Kit and method for detecting target nucleic acid

Abstract

The invention also relates to the use of a kit or method according to the invention for detecting a target nucleic acid and for diagnosing a disease or condition. The kits and methods according to the invention are particularly useful for detecting point mutations on target nucleic acids.

Description

Technical Field

[0001] The subject matter of the present invention is a kit and a sandwich-type method using said kit for the sensitive and robust detection of target nucleic acids in a sample. The present invention also relates to the use of the kit or method according to the present invention, for example for the molecular diagnosis of diseases or disorders. The present invention belongs to the field of molecular biology assays. Background Art

[0002] Nucleic acids are considered important markers for diseases such as cancer or infections. However, their detection can be difficult due to their extremely low concentration.

[0003] Disease-related nucleic acid biomarkers usually exist at ultra-low abundances. Therefore, precise and robust detection of analytes mainly in large amounts of biological fluids is very important for biological research, precision medicine, and early diagnosis. The COVID-19 pandemic has revealed the urgent need for rapid, highly sensitive, and highly specific detection methods for controlling the rapidly evolving pandemic. In clinical diagnosis, direct nucleic acid detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has not been applied because of the challenge of detecting ultra-low concentration RNA molecules in suspension. Direct detection is limited by strong backgrounds from non-specific proteins, nucleic acids, or other biomolecules.

[0004] In molecular diagnosis, there are two types of assays each with specific advantages: (1) target amplification assays and (2) signal amplification assays. Target amplification assays such as PCR are sensitive but require target extraction and purification, enzymatic reactions, and are more prone to false positives. In contrast, signal amplification assays are simpler because they tend to skip nucleic acid purification and enzymatic amplification. Therefore, there is less material loss and a lower false positive rate. However, signal amplification assays are less sensitive compared to PCR-based target amplification methods. Many efforts have been devoted to developing ultrasensitive signal amplification assays for detecting unamplified nucleic acids. Compared with RT-PCR, potential portable microfluidic devices have shown specific and sensitive detection of Ebola nucleic acids (Cai, H. et al., 2015). Interestingly, some of these works have been able to detect target nucleic acids directly from whole blood lysates (Ngo, H. T. et al., 2018) (Zheng, Z. et al., 2006).

[0005] To date, the recommended method for COVID-19 diagnosis is based on quantitative reverse transcription polymerase chain reaction (qRT-PCR) that requires nucleic acid extraction from throat swabs and a target amplification procedure. Such procedures require technicians, specific equipment, and long processing times (>2 h), making on-site SARS-CoV-2 nucleic acid testing difficult to implement. In addition, the high demand for commercial RNA extraction kits has led to shortages of these reagents, so several diagnostic workflows that bypass the long intermediate RNA extraction step have been developed. In the past decade, several studies have shown the performance of magnetic beads in extracting nucleic acids from biological fluids prior to molecular detection. Magnetic beads consist of iron oxide nanoparticles embedded in a polymer matrix and are able to separate nucleic acids differently according to surface functionalization. This functionalization affects the binding kinetics and compatibility with molecular detection strategies. For example, silica-coated beads bind all nucleic acids non-selectively by electrostatic interactions, so they are mainly used in detection methods that tolerate high concentrations of non-target background nucleic acids, such as RT-PCR. Oligonucleotide-conjugated beads, such as oligo(dT) beads or beads with surface functionalization of specific sequences, are used for mRNA target extraction and have high recovery rates. This purification can be used manually, within microfluidic chips, or in automated robotic setups. In addition to the separation and elution of nucleic acids, several specific nucleic acid sequences have been directly detected on the bead surface by hybridization assays using different materials (such as biotin-avidin, protein-enzyme, fluorescent dyes, quantum dots, etc.). However, most of these methods are limited by low signal intensity or rapid photobleaching (Lim et al., 2009).

[0006] EP3536806 “Oligonucleotide-functionalized hydrophobic polymer nanoparticles” describes nanoparticles containing a hydrophobic polymer and a luminescent component, as energy-providing elements, which can be used to detect target nucleic acids. The nanoparticles can be conjugated with oligonucleotides that are complementary to specific targets or non-specific sequences of targets. Target-specific oligonucleotides are conjugated to the energy-providing elements, and their nucleotide sequences are complementary to another nucleotide sequence labeled with an excitation energy acceptor compound. A FRET (fluorescence resonance energy transfer) effect occurs between the acceptor compound and the donor compound in the nanoparticles. The lower limit of detection using these nanoparticles in the method is a concentration of 5 pM of target nucleotides.

[0007] WO 2017 / 220453 describes the use of magnetic beads to detect mutations. The magnetic bead-binding probe hybridizes to one end of the target nucleic acid, and the surface-binding probe hybridizes to the other end of the target nucleic acid. Stringency is applied to the hybridization complex by magnetic force and / or temperature.

[0008] There is still a need for simple, rapid, sensitive, and robust methods for detecting ultra-low-abundance nucleic acids. Summary of the Invention

[0009] In this work, the inventors aimed to develop a sandwich hybridization method for detecting low-abundance nucleic acid molecules from a sample. The method according to the invention involves hybridization in which the nucleic acid of interest is detected by dual direct hybridization using different complementary oligonucleotides that specifically bind to different regions of the target nucleic acid.

[0010] The method according to the invention combines:

[0011] · Solid-surface-based hybridization of a sequence-specific probe with the target in solution to capture the target nucleic acid molecule, where the capture probe is immobilized on a solid substrate to allow direct separation and enrichment of the target molecule from the original sample with minimal handling, and

[0012] · At least a second sequence-specific capture that allows direct detection on the solid surface by binding to a super-bright luminescent particle.

