmolecules on the plasmonic surfaces.

[0049] The dual set formed by the 1stand 2ndmolecules may for instance consist, on the one hand, of aptamers of single-stranded DNA, RNA, or XNA, and, on the other hand, of molecule fragments of polynucleotides, which are complementary to the aptamers. Xeno nucleic acids XNA are synthetic nucleic acid analogues, the backbone of which differs from the ribose and deoxyribose found in the nucleic acids of naturally occurring RNA and DNA. Preferred is to use aptamers of single-stranded DNA, while the molecule fragments are complementary DNA fragments.

[0050] For example, the nanoscale plasmonic surfaces may be functionalized with DNA aptamers. That is, DNA aptamers can be used as receptors on the nanoscale elements, while the reporter molecules are located on a complementary DNA (cDNA) fragment, as assumed in FIGS. 5A - 9D. The cDNA fragments can bind to the DNA aptamers, like the analyte molecules. However, and as noted earlier, converse configurations can be contemplated, too, as illustrated in FIGS. 10A - 10B. In that case, cDNA fragments 107 are immobilized on the plasmonic surfaces, while the aptamers 207 are in the liquid solution and carry the Raman reporter molecules 257.

[0051] In all cases, the 1stmolecules, which are immobilized on the plasmonic surfaces, can be used to control the distance of the Raman reporter molecules to the plasmonic surfaces, something that makes it possible to tune the assay sensitivity. More precisely, the distance of the Raman reporter molecules to the plasmonic surfaces is determined by the types of 1stand 2ndmolecules considered, the location of the Raman reporter molecules on the 2ndmolecules, and the interaction between the 1stand 2ndmolecules. Also, when designing the assay, the affinity of the 1stmolecules (e.g., aptamers) to its analyte (the 3rdmolecules) can be optimized for tuning the sensitivity range.

[0052] By definition, the 2ndand 3rdmolecules are always in a liquid solution when contacting the nanoscale plasmonic surfaces. The fact that the 3rdmolecules (analyte) can bind to either the 1stmolecules (only) or the 2ndmolecules (only) results in a competition, which limits (i.e., restricts) the extent to which the 2ndmolecules can bind to the 1stmolecules on the plasmonic surfaces. Any molecule in excess remains in the liquid solution; the liquid solution remains in contact with the plasmonic surfaces when measuring the Raman signal. This feature of the present approach is what makes it possible to get rid of the usual intermediary steps.

[0053] Indeed, the present approach does not require any separation step (centrifugation, washing, etc.) to remove the unbound 2ndmolecules (e.g., cDNA fragments). The reason is that the latter do not produce a signal when they are in solution, i.e., away from the plasmonic surfaces, because the SERS signal intensity is highly dependent on the distance of the Raman reporter molecules to the plasmonic surfaces. Rather, the present approach makes it possible to mix every components and directly measure the resulting SERS signal, without requiring any intermediary step. In that sense, the assay can be considered a one- step assay.

[0054] The Raman reporter molecules (also called Raman tags, or Raman reporters) will preferably have a large Raman cross section, to favor detection. Such molecules may notably include Nile blue, methylene blue, as well as other chromophores, or fluorophore molecules, such as Rhodamine B. Other organic molecules could be used as suitable Raman reporters, such as 4-mercaptobenzonitrile and 4-mercaptobenzoic acid. Such Raman reporter molecules have a distinct Raman scattering fingerprint, which permits to measure a loss of Raman signal (step S40) when analytes (3rdmolecules) compete with the 2ndmolecules. For example, in absence of the analytes (see, e.g., FIG. 5A), the cDNA fragments 200 can bind to all of their counterpart aptamers 100 on the nanoparticles P and bring the reporter molecules 250 in the vicinity of the plasmonic surface P, yielding a high-intensity SERS signal, step S35. However, when the 3rdmolecules 300 are present (FIG. 5B), they bind to the aptamers 100 and thus "occupy" the aptamers. Now, this prevents the cDNA fragments 200 from binding with the aptamers 100, thereby lowering the SERS signal intensity. Depending on the binding affinities involved, the 3rdmolecules 300 may even expel the 2ndmolecules 200 from the aptamers 100. Comparing (step S40) the two signals allows information as to the 3rdmolecules to be retrieved, such as an amount, a number concentration, or a mass concentration of such molecules.

[0055] In general, one may want to match the binding affinity between the 1stmolecules (e.g., aptamers) and the 2ndmolecules (e.g., cDNA) with the binding affinity between the 3rdmolecules and the 1stmolecules for best performance. That is, in embodiments, the binding affinity between the 1stand 2ndmolecules is equal to the binding affinity between the 1stand 3rdmolecules, subject to ±10 % of the average binding affinity between the two pairs of categories considered. The exact binding affinity values depends on the desired dynamic range of the assay. Note, the above characterization of the binding affinity match assumes that each category of molecules (i.e., the 1st, 2nd, and 3rdmolecules) include molecules of the same type. This, however, is not a strict requirement, as further discussed later.