[0013] Using solid-surface-based hybridization to capture the target nucleic acid molecule involves a solid surface, which can be the surface of an immobilized surface or a solid particle, such as a bead, e.g., a magnetic bead or a glass bead. Separation of the complex bead / target can be achieved, for example, by applying magnetic force or gravity, centrifugation, or filtration, depending on the nature of the solid surface. This separation is important for removing excess non-bound DNA-modified nanoprobes and / or super-bright luminescent particles. It is also useful for separating the target nucleic acid of interest even from cell lysates without the need for RNA extraction. Detection of the fluorescence signal is performed directly on the solid surface by fluorescence quantification because the fluorescent particles bind to the target by complementary hybridization.

[0014] The kits and methods of the invention are suitable for detecting target nucleic acids in samples such as environmental samples or biological samples. The kits and methods according to the invention are also suitable for diagnostic applications.

[0015] The advantages of the kits and methods according to the invention are their extremely high sensitivity, their robustness and simplicity, and that it is an enzyme-free amplification detection assay.

[0016] In fact, the kits and methods according to the invention allow precise and robust detection of nucleic acids with a detection limit of about 1 fM. The extreme brightness of the luminescent particles is crucial for achieving a high signal-to-noise ratio for rapid detection of the target by simple techniques.

[0017] In addition, like the detection kits and methods according to the prior art, low-abundance target nucleic acid molecules can be detected and separated without the need for a supplementary nucleic acid extraction step.

[0018] The target nucleic acid molecule can be any nucleic acid molecule, including double-stranded DNA molecules, single-stranded DNA molecules, double-stranded RNA molecules, and single-stranded RNA molecules, and includes nucleic acid molecules of short and long lengths. Point mutations can be detected using the kits or methods of the present invention.

[0019] The ultra-bright particle brightness and high concentration of complementary oligonucleotides attached to the beads and ultra-bright luminescent particles are a great advantage of the kits and tests of the present invention to improve the detection limit of nucleic acids. In fact, when using a fluorescence plate reader, the detection limit of the assay of the present invention is about 1 fM. Compared with prior art tests, this represents a significant improvement in the detection limit. The kits and tests according to the present invention are capable of detecting RNA and DNA of different lengths and can be used to detect nucleic acids in a sample. For example, the kits and tests according to the present invention can be used for diagnosing diseases or for detecting pathogens, such as parasites, in environmental samples.

[0020] The present invention enables the steps of detecting a target nucleic acid and extracting the target nucleic acid from its original medium to be carried out in a limited number of steps. The proposed assay is very sensitive because its detection limit is about 1 fM. The present invention does not require amplification, especially by PCR, nor does it require the use of enzymes. The present invention enables the detection of nucleic acids from short microRNAs to long nucleic acids using direct detection of luminescence signals.

[0021] Finally, the fluorescence signal generated by the test according to the present invention can be read by using a plate reader, which accelerates the detection of nucleic acids in large-scale samples.

[0022] In a particular embodiment, the method according to the present invention utilizes the synergistic effect of combining capture using hybridization on a solid surface based on beads, especially magnetic beads, with ultra-bright fluorescent particles.

[0023] In another particular embodiment, the kits and methods according to the present invention utilize the synergistic effect of combining capture using hybridization on a solid surface with detection by using ultra-bright fluorescent DNA-nanoparticles (DNA NPs), as disclosed in, for example, WO 2017 / 220453.

[0024] In a first aspect, the present invention relates to a kit for sandwich test detection of a target nucleic acid in a sample. In a second aspect, the present invention relates to a method for sandwich test detection of a target nucleic acid in a sample using the kit of the present invention. Detailed Description

[0025] In a first aspect, the present invention relates to a kit for the detection of a target nucleic acid in a sample, wherein the kit comprises at least:

[0026] i) Probe P1, which comprises or consists of a nucleic acid fragment NA1 linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of a region T1 of a target nucleic acid.

[0027] ii) A functional unit F2 bound to a solid surface, wherein F2 exhibits a high affinity for F1 or is covalently bound to F1.

[0028] iii) Probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, said super-bright luminescent particle exhibiting a brightness of at least 10 7 M -1 cm -1 , preferably at least 2×10 7 M -1 cm -1 , at least 5×10 7 M -1 cm -1 , at least 10×10 7 M -1 cm -1 or at least 40×10 7 M -1 cm -1 of brightness.

[0029] Figure 1A shows a non-limiting illustration of the elements of the kit according to the invention and their respective interactions.

[0030] "Kit" refers to a kit comprising two or more separate components, said components being included together in a conventional material such as a package. These separate components interact for a specific purpose to achieve a specific result. It should be understood that the terms "comprising" or "including" are not restrictive. The term "consisting of" is considered a preferred embodiment of the term "comprising".

[0031] The term "sample" refers to any small amount of medium that is likely to contain a target nucleic acid. Among various samples, one can list biological samples including cells or tissues from any source. One can also list environmental samples provided by extraction or removal from an environmental matrix such as water.

[0032] In a particular embodiment, the invention relates to a kit for in vitro detection of a target nucleic acid in a sample.

[0033] The expression "in vitro" has its conventional meaning and refers to an experiment performed on a biomolecule or a living cell that has been extracted from its normal environment and provided as a sample to be analyzed.

[0034] The term "nucleic acid" refers to a polymer of deoxyribonucleotides or ribonucleotides, and encompasses natural nucleotides and known natural nucleotide analogs that can act in a manner similar to naturally occurring nucleotides. Non-limiting examples of such natural nucleotide analogs are locked nucleic acids (LNAs) and 2'-O-methylated nucleotides. The target nucleic acid molecules detected by the kits or methods according to the present invention are any nucleic acid molecules, including single-stranded DNA molecules, double-stranded DNA molecules, single-stranded RNA molecules, and double-stranded RNA molecules, including microRNA molecules.

[0035] The nucleic acid fragment of probe P1 or probe P2 is preferably an oligonucleotide. "Oligonucleotide" refers to a nucleic acid comprising or consisting of a sequence of at least 5 nucleotides, preferably a nucleic acid comprising or consisting of a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 to at least 150 nucleotides.

[0036] "Complementary" means that the nucleic acid sequence is capable of forming a stable hybridization complex under appropriate stringent conditions. Sequence-specific hybridization uses an oligonucleotide probe complementary to the sequence of interest to capture the sequence of interest by hybridization. "Hybridization" refers to the formation of a stable complex between complementary nucleic acid molecules. Hybridization can occur between partially complementary molecules or between fully complementary molecules. The stability of the formed hybridization complex depends on the reaction conditions and the proportion of complementary nucleic acid sequences in the nucleic acid molecules. The sequence of the oligonucleotide is designed to ensure the formation of a stable hybridization complex.

[0037] Preferably, the nucleotide sequence of probe P1, probe P2, or probe P3 complementary to a nucleic acid has a length of at least 8 nucleotides, preferably at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 to at least 150 nucleotides.

[0038] In probe P1, the nucleotide acid fragment NA1 is bound to the functional unit F1 by a covalent bond or a non-covalent bond, preferably by a covalent bond. In probe P2, the nucleotide acid fragment NA2 is bound to the ultra-bright luminescent particle (ULP) by a covalent bond or a non-covalent bond, preferably by a covalent bond.

[0039] In a particular embodiment of the kit according to the present invention, probe P1 comprises or consists of: a nucleic acid fragment NA1 and a functional unit F1, wherein NA1 and F1 are bound by a linker. In another particular embodiment of the kit according to the present invention, probe P2 comprises or consists of: a nucleic acid fragment NA2 and a ULP, wherein NA2 and ULP are bound by a linker.

[0040] "Linker" refers to any suitable compound selected by those skilled in the art, including:

[0041] - Non-coding nucleotide sequences selected from DNA linkers, such as "A10" or "A20" linkers (if present, the DNA linkers contain nucleotides that do not participate in complementary hybridization sequences) and / or

[0042] - Non-nucleic acid elements, such as chemical elements, like tetraethylene glycol (TEG).

[0043] "Functional unit" refers to any molecule, biomolecule or chemical group capable of selectively forming non-covalent or covalent bonds with another functional unit.

[0044] Among various functional units commonly used by those skilled in the art, one can list biotin (BIO), avidin (A), neutravidin (N) and streptavidin (SV). Biotin is known for its high affinity for avidin, neutravidin and streptavidin. The functional unit can also be a complementary nucleic acid sequence of at least 12 bases.

[0045] In a particular embodiment, the first functional unit is biotin (BIO), wherein the second functional unit is selected from streptavidin (SV) and streptavidin analogs, particularly selected from avidin (A) and neutravidin (N).

[0046] In a first embodiment of the present invention, F2 is linked to F1 by non-covalent bonds and exhibits a high affinity for F1. "Non-covalent bond" refers to a bond that does not involve electron sharing but involves other electromagnetic interactions. Non-covalent bonds include hydrophobic effects, van der Waals forces, electrostatic forces and π effects. Among various non-covalent bonds, one can list, for example, the non-covalent π bond between complementary nucleic acid molecules. "High affinity" refers to the binding complex affinity, characterized by a kD of at least 1 nM (upper limit), preferably at least 1 pM. In a particular example, the first functional unit is biotin (BIO) and the second functional unit is streptavidin, neutravidin or avidin. In this embodiment, F1 and F2 are capable of forming a high-affinity binding complex together through non-covalent (non-electron-sharing) interactions. For example, the very high affinity between biotin and streptavidin allows the formation of a stable non-covalent complex, and this stability can even be observed when physical external forces are applied to the complex. In a more particular embodiment, the functional unit F1 is biotin and the functional unit F2 is avidin or streptavidin.

[0047] In a second embodiment of the present invention, F2 is linked to F1 by a covalent bond. A "covalent bond" refers to a chemical bond in which electrons are shared and form at least one electron pair between two atoms. For a covalent bond, the functional unit is a chemically reactive group. In a particular embodiment, the functional group can be an azide group, which can react with a derivative of acetylene by a "click" cycloaddition reaction. In a particular embodiment, the functional group can be a carboxylic acid ester that can react with an amine to form a covalent amide bond.

[0048] In the kit or method according to the present invention, F1 is covalently or non-covalently bound to the nucleic acid fragment NA1 and is capable of covalently or non-covalently binding to F2.

[0049] The functional unit F2 is bound to the solid surface by any technique known to those skilled in the art, either by a covalent bond or a non-covalent bond. For example, to functionalize a glass surface with streptavidin, a well-established method is based on adsorbing BSA-biotin, further binding streptavidin, and finally adding the corresponding DNA capture with a biotin unit ( Figure 2A ).

[0050] In a first particular embodiment of the kit according to the present invention, the functional unit F2 is bound to a solid immobilized surface, such as: a glass surface, a reaction support surface, a porous plate surface, a microfluidic device surface, or any surface suitable for covalent or non-covalent functionalization.