[0056] The binding affinity reflects the strength of the binding interaction between a single biomolecule (e.g., a DNA aptamer) and its binding partner (e.g., cDNA or analyte molecule). The binding affinity is typically measured as an equilibrium dissociation constant, noted KD, which has molar units. There are several methods to measure the binding affinity, for example methods based on surface plasmon resonance or methods based on electrochemistry.

[0057] In preferred embodiments, the 2ndmolecules are labelled with the Raman reporter molecules in such a manner as to ensure, together with the 1stmolecules on the plasmonic surfaces P, an adequate average distance between the Raman reporter molecules and the plasmonic surfaces P. As said, the location of the Raman reporter molecules on the 2ndmolecules, the types of, and the interaction between, the 1stand 2ndmolecules, determine the distances between the Raman reporter molecules and the plasmonic surfaces. In general, the molecules will be designed (or selected) so as to give rise to an average distance that is preferably between 0.1 nm and 20 nm. More preferably, this average distance will be between 0.1 nm and 10 nm, or, even, between 0.1 and 5 nm, to increase the intensity of the SERS signals. For instance, different combinations of 1stand 2ndmolecules may give rise to difference distances between the Raman reporter molecules and the plasmonic surfaces P3, as illustrated in FIG. 11, something that can be exploited to compensate for weaker reporter signals, as discussed later in respect of particular embodiments. As indicated earlier, the plasmonic surfaces can be surfaces of patterned nanostructures or surfaces of nanoparticles P. In general, use can be made of SERS surfaces that are made from gold (Au), silver (Ag), or another metal. In preferred embodiments, the nanoscale elements are made of gold. However, such metals are prone to contamination during fabrication, storage, and / or exposure to solutions and samples. Contamination of a surface is hard to predict as some contaminants may replace others over time. Surface contamination can negatively impact the sensitivity and reproducibility of SERS measurements due to the strong distance dependence between the analyte and SERS surfaces. Thus, the plasmonic surfaces are preferably oxidized or coated with, e.g., silica or polymers. This will further help prevent nanoparticle aggregation and keep the colloidal dispersion stable.

[0058] Practical applications of the present approach will often use nanoparticles, which can for instance be nanorods, as assumed in the accompanying drawings. The 2ndand 3rdmolecules can be brought S31 - S33 into contact with the nanoparticles by mixing S32 - S33 liquid solutions of the nanoparticles P, the 2ndmolecules, and the 3rdmolecules. In that case, the nanoparticles form a colloidal suspension in the resulting liquid solution. The nanoparticles P can be dimensioned to enhance the detection by the Raman detector for a specific wavelength of electromagnetic radiation. Typically, such particles can have diameters ranging from 20 nm to 100 nm. They can be spherical or elongated. Typical length-to-width ratios are of about 1 :3 up to 1:5 (width:length). Such dimensions give rise to a SERS enhancement. Meanwhile, such nanoparticles can harbor large amounts of aptamers. For instance, in embodiments, the number of aptamers that functionalize each nanoparticle P is, on average, of between 100 to 10000. Suitable nanoparticles are commercially available (in various sizes), including particles that are already functionalized, e.g., from Nanopartz™ (https: / / www.nanopartz.com / ). So are single strands of polynucleotides, including labelled polynucleotides, which can be purchased from, e.g., biomers.net (registered trademark, https: / / biorners.net / ). Nanoparticles can also be functionalized using any suitable method, whether commercially available or not.

[0059] One may possibly mix respective liquid solutions 41, 42, 43 of the nanoparticles P, the 2ndmolecules, and the 3rdmolecules, to bring the 2ndmolecules and the 3rdmolecules into contact with the nanoparticles P, as suggested by the setup shown in FIG. 1. One may further pre-mix some of the liquid solutions, e.g., using cascaded microfluidic chips 10, 20, as shown in FIG. 2, to compensate for discrepancies in the binding affinities, if necessary. For example, one may pre-mix S323 liquid solutions 41, 42 of the nanoparticles P and the 3rdmolecules (analyte) to obtain a first liquid solution, prior to mixing S33 a second solution 43 of the 2ndmolecules (e.g., cDNA) with the first liquid solution. In this example, the goal is to favor interactions between the 1stmolecules and the 3rdmolecules, hence giving the analyte a head start.