[0051] In a second particular embodiment of the kit according to the present invention, the functional unit F2 is bound to a surface that is a solid particle surface. The solid particle can be, for example, a magnetic bead or a glass bead. Those skilled in the art can select any suitable bead commonly used in such kits. The solid particles are easily present in the form of a suspension and are sensitive to the applied physical external forces.

[0052] The solid particles of the kit according to the present invention respond to physical external forces. Among various physical forces, one can list the following examples:

[0053] - Gravity, which is applicable to the case of a centrifugation step,

[0054] - Electromagnetic force, which is applicable to the case where a magnet exists, or

[0055] - Shearing force, which is generated by flow or filtration.

[0056] In a particular embodiment, the kit according to the present invention includes magnetic beads covalently coupled to the functional unit F2. The magnetic beads coupled to the functional unit F2 are sensitive to magnetic force.

[0057] The super-bright luminescent particles exhibit a very high level of brightness. Brightness is defined as the product of the extinction coefficient and the fluorescence quantum yield. In the kits and methods according to the present invention, the super-bright luminescent particles (ULP) are characterized by a brightness of at least 10 7 M - 1 cm -1 , preferably at least 2×10 7 M -1 cm -1 , at least 5×10 7 M -1 cm -1 , at least 10×10 7 M -1 cm -1 , at least 40×10 7 M -1 cm -1 of any particles.

[0058] The brightness of the particles can also be defined by the ratio of the dye weight to the total particle weight. For the super-bright particles in the kits or methods according to the present invention, the ratio is preferably at least 1 wt% dye loading, preferably at least 5 wt% dye loading, at least 10 wt% dye loading, at least 20 wt% dye loading, at least 30 wt% dye loading, at least 40 wt% dye loading or at least 50 wt% dye loading.

[0059] In the kits according to the present invention, the super-bright luminescent particles (ULP) can be selected from any luminescent particles known to those skilled in the art. The particles are preferably selected from: fluorescent particles, phosphorescent particles, chemiluminescent particles and bioluminescent particles. In a particular embodiment, the super-bright luminescent particles are super-bright fluorescent particles.

[0060] In a particular embodiment of the kits according to the present invention, the probe P2 comprises or consists of a nucleic acid fragment NA2 linked to a super-bright fluorescent particle, the super-bright fluorescent particle exhibiting at least 10 7 M -1 cm -1 , preferably at least 2×10 7 M -1 cm -1 , at least 5×10 7 M -1 cm -1 , at least 10×10 7 M -1 cm -1 or at least 40×10 7 M -1 cm -1Brightness. More particularly, the kit according to the invention comprises a probe P2 which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright fluorescent polymeric nanoparticle, said super-bright fluorescent polymeric nanoparticle exhibiting at least 10 7 M -1 cm -1 、preferably at least 2×10 7 M -1 cm -1 、at least 5×10 7 M -1 cm -1 、at least 10×10 7 M -1 cm -1 or at least 40×10 7 M -1 cm -1 brightness.

[0061] In another particular embodiment, the super-bright luminescent particle is a super-bright luminescent nanoparticle. In an even more particular embodiment, the super-bright luminescent particle is a super-bright luminescent polymeric nanoparticle.

[0062] Dye-loaded polymeric nanoparticles are a promising sensitive alternative for detecting nucleic acids in solution due to their high brightness and modularity. Previously, to ensure high brightness while addressing the quenching problem caused by aggregation of the encapsulated dye in these NPs, the concept of ion dye isolation by large volumes of hydrophobic counterions was proposed, resulting in NPs that are approximately 100 times brighter than similarly sized semiconductor quantum dots (QDs) (Melnychuk & Klymchenko, 2018), (Reisch et al., 2014), (Reisch et al., 2017). Additionally, a charge-controlled nanoprecipitation method with hydrophobic polymers bearing a small number of charged groups was introduced to achieve controlled sizes of polymeric NPs (Reisch et al., 2015). A strategy based on charged amino acids for exposing azide groups on the NP surface allows further modification with nucleic acids by click chemistry. The resulting DNA-NP conjugates generate super-bright nanoprobes that can be used for amplified detection of DNA / RNA in solution with a detection limit in the picomolar range, as well as for amplified detection of DNA / RNA on surfaces with single-molecule sensitivity and are compatible with smartphone-based sensing. Moreover, these DNA-functionalized NPs have been validated for detecting microRNAs in cell extracts and can directly detect intracellular RNA by RNA-fluorescence in situ hybridization protocols. Their application for amplified enzyme-free detection of RNA in solution using total RNA extracts from cells or serum media by a simple one-step protocol has been explored.

[0063] Polymeric nanoparticles loaded with super-bright dyes refer to particles loaded with fluorescent dyes and containing at least 200, 500, 1000, 2000, or 10,000 fluorophore molecules, where one fluorophore molecule exhibits a brightness of at least 4×10 4 M -1 cm -1 . In particular, the super-bright particles can be composed of polymers. Non-limiting examples of polymers are: polymethacrylates, aliphatic polyesters, polystyrenes, polyurethanes. Examples of polymethacrylates include, but are not limited to, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate). Examples of polymethacrylate derivatives are poly(methyl methacrylate-co-methacrylic acid) (PMMA-MA), poly(methyl methacrylate-co-2-methacrylamidoethanesulfonic acid) (PMMA-SO3). Examples of aliphatic polyesters can be listed as, but are not limited to, polycaprolactone (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA).

[0064] According to a more particular embodiment, the kit according to the invention comprises fluorescent nanoparticles loaded with super-bright dyes, in particular as described in EP 3 536 806.