[0060] So far, the description implicitly assumes that each category of molecules (i.e., the 1st, 2nd, and 3rdmolecules) includes molecules of the same type. That is, the above embodiments refer to one type of 1stmolecules, one type of 2ndmolecules, and one type of 3rdmolecules, which interact at a time. For example, in FIGS. 5A and 5B, the nanoparticles P (e.g., gold nanorods) harbor a single type of 1stmolecules 100 (e.g., DNA aptamers), which interact with a single type of 2ndmolecules 200 (e.g., cDNA fragments labelled with reporter molecules 250, such as Nile blue, methylene blue, or fluorophore molecules) and a single type of 3rdmolecules 300 (e.g., small organic molecules, DNA fragments, proteins). The 2ndand 3rdmolecules compete to bind to the 1stmolecules, hence giving rise to a loss of SERS signal intensity, at least in a given region of the Raman spectrum. That is, in absence of analyte 300 (FIG. 5A), the 2ndmolecules 200 can potentially bind to all the available aptamers 100. Conversely, when analyte molecules 300 are present (FIG. 5B), these molecules 300 can bind to the aptamers 100, instead of the cDNA fragments. As fewer Raman reporter molecules 250 are now present in the vicinity of the plasmonic surfaces P, the corresponding Raman signal drops in intensity. Application examples are discussed in section 2.

[0061] However, the 1st, 2nd, and 3rdmolecules should more generally be regarded as categories of molecules that may include, each, one or several types of molecules. For instance, the nanoscale plasmonic surfaces can be functionalized with 1stmolecules of multiple types, see, e.g., numeral references 101 - 106, 108 - 110, in FIGS. 6A - 9D, and 11. Accordingly, the 2ndmolecules may include single strands of complementary polynucleotides that can bind to each of the multiple types of the 1stmolecules. In particular, the 2ndmolecules may include several types of molecules (as denoted by the numeral references 201 - 206, 208 - 210, in FIGS. 6A - 9D, and 11), where the multiple types of second molecules may respectively bind to the multiple types of first molecules, as assumed in FIGS. 6A - 9D, and 11.

[0062] Various scenarios can be contemplated. In the example of FIGS. 7A - 7C, the 3rdmolecules are molecules 303 of a single type, while two different types of 1stmolecules 103, 104 and two different types of 2ndmolecules 203, 204 are involved. Still, the two different types 103, 104 of 1stmolecules have different binding affinities to the 3rdmolecules 303, which allows a broader dynamic range to be achieved. For example, molecules 103 (with high affinity) can capture low concentrations of molecules 303 to generate a detectable signal. However, this approach would suffer from signal saturation when the concentration of molecules 303 increases. Still, molecules 104 (with low affinity) can start capturing 303 at higher concentrations, generating a detectable signal for the higher concentration ranges when molecules 103 are fully occupied. Such a scenario can for example be exploited in an assay that includes a selection of aptamers having different affinities to a known target molecule.

[0063] The example of FIGS. 8A - 8D involves 3rdmolecules 305 of a single type, together with two different types of 1stmolecules 105, 106 and two different types of 2ndmolecules 205, 206. In this example, the molecule 105 is a DNA aptamer, while the molecule 106 is a non-analyte-sensing DNA probe. That is, only the aptamer 105 can bind to the analyte 305, whereas the DNA probe 106 cannot. Meanwhile, the 2ndmolecules (cDNA fragments in this example) 205, 206 can respectively bind to the molecules 105, 106. For completeness, the cDNA fragments 205, 206 are labelled with distinct Raman reporter molecules 255, 256.

[0064] The non-analyte-sensing DNA probe 106 is used for purposes internal calibration and assay signal normalization. FIGS. 8A and 8B illustrate the behavior of the nanoparticle in poor conditions. In absence of analyte (FIG. 8A), the cDNA fragments 205, 206 bind to a few complementary single strands 105, 106, respectively, while some of the immobilized molecules 105, 106 remain unoccupied. In the presence of analyte 305 (FIG. 8B), however, some of the aptamers 105 may bind to analyte molecules 305. FIGS. 8C and 8D illustrate the behavior of the nanoparticle P in good conditions. In absence of analyte (FIG. 8C), the cDNA fragments 205, 206 respectively bind to most of the complementary molecules 105, 106, whereby most, if not all, of the immobilized molecules 105, 106 are occupied. In the presence of analyte 305 (FIG. 8D), such molecules 305 compete with the cDNA fragments 205 and may even substitute to the cDNA fragments 205 to bind to the aptamers 105.

[0065] Thus, FIGS. 8A - 8D illustrates a scenario, in which the 1stmolecules include two types 105, 106 of molecules, each being immobilized on the nanoscale plasmonic surfaces P, where each of the two types 105, 106 of molecules has a binding affinity with the 2ndmolecules. However, only one 105 of the two types of molecules 105, 106 has a binding affinity with the 3rdmolecules 305, while the other one 106 has no binding affinity with the 3rdmolecules 305.

[0066] This makes it possible to calibrate and / or normalize the assay, thanks to the signals obtained through the hybridization of the molecules 106 and 206 and the corresponding Raman reporter molecules 256. That is, the loss of Raman signal corresponding to the Raman reporter 255 (hybridization of the molecules 105 and 205) can be compared with the signal due to the Raman reporter molecules 256, hence a possible calibration or normalization. The property of the 3rdmolecules can for example be determined S40 by comparing the Raman signal due to the Raman reporter molecules 255 with the Raman signal due to the Raman reporter molecules 256. In each case, the signal measured is due to a given type of 2ndmolecules that effectively bind to the corresponding type of 1stmolecules.

[0067] More generally, the assay may include several types of non-analyte-sensing DNA fragments for internal calibration and assay signal normalization. The non-analyte-sensing DNA fragments are immobilized on the plasmonic surfaces along with aptamers and play the role of controls, which can serve for internal calibration. The hybridization of control molecules is not affected by the presence of analytes, and therefore the signal due to the corresponding reporter molecules can be used to calibrate the assay.

[0068] A different normalization scenario is one in which the property sought is determined S40 by comparing the Raman signal due to the Raman reporter molecules 250 with a previous Raman signal, i.e., the signal that is primarily caused by the Raman reporter molecules 250 labelling those 2ndmolecules 200 that have bounded to the 1stmolecules 100 in absence of the 3rdmolecules, as illustrated in FIGS. 5A and 5B. In both cases, however, the reference Raman signals are obtained in absence of competition between the 3rdmolecules (analyte) and (one type of) the 2ndmolecules. More generally, the assay can be calibrated S20 by measuring one or more reference Raman signals, as primarily caused by Raman reporter molecules in absence, or in presence of known amounts, of the 3rdmolecules.

[0069] The examples shown in FIGS. 5A, 5B, 7A - 7C, and 8A - 8D, involve a single type of 3rdmolecules (analyte). In other embodiments, the 3rdmolecules include analyte molecules of multiple types, as illustrated in FIGS. 6A - 6B, and 9A - 9D. The goal is to achieve a multiplexed assay, where several analyte molecules are concurrently analyzed. To this aim, the relevant Raman signals may have to be compared with carefully chosen reference signals.

[0070] The multiple types 101, 102 of 1stmolecules will normally have respective (i.e., distinct) binding affinities with the multiple types 301, 302 of the 3rdmolecules involved. Meanwhile, the 2ndmolecules are molecules of multiple types 201, 202, too, which are labelled with respective (i.e., distinct) Raman reporter molecules 251, 252. And the molecules 201, 202 have respective (i.e., distinct) binding affinities with the multiple types of 1stmolecules 101, 102. Now, such affinities will preferably match the binding affinities of the 3rdmolecules 301, 302 with the 1stmolecules 101, 102, to enable a fair competition and ease the subsequent quantification. Again, the binding affinities may match subject to ± 10 % of the average binding affinities, considered two-by-two. This way, the method can be performed as a multiplexed, competitive assay for detecting properties of each of the multiple types 301, 302 of the 3rdmolecules. This is achieved by measuring S34, S35 current Raman signals as primarily caused by the Raman reporter molecules 251, 252 of the 2ndmolecules 201, 202 that effectively bind to the 1stmolecules 101, 102. Again, such signals may be compared to previous Raman signals, as obtained in absence of competition. Alternatively, one may rely on non-analyte-sensing molecules, as explained earlier.

[0071] FIGS. 6A, 6B and 9A - 9D illustrate similar scenarios. The main difference is that, in FIGS. 6A, 6B, different types of 1stmolecules 101, 102 are immobilized on same nanoparticles P, whereas the different types of 1stmolecules 101, 102 are immobilized on respective nanoparticles Pl, P2 in FIGS. 9A - 9D. That is, the nanoparticles Pl, P2 include multiple groups of nanoparticles Pl, P2, where the nanoparticles in each group Pl, P2 are functionalized with respective types of 1stmolecules 101, 102. For example, each group of nanoparticles may harbor a respective type of aptamer. On the contrary, each nanoparticle P harbors several types of aptamers in the example of FIGS. 6A, 6B. In both cases, however, the assay includes a selection of aptamers having affinities to different target molecules for a multiplexed detection of analytes.