[0065] In a particular embodiment of this first aspect, the invention relates to a kit for detecting a target nucleic acid in a sample, wherein the kit comprises at least:

[0066] i) A probe P1, which comprises or consists of a nucleic acid fragment NA1 linked to a functional unit F1, wherein the nucleic acid fragment NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of a region T1 of the target nucleic acid molecule,

[0067] ii) A functional unit F2 bound to a solid surface,

[0068] iii) A probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a luminescent particle loaded with a super-bright dye, wherein the nucleic acid fragment NA2 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of a region T2 of the target nucleic acid.

[0069] In another particular embodiment of the invention, the kit of the invention comprises at least:

[0070] i) At least one probe P1, which comprises or consists of a nucleic acid fragment NA1 covalently linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of a region T1 of the target nucleic acid,

[0071] ii) A functional unit F2 bound to a solid surface,

[0072] iii) At least one probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, and

[0073] iv) At least one probe P3, which comprises or consists of: a first part, which comprises or consists of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of a region T3 of the target nucleic acid, wherein said first part and said second part are joined by a nucleotide linker.

[0074] Figure 1B shows a non-limiting illustration of the elements of the kit according to the invention and their respective interactions.

[0075] In this embodiment of the kit according to the invention, the probe P3, also referred to as the "post-it capture sequence" or the "post-it probe", comprises at least two parts:

[0076] - A first part, which comprises or consists of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2 of the probe P2, and

[0077] - A second part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of a region T3 of the target nucleic acid, wherein T3 is different from T1,

[0078] wherein said first part and said second part are joined by a nucleotide linker.

[0079] The first part and the second part of P3 are preferably oligonucleotides, and the probe P3 is preferably a nucleic acid comprising or consisting of a sequence of at least 10 nucleotides, preferably comprising or consisting of a sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 80, 100, 120 or 150 nucleotides.

[0080] In a particular embodiment, the first part and the second part of P3 are joined by a linker, the length of which is preferably at least 5 nucleotides, preferably at least 10, 15, 20 or 40 nucleotides.

[0081] According to this particular embodiment, the nucleotide sequence of the nucleic acid fragment NA2 of the probe P2 is independent of the nucleotide sequence of the target nucleic acid. This embodiment represents an advantage of including the probe P2, which comprises the nucleic acid fragment NA2, the nucleotide sequence of which is independent of the target to be detected.

[0082] According to another particular embodiment, the kit of the present invention may include at least two P1 probes, such as 2, 3, 4, 5 or n P1 probes, where n is the total number of P1 probes in the kit. Each of the P1 probes is designated as P1-1, P1-2 or P1-n, and comprises a nucleic acid fragment NA1 designated as NA1-1, NA1-2 or NA1-n respectively, whose nucleotide sequence is complementary to the nucleotide sequence T1-1, T1-2 or T1-n of the target nucleic acid. The kit according to the present invention may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 probes.

[0083] According to this embodiment, the kit of the present invention includes at least:

[0084] i) at least two probes P1, which comprise or consist of a nucleic acid fragment NA1 covalently linked to a functional unit F1, where NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of the region T1 of the target nucleic acid,

[0085] ii) a functional unit F2 bound to a solid surface,

[0086] iii) at least one probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, and

[0087] iv) at least one probe P3, which comprises or consists of: a first part, which comprises or consists of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of the region T3 of the target nucleic acid, where the first part and the second part are joined by a nucleotide linker.

[0088] According to another particular embodiment, the kit of the present invention may include at least two, three or more than three probes P3. Each of the P3 probes differs from the other P3 probes in the nucleotide sequence of its first part and / or the nucleotide sequence of its second part.

[0089] For example, the probe P3-NA4-1 comprises a nucleic acid fragment NA4-1, which comprises or consists of a nucleotide sequence complementary to the nucleotide sequence NA2-1 on the P2 probe.

[0090] For example, the probe P3-NA3-1 comprises a nucleic acid fragment NA3-1, which comprises or consists of a nucleotide sequence complementary to the nucleotide sequence T3-1 on the target nucleic acid.

[0091] According to this embodiment, the kit of the present invention may include 1, 2, 3, or n different P3 probes, where n represents the number of the P3 probes. The kit according to the present invention may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 P3 probes.

[0092] According to this embodiment, the kit of the present invention at least includes:

[0093] i) At least one probe P1, which comprises or consists of a nucleic acid fragment NA1 covalently linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of a region T1 of the target nucleic acid,

[0094] ii) A functional unit F2 bound to a solid surface,

[0095] iii) A probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, and

[0096] iv) At least two probes P3, which comprises or consists of: a first part, which comprises or consists of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of a region T3 of the target nucleic acid, wherein the first part and the second part are bound by a nucleotide linker.

[0097] The nucleotide sequences of multiple P1 probes and multiple P3 probes can respectively bind to different regions of the target nucleic acid, wherein the target nucleic acid is a long nucleic acid molecule, and "long" means a target nucleic acid of at least 35 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 1000 nucleotides, and more than 1000 nucleotides. The presence of the 1, 2, 3, or n probes allows the detection of such long nucleic acid molecules, which are intact or in fragment form in the sample.

[0098] According to another particular embodiment, the kit of the present invention may include at least two probes P2, such as 2, 3, 4, 5, or n probes P2, where n is the total number of P2 probes in the kit.

[0099] Each of the P2 probes is designated as P2-1, P2-2, or P2-n, and comprises a nucleic acid fragment NA2 respectively designated as NA2-1, NA2-2, or NA2-n, whose nucleotide sequences are respectively complementary to the sequences of the nucleotide fragments NA4-1, NA4-2, or NA4-n of the probe P3. The kit according to the present invention may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 probes P2.