[0072] In the example of FIGS. 10A, 10B, cDNA fragments 107 are immobilized on the plasmonic surfaces P, in lieu of aptamers. That is, the nanoparticles are functionalized with cDNA fragments, while aptamers 207 play the role of the 2ndmolecules, which are in the liquid solution that comes into contact with the plasmonic surfaces. Accordingly, the aptamers 207 are labelled with the Raman reporter molecules 257. The cDNA fragments 107 can bind to the DNA aptamers 207 (FIG. 10A), but not to the analyte molecules 307. However, when the analyte molecules 307 are present (FIG. 10B), they can bind to the aptamers 207 in solution. From this point on, the aptamers 207 can no longer bind to the cDNA molecules 107, hence a competition.

[0073] FIG. 11 illustrates how different types of pairs of 1stand 2ndmolecules can result in different average distances between the Raman reporter molecules 258, 259, 260 and the plasmonic surface P3. The latter is assumed to be the surface of a patterned nanostructure in this example. Namely, three types 108, 109, 110 of 1stmolecules are involved, which can respectively bind to three respective types 208, 209, 210 of 2ndmolecules. The latter are labelled with respective Raman reporter molecules 208, 209, 210. The distinct types of 1stand 2ndmolecules may have different sizes. In addition, the locations of the Raman reporter molecules on the 2ndmolecules can vary from one type to the other. As a result, the 1stand 2ndmolecule ensure that distinct average distances are obtained between the respective groups 258, 259, 260 of Raman reporter molecules and the nanoscale plasmonic surfaces P3. This makes it possible to adjust the signal strengths of the Raman reporter molecules. For example, some of the Raman signals may be too weak (e.g., reporter C), should the corresponding reporters all be at the same average distance. In such a case, one may prefer using a cDNA fragment that will hybridize an aptamer designed so as ensure a shorter average distance to the plasmonic surface P3. More generally, one may adapt the types of molecules in accordance with the Raman signal intensity to adjust the distances of the reporters, see, e.g., reporters A, B, and C, in FIG. 11.

[0074] Referring more specifically to FIGS. 1 and 2, another aspect of the invention is now described in detail, which concerns a system 1 for performing a competitive assay using SERS. Functional aspects of such a system have already been described, if only implicitly, in reference to the present methods. Accordingly, the system 1 is only briefly described in the following.

[0075] Consistently with the present methods, the system 1 is based on nanoscale plasmonic surfaces P, which are functionalized with 1stmolecules. As seen in FIG. 1, the system 1 includes a device 2 and liquid supplies 41 - 43, which connect to the device 2. The system 1 further includes a flow control system 30, 35, which is operatively connected to the liquid supplies 41 - 43 to bring the 2ndand 3rdmolecules into contact with the nanoscale plasmonic surfaces P in the device 2. Irrespective of the number of liquid solutions initially involved (three in the examples of FIGS. 1 and 2), the 2ndand 3rdmolecules will, at some point, reside in a same liquid solution, which either includes the nanoscale plasmonic surfaces (e.g., nanoparticles in suspension) or comes to wet surfaces of nanostructures, as previously explained.

[0076] The liquid supplies 41 - 43 contain one or more liquid solutions of the 2ndand 3rdmolecules. The 2ndand 3rdmolecules may initially form part of distinct liquid solutions 41, 43, as assumed in FIG. 1. The liquid supplies may further include a solution 42 of functionalized nanoparticles P, as noted earlier.

[0077] As before, the 2ndmolecules are labelled with Raman reporter molecules. The 1stmolecules and the 2ndmolecules form a dual set of complementary single strands of polynucleotides. I.e., the 2ndmolecules can bind to the 1stmolecules. The 3rdmolecules are analyte molecules that can bind either to the 1stmolecules or to the 2ndmolecules. Thus, when a liquid solution containing the 2ndand 3rdmolecule comes into contact with the plasmonic surfaces, a competition occurs, which competitively limits the extent to which the 2ndmolecules can bind to the 1stmolecules.

[0078] The system 1 further includes a Raman detector 71, 72, which is configured in the system 1 to measure a current Raman signal, as primarily caused by the Raman reporter molecules labelling the 2ndmolecules that effectively bind to the 1stmolecules, in operation.

[0079] Finally, the system 1 includes a processing unit 3, 4, which is configured to (cause to) determine a property of the 3rdmolecules by comparing the current measured Raman signal with one or more reference Raman signals, as previously discussed. This processing unit 3, 4 may include one or more computers (or other information processing units). In embodiments, the system 1 includes a local processing unit 4, which collects data from the detector 71, 72 and processes such data to determine this property. In variants, the system 1 includes a local processing unit 4 (e.g., a smartphone), which merely collects data from the detector 71, 72 and transmits the data to a remote server 3, for the latter to process the data and obtain the information. In both cases, the collected data may be stored remotely.

[0080] The device 2 is in fluidic communication with the liquid supplies, such that the liquid supplies 41 - 43 can bring liquid solutions of the 2ndmolecules and the 3rdmolecules into contact with nanoscale plasmonic surfaces in the device 2. The device 2 may for instance include a microtiter plate. If necessary, distinct combinations of liquids may possibly be brought to respective wells of the microtiter plate, to allow several reactions in parallel. In variants, the device includes one or more microfluidic chips 10, 20.

[0081] For example, as seen in FIG. 2, the device may include two connected microfluidic chips 10, 20. As in the example of FIG. 1, the plasmonic surfaces are surfaces of functionalized nanoparticles P, which initially form a colloidal suspension. The liquid 42 is pre-mixed with the liquid 41 containing the analyte A, thanks to a first microfluidic chip 20, whose input channels 21, 22 join in a single channel 24 to allow the two liquids to mix, hence giving a head start to the analyte A. The resulting liquid then enters a second microfluidic chip 10, through an input channel 12, while the suspension of nanoparticles P enters through the input channel 11. The two input channels 11, 12 join in a main channel 14, where the liquids mix. Thus, all the liquid supplies 41 - 43 eventually connect to the main channel 14, which gives rise to a competition between the analyte A and the cDNA coming from liquid supply 43.

[0082] The main channel 14 leads to a detection area 18. The flow control system 30, 35 is operatively connected to the liquid supplies 41 - 43 to inject liquids from the liquid supplies into the main channel 14 and control flows of the injected liquids. This permits to achieve a laminar flow of the mixed solution in the main channel 14. In addition, various other microstructures (e.g., defining posts, vents, trenches, posts, mesas, electrodes, capillary pumps, loading / offloading areas, anti-wetting structures, flow resistors, etc.) and flow control elements (e.g., valves, flow sensors) may be present, which are not shown, for simplicity.

[0083] The microchannels 11, 12, 14, 21, 22, 24 can for instance be formed as grooves on one or more surfaces of a microfluidic chips or a microfluidic cartridge. That is, instead of cascading microfluidic chips, the channels may be defined on or in the same microfluidic device, designed as a microfluidic cartridge. The Raman spectrometer typically includes an electromagnetic radiation source 71 and a detector 72. The electromagnetic radiation source 71 is configured to irradiate a portion of the mixed solution at the level of a detection area 18. The detector 72 is configured to detect electromagnetic radiation emitted by the irradiated liquid portion at the level of the detection area 18. The recorded spectra are passed to the unit 4 and possibly processed remotely, at the unit 3.

[0084] The setups shown in FIGS. 1 and 2 can be used to perform methods as captured in the flowcharts of FIGS. 3 and 4. As seen in the flow of FIG. 3, the system 1 is set up at step S10 and the assay is calibrated at step S20, whereby one or more reference signals may be recorded, e.g., for known amounts (or concentrations) of analyte molecules and cDNA molecules. If necessary, a normalization step is performed at step S25, whereby a signal is recorded in absence of analyte (see, e.g., FIG. 5A). In variants, the normalization signal is acquired during the assay itself, which is performed at step S30, e.g., using non-analytesensing aptamers, see, e.g., FIGS. 8A - 8D. The data are collected by the unit 4. Once all necessary spectra have been recorded, properties of the analyte(s) are determined at step S40, e.g., at a remote processing unit 3.

[0085] The steps governing the assay are shown in FIG. 4. The assay starts at step S31. The various liquid solutions are injected and mixed at step S32. Namely, the solution of functionalized nanoparticles P is injected S321 concomitantly S322 with the solution containing analyte molecules A, in order to premix S323 the two solutions and give head start to the analyte. The solution of cDNA molecules is injected S324 in parallel. All liquid flows are continuously controlled, while injecting the liquids. A liquid solution containing the analyte, the cDNA molecules, and a dispersion of nanoparticles, is finally obtained at step S33. This solution may reach a detection area, where it is irradiated S34, and a Raman signal is concomitantly detected S35.

[0086] A specific band of the signal may then be selected for processing, with a view to determining the property(ies) of interest, as illustrated in FIGS. 12A - 14B.

[0087] The above embodiments have been succinctly described in reference to the accompanying drawings and may accommodate a number of variants.

[0088] 2. Examples of applications

[0089] Two experiments are reported below. The first experiment concerns the detection of gentamicin below [1 pM] in mock plasma following a competitive assay using commercial nanoparticles coated with an aptamer. In detail, commercial gold nanorods functionalized with gentamicin aptamers (AuNR-aptamer) are exposed to gentamicin for 20 min. Molecules of cDNA, labelled with Nile blue (cDNA-NB), are then added. A SERS spectrum is collected 2 min later, which is shown in FIG. 12A. FIG. 12B magnifies the dotted region of FIG. 12A. FIG. 12B shows that different concentrations of gentamicin can adequately be detected. I.e., the assay can be calibrated using the curves shown in FIG. 12B, to allow a subsequent experiment with an unknown concentration of gentamicin.