[0100] According to this embodiment, each of the P2 probes may also differ from the other P2 probes due to the nature of the super-luminescent particles. In fact, by using the kit according to the present invention and using P2 probes containing different ULP, multiplex detection can be achieved.

[0101] When there are two or more different P3 probes in a particular embodiment, the kit according to the present invention may include one or more different P2 probes. Conversely, when there are two or more different P2 probes in a particular embodiment, the kit according to the present invention must include two or more different P3 probes because each of the P3 probes should be suitable for hybridizing with each of the P2 probes.

[0102] According to this embodiment, the kit of the present invention includes at least:

[0103] i) at least one probe P1, which comprises or consists of a nucleic acid fragment NA1 covalently linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of a region T1 of the target nucleic acid,

[0104] ii) a functional unit F2 bound to a solid surface,

[0105] iii) at least one probe P2, preferably at least two probes P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, and

[0106] iv) at least one P3 probe, which comprises or consists of: a nucleotide sequence complementary to the nucleotide sequence of NA2 and a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of a region T3 of the target nucleic acid.

[0107] This is important for the simultaneous multiplex detection of different regions of nucleic acids. In this case, the ULP preferably has different spectral characteristics.

[0108] The kit according to the present invention may also include any suitable elements for performing the detection reaction, such as buffers.

[0109] The second aspect of the present invention relates to a method for detecting a target nucleic acid molecule in a sample prepared from a biological sample by a sandwich test, the method comprising at least the following steps:

[0110] a) contacting at least the following under conditions suitable for hybridization of complementary nucleic acid sequences and suitable for non-covalent complex formation:

[0111] - Probe P1, which comprises or consists of a first nucleic acid fragment NA1 linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid,

[0112] - A functional unit F2 bound to a solid surface, wherein the functional unit F2 exhibits a high affinity for F1 or is covalently bound to F1, and

[0113] - Probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, the super-bright luminescent particle exhibiting at least 10 7 M -1 cm -1 、preferably at least 2×10 7 M -1 cm -1 、at least 5×10 7 M -1 cm -1 、at least 10×10 7 M -1 cm -1 or at least 40×10 7 M -1 cm -1 of brightness,

[0114] b) Applying a physical external force to the mixture of step a) to separate the formed complex, and

[0115] c) Measuring the luminescence emission intensity associated with the non-covalent complex of the nucleic acid.

[0116] Thus, the present invention relates to a method for detecting a target nucleic acid in a sample belonging to the sandwich-type test category. In the method according to the present invention, the physical force applied is suitable for the nature of the solid surface of the kit and can be easily selected by a person skilled in the art of testing.

[0117] In a particular embodiment, the present invention relates to a method for in vitro detection of a target nucleic acid in a sample.

[0118] Among various physical forces, the following examples can be cited:

[0119] - Gravity, which is applicable in the case of a centrifugation step,

[0120] - Electromagnetic force, which is applicable in the case where a magnet is present, or

[0121] - Shearing force, which is generated by flow or filtration.

[0122] In particular embodiments, the method according to the invention comprises applying a centrifugation step, applying an electromagnetic force or at least applying a filtration step. In the method according to the invention, "applying a physical force" comprises using one, two, three or more than three steps of said physical force.

[0123] In particular embodiments, the invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein the functional unit F2 is bound to a solid surface, which is the surface of a solid particle. In a more particular embodiment, the solid particle is a magnetic bead or a glass bead.

[0124] In the method according to the invention, the ultra-bright luminescent particle (ULP) is characterized by a brightness of at least 10 7 M -1 cm -1 、preferably at least 2×10 7 M -1 cm -1 、at least 5×10 7 M -1 cm -1 、at least 10×10 7 M -1 cm -1 、at least 40×10 7 M -1 cm -1 of any luminescent particle, or is characterized by a dye loading of at least 1 wt% of the ratio of the dye weight to the total particle weight, preferably at least 5 wt% dye loading, at least 10 wt% dye loading, at least 20 wt% dye loading, at least 30 wt% dye loading, at least 40 wt% dye loading or at least 50 wt% dye loading.

[0125] In the method according to the invention, the ultra-bright luminescent particle (ULP) can be selected from any luminescent particle known to those skilled in the art. The particles are preferably selected from: fluorescent particles, phosphorescent particles, chemiluminescent particles and bioluminescent particles. In particular embodiments, the ultra-bright luminescent particle is an ultra-bright fluorescent particle.

[0126] In a particular embodiment of the method according to the invention, the probe P2 comprises or consists of a nucleic acid fragment NA2 linked to an ultra-bright fluorescent particle, the ultra-bright fluorescent particle exhibiting a brightness of at least 10 7 M -1 cm -1 More particularly, the method according to the invention comprises a probe P2 which comprises or consists of a nucleic acid fragment NA2 linked to an ultra-bright fluorescent polymeric nanoparticle, the ultra-bright fluorescent polymeric nanoparticle exhibiting a brightness of at least 10 7 M -1 cm -1 of brightness.

[0127] In another particular embodiment, the super-bright luminescent particles are super-bright luminescent nanoparticles. In an even more particular embodiment, the super-bright luminescent particles are super-bright polymeric nanoparticles.

[0128] According to a more particular embodiment, the method according to the invention comprises fluorescent nanoparticles loaded with a super-bright dye, in particular as described in EP 3 536 806.

[0129] In a particular embodiment of the method according to the invention, the probe P2 comprises or consists of a nucleic acid fragment NA2 linked to a super-bright fluorescent particle, the super-bright fluorescent particle exhibiting a brightness of at least 10 7 M -1 cm -1 and the nucleic acid fragment NA2 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of the region T2 of the target nucleic acid.