[0090] FIGS. 13A - 13D illustrate how an assay architecture according to embodiments can be used for specific detection towards gentamicin, not penicillin. Commercial AuNR-aptamer are exposed to gentamicin or penicillin for 20 min. Molecules of cDNA, labelled with Nile blue (cDNA-NB), are then added. In each case, a SERS spectrum is collected 2 min later, which is shown in FIG. 13A and 14A, respectively for 5 pM and 50 pM of added gentamicin and penicillin. FIGS. 13B and 14B magnify the dotted regions of FIGS. 13A and 14A, respectively. FIGS. 13B and 14B show that the assay generates a signal specific to gentamicin but not penicillin even at high concentrations of 5 pM and 50 pM

[0091] While the present invention has been described with reference to a limited number of embodiments, variants, and the accompanying drawings, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the present invention. In particular, a feature (devicelike or method-like) recited in a given embodiment, variant or shown in a drawing may be combined with or replace another feature in another embodiment, variant, or drawing, without departing from the scope of the present invention. Various combinations of the features described in respect of any of the above embodiments or variants may accordingly be contemplated, that remain within the scope of the appended claims. In addition, many minor modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from its scope. Therefore, it is intended that the present invention is not limited to the particular embodiments disclosed, but that the present invention will include all embodiments falling within the scope of the appended claims. In addition, many other variants than explicitly touched above can be contemplated. For example, the system 1 may include other types of devices 2 or additional microfluidic chips.

Claims

1. CLAIMSWHAT IS CLAIMED IS:

1. A method of performing a competitive assay using surface-enhanced Raman spectroscopy based on nanoscale plasmonic surfaces (P) functionalized with first molecules (100 - 110), wherein the method comprises: bringing (S31 - S33) second molecules (200 - 210) and third molecules (300 - 307) in a liquid solution into contact with the nanoscale plasmonic surfaces (P), wherein the first molecules and the second molecules form a dual set of complementary single strands of polynucleotides, whereby the second molecules can bind to the first molecules, the second molecules are labelled with Raman reporter molecules (250-260), and the third molecules are analyte molecules that can bind either to the first molecules or to the second molecules, thereby competitively limiting the extent to which the second molecules can bind to the first molecules; measuring (S34, S35) a current Raman signal primarily caused by the Raman reporter molecules labelling the second molecules that effectively bind to the first molecules; and determining (S40) a property of the third molecules by comparing the current measured Raman signal with one or more reference Raman signals.

2. The method according to claim 1, wherein the third molecules can bind to the first molecules but cannot bind to the second molecules, whereby the second molecules and the third molecules compete to bind to the first molecules upon bringing the second molecules and the third molecules into contact with the nanoscale plasmonic surfaces.

3. The method according to claim 1, wherein the third molecules can bind to the second molecules but not to the first molecules, whereby the second molecules cannot bind to the first molecules once bounded by the third molecules.

4. The method according to any one of claims 1 to 3, wherein said dual set consists of, on the one hand, aptamers of single-stranded DNA, single-stranded RNA, or singlestranded XNA, and,on the other hand, molecule fragments of polynucleotides, wherein said fragments are complementary to said aptamers.

5. The method according to claim 4, wherein said aptamers are aptamers of single-stranded DNA, and said molecule fragments are complementary DNA fragments.

6. The method according to claim 5, wherein the nanoscale plasmonic surfaces (P) are functionalized with said aptamers.

7. The method according to any one of claims 1 to 6, wherein the second molecules are labelled with the Raman reporter molecules in such a manner as to ensure, together with the first molecules that functionalize the nanoscale plasmonic surfaces (P), that an average distance between the Raman reporter molecules and said nanoscale plasmonic surfaces (P) is between 0.1 nm and 20 nm, preferably between 0.1 nm and 10 nm, and more preferably between 0.1 and 5 nm.

8. The method according to any one of claims 1 to 7, wherein the nanoscale plasmonic surfaces (P) are surfaces of nanostructures or nanoparticles (P), and said nanostructures or nanoparticles comprise gold, which is preferably oxidized, or coated with silica or with a polymer.

9. The method according to any one of claims 1 to 8, wherein the nanoscale plasmonic surfaces are surfaces of nanoparticles (P), and the second molecules and the third molecules are brought (S31 - S33) into contact with the nanoscale plasmonic surfaces (P) by mixing (S32 - S33) liquid solutions of the nanoparticles (P), the second molecules, and the third molecules, for the nanoparticles to form a colloidal suspension in a resulting liquid solution.