[0130] In another particular embodiment, the invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein step a) comprises contacting at least the following:

[0131] - at least one probe P1, which comprises or consists of a nucleic acid fragment NA1 linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of the region T1 of the target nucleic acid,

[0132] - a functional unit F2 bound to a solid surface, wherein F2 exhibits a high affinity for F1 or is covalently bound to F1, and

[0133] - a probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle,

[0134] - at least one probe P3, which comprises or consists of: a nucleotide sequence complementary to the nucleotide sequence of the region T3 of the target nucleic acid, wherein T3 is different from T1, and a nucleotide sequence complementary to the nucleotide sequence of NA2, and

[0135] - thereby forming a mixture.

[0136] In another particular embodiment, the invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein step a) comprises contacting at least the following:

[0137] - at least two probes P1, wherein each of the probes P1 comprises or consists of: a nucleic acid fragment NA1, wherein each of the NA1 is designated as NA1-1 or NA1-2 and is complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid, respectively.

[0138] - Probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle,

[0139] - At least one probe P3, which comprises or consists of:

[0140] * A nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, where T3 is different from T1, and

[0141] * A nucleotide sequence complementary to the nucleotide sequence of NA2, and

[0142] - Thereby forming a mixture.

[0143] In another specific embodiment, the present invention relates to a method for detecting a target nucleic acid molecule in a sample, wherein step a) comprises contacting at least the following substances:

[0144] - At least two probes P1, wherein each of the probes P1 comprises or consists of: a nucleic acid fragment NA1, wherein each of the NA1 is designated as NA1-1 or NA1-2 and is complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid, respectively.

[0145] - Probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle,

[0146] - At least two probes P3, wherein each of the probes P3 comprises or consists of:

[0147] * A nucleic acid fragment NA3, which comprises or consists of nucleotide sequences NA3-1 or NA3-2 that are complementary to the nucleotide sequences T3-1 and T3-2 of the target nucleic acid, respectively, where T3-1 or T3-2 is different from T1, and

[0148] * A nucleotide sequence complementary to the nucleotide sequence of NA2,

[0149] And

[0150] - Thereby forming a mixture.

[0151] In a third aspect, the present invention relates to the use of the kit according to the present invention for detecting a target nucleic acid.

[0152] In said third aspect, the present invention also relates to the use of the method according to the present invention for detecting a target nucleic acid.

[0153] The kits and methods according to the invention are suitable for detecting target nucleic acids of variable length. In fact, the kits and methods according to the invention are suitable for detecting target nucleic acids, such as microRNAs or long-length RNAs, single-stranded DNAs or double-stranded DNAs of different lengths. Thus, the kits and methods according to the invention can be used for the detection of target nucleic acids preferably having a length of at least 15 nucleotides.

[0154] In a fourth aspect, the invention relates to an in vitro diagnostic method for a pathology or disorder, wherein the method comprises using a kit or method according to the invention to detect and / or extract a target nucleic acid specific for the pathology or disorder.

[0155] In particular, the invention relates to an in vitro diagnostic method for a pathology caused by the presence of the SARS Cov-2 virus, wherein the method comprises using a kit or method according to the invention to detect and / or extract a target nucleic acid from the virus.

[0156] The invention is illustrated in more detail by the following figures and examples. Description of the Drawings

[0157] Figures 1A and 1B show particular embodiments of the nucleic acid detection method according to the invention. Figure 1A shows a super-bright luminescent particle (ULP) functionalized with a nucleic acid fragment NA2 capable of directly hybridizing to a target nucleic acid region T2. Figure 1B shows a super-bright luminescent particle (ULP) functionalized with a nucleic acid fragment NA2 capable of binding to a target nucleic acid region T2 via an intermediate or "sticky note" probe P3.

[0158] Figure 2A Shows a particular embodiment of the invention in the presence of a target nucleic acid, wherein: the P1 probe is immobilized on a solid surface and comprises a functional unit F1 and a nucleic acid fragment NA1, and the P2 probe comprises a nucleic acid fragment NA2 and a super-bright particle (NP). Figure 2B Shows the quantification of the number of particles (SEQ ID N°6) upon increasing DNA target concentration (0 pM, 0.001 pM, 0.01 pM, 0.1 pM, 1 pM and 10 pM). PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and imaging.

[0159] Figure 3A shows a particular embodiment of the invention in the presence of a target nucleic acid, wherein: the P1 probe is to be immobilized on suspended solid particles and comprises a functional unit F1 and a nucleic acid fragment NA1, and the P2 probe comprises a nucleic acid fragment NA2 and a super-bright particle (NP). Figure 3B shows the quantification of the fluorescence intensity (SEQ ID N°6) upon increasing DNA target concentration (0 pM, 0.001 pM, 0.01 pM, 0.1 pM, 1 pM and 10 pM). PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection.

[0160] Figures 4A and 4B show the detection of target nucleic acids of different lengths. Figure 4A shows the quantification of the number of particles of 100-nucleotide RNA targets (left columns for each concentration) or 1000-nucleotide RNA targets (right columns for each concentration) at increasing concentrations (0 pM, 1 pM, 10 pM, 100 pM, and 1000 pM) by detection on an immobilized glass surface. Figure 4B shows the quantification of the fluorescence intensity of 100-nucleotide-long RNA targets (left columns for each concentration) or 1000-nucleotide-long RNA targets (right columns for each concentration) at increasing spiked concentrations (0 pM, 1 pM, 10 pM, 100 pM, and 1000 pM) by magnetic bead sandwich assay in 2.5 μg total RNA extract. RNA secure 1x buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection. Figure 4C shows 100-nucleotide RNA targets and 1000-nucleotide RNA targets with different positions of the target sequences complementary to DNA-NP. Figure 4D shows the possible difficulties of direct hybridization of DNA-NP with target sequences not located at the target ends.