10. The method according to claim 9, wherein a number of the aptamers that functionalize each of the nanoparticles (P) is, on average, of between 100 to 10000.

11. The method according to claim 9 or 10, wherein bringing the second molecules and the third molecules into contact with the nanoparticles (P) comprisespre-mixing (S323) liquid solutions of the nanoparticles and the third molecules to obtain a first liquid solution, prior to mixing (S33) a second solution of the second molecules with the first liquid solution.

12. The method according to any one of claims 1 to 11, wherein the first molecules are molecules of a single type, the second molecules are molecules of a single type, and the third molecules are molecules of a single type, and a binding affinity between the second molecules and the first molecules is equal to a binding affinity between the third molecules and the first molecules, subject to ± 10 % of an average binding affinity between the first molecules and each of the second molecules and the third molecules.

13. The method according to any one of claims 1 to 11, wherein the nanoscale plasmonic surfaces (P) are functionalized with first molecules of multiple types, and the second molecules include single strands of complementary polynucleotides that can bind to each of the multiple types of the first molecules, the second molecules preferably including molecules of multiple types, where the multiple types of the second molecules can respectively bind to the multiple types of the first molecules.

14. The method according to claim 13, wherein the third molecules are molecules of a single type, and the multiple types of the first molecules have different binding affinities to the third molecules.

15. The method according to claim 13, wherein the third molecules include analyte molecules of multiple types, the multiple types of the first molecules have respective binding affinities with the multiple types of the third molecules, the second molecules are molecules of multiple types, which are labelled with respective Raman reporter molecules and have binding affinities with the multiple types of the first molecules that respectively match binding affinities of the multiple types of the third molecules with the multiple types of the first molecules, and the method is performed as a multiplexed, competitive assay for detecting properties of each of the multiple types of the third molecules, by measuring (S34, S35) current Raman signals as primarily caused by the respective Raman reporter molecules labelling the second molecules that effectively bind to the first molecules.

16. The method according to claim 15, wherein the second molecules of said multiple types are labelled with said respective Raman reporter molecules in such a manner as to ensure, together with the first molecules of the multiple types, distinct average distances between the Raman reporter molecules and said nanoscale plasmonic surfaces (P).

17. The method according to any one of claims 13 to 16, wherein the nanoparticles (P) include multiple groups of nanoparticles (P), and nanoparticles in each of the groups are functionalized with a respective one of the multiple types of the first molecules.

18. The method according to any one of claims 1 to 17, wherein the method further comprises calibrating (S20) the assay by measuring (S20) said one or more reference Raman signals, as primarily caused by the Raman reporter molecules in absence, or in presence of known amounts, of the third molecules.

19. The method according to any one of claims 1 to 18, wherein the first molecules include two types of molecules, each being immobilized on at least some of the nanoscale plasmonic surfaces, and each of the two types of the first molecules has a binding affinity with the second molecules but only one of the two types of the first molecules has a binding affinity with the third molecules, while the other one of the two types of the first molecules has no binding affinity with the third molecules.

20. The method according to any one of claims 1 to 19, wherein the method is free of any separation or washing step.

21. The method according to any one of claims 1 to 20, wherein said Raman reporter molecules include organic molecules, which preferably include one or each of Nile blue and methylene blue.

22. A system (1) for performing a competitive assay using surface-enhanced Raman spectroscopy based on nanoscale plasmonic surfaces (P) functionalized with first molecules (100 - 110), wherein the system comprises: a device (2, 10, 20); liquid supplies (41 - 43), which connect to the device (2, 10, 20); a flow control system (30, 35), which is operatively connected to the liquid supplies (41 - 43) to bring second molecules (200- 210) and third molecules (300- 307) in a liquid solution into contact with the nanoscale plasmonic surfaces (P) in the device (2), whereinthe first molecules (100 - 110) and the second molecules (200- 210) form a dual set of complementary single strands of polynucleotides, whereby the second molecules can bind to the first molecules, the second molecules are labelled with Raman reporter molecules (250 - 260), and the third molecules (300 - 307) are analyte molecules that can bind either to the first molecules or to the second molecules, thereby competitively limiting the extent to which the second molecules can bind to the first molecules; a Raman detector (71, 72), which is configured in the system to measure a current Raman signal primarily caused by the Raman reporter molecules labelling the second molecules that effectively bind to the first molecules; and a processing unit (3, 4), which is configured to cause to determine a property of the third molecules by comparing the current measured Raman signal with one or more reference Raman signals.

23. The system (1) according to claim 22, wherein the device (2, 10, 20) includes one or more of: a set of one or more microfluidic chips (10, 20), a microfluidic cartridge, and a microtiter plate.

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