[0161] Figure 5A Shows a particular embodiment of the invention in the presence of a target nucleic acid, wherein: the P1 probe (biotin capture) is immobilized on suspended solid particles, which contain a functional unit F1 and a nucleic acid fragment NA1 that hybridizes to the target. The P2 probe contains a nucleic acid fragment NA2 and a super-bright particle (NP). The P3 probe (DNA sticky note) hybridizes to the target nucleic acid in part and to the nucleic acid fragment NA2 and the super-bright particle (NP) in part. The right downward arrow shows that there is no steric hindrance at the target ends after DNA-NP hybridization. Figure 5B Shows the quantification of the fluorescence intensity of a 48-nucleotide DNA target (SEQ ID N°6) at increasing spiked concentrations (0 pM, 0.01 pM, 0.1 pM, 1 pM, and 10 pM) by magnetic bead sandwich assay in 2.5 μg total RNA extract, which uses direct hybridization of DNA-NP (white columns) or indirect hybridization through a sticky note probe (P3) (black columns). PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection.

[0162] Figure 6A and Figure 6B Shows the influence of NP properties on the sensitivity of sandwich hybridization. Figure 6A Shows the influence of NP size: the quantification of the fluorescence intensity of a 48-nucleotide DNA target (SEQ ID N°6) at increasing spiked concentrations (0 pM, 0.1 pM, 1 pM, and 10 pM) by magnetic bead sandwich assay in 2.5 μg total RNA extract, which uses DNA-NPs with a nuclear size of 20 nm or 50 nm for indirect hybridization through the P3 probe.Figure 6B Shows the influence of dye loading: quantification of the fluorescence intensity of a 48-nucleotide DNA target (SEQ ID N°6) at increasing spiked concentrations (0 pM, 0.1 pM, 1 pM, and 10 pM) in 2.5 μg of total RNA extract, detected by magnetic bead sandwich assay using 0.1 wt%, 1 wt%, 10 wt%, and 50 wt% of dye-loaded DNA-NPs by indirect hybridization with a sticky note probe (P3 probe). RNA secure 1x buffer containing 0.01 g / L of Tween 80 was systematically used for incubation and detection.

[0163] Figure 7A and Figure 7B Shows the detection method for long RNA targets. Figure 7A Shows the workflow for long RNA sandwich detection by capturing DNA-NPs in indirect hybridization suspension beads with a sticky note probe (P3). Figure 7B Shows the quantification of the fluorescence intensity of a 100-nucleotide RNA target (SEQ ID N°23) or a 1000-nucleotide RNA target (SEQ ID N°24) at increasing spiked concentrations (0 pM, 0.01 pM, 0.1 pM, 1 pM, and 10 pM) in 2.5 μg of total RNA extract, detected by magnetic bead sandwich assay using DNA-NPs with a nuclear size of 50 nm by indirect hybridization with a sticky note probe (P3), and capturing biotin with or without a TEG motif in 20 adenine linkers. RNA secure 1x buffer containing 0.01 g / L of Tween 80 was systematically used for incubation and detection.

[0164] Figure 8 Shows the detection of SARS-CoV-2 RNA in RNA extracted from clinical samples. Using DNA-NPs with a nuclear size of 50 nm and three biotin captures (P1) and three sticky note probes (P3) captures, fluorescence intensity quantification was performed when detecting an infected sample CS7 (CT = 18.75) after multiple dilutions in a negative sample CS1 (CT = 37), while keeping the total RNA (0.25 μg) constant. RNA secure 1x buffer containing 0.01 g / L of Tween 80 was systematically used for incubation and detection.

[0165] Figures 9A to 9C Represents the direct detection of SARS-CoV-2 RNA directly from clinical samples without RNA extraction. Figure 9A and Figure 9BShows the fluorescence intensity quantification in the detection of SARS-CoV-2 RNA in clinical samples from two different sources (CS series and CH series) using DNA-NPs with a core size of 50 nm and three biotin captures (P1) and three sticky note probes (P3). Figure 9C Shows multiple dilutions of negative CS2 samples and positive CS6 samples in buffer. RNA secure 1x buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection.

[0166] Figure 10 Shows the concentration-dependent response of the assay on the glass surface for detecting microRNA targets: miR200a (left column) and miR21 (right column). The target concentration was from 0 pM to 100 pM, and the quantification of NPs was from 0 to 25000. PBS buffer containing 0.01 g / L Tween 80 was systematically used for incubation and detection.

[0167] Examples:

[0168] Example 1: Detection of short nucleic acids

[0169] 1.1. Materials and methods

[0170] Chemicals were purchased from Sigma Aldrich, Alfa Aesar or Thermofisher Scientific. The polymer PMMA-AspN3-5% was synthesized as described previously (Melnychuk et al., 2020) (Melnychuk and Klymchenko, 2018). Rhodamine B octadecyl ester tetrakis(pentafluorophenyl)borate (R18 / F5) was synthesized by ion exchange and purified by column chromatography as described previously (Reish et al., 2014).

[0171] Preparation of NPs

[0172] NP-PEMA-MA-5%

[0173] Using a micropipette, 50 μL of the polymer acetonitrile solution (2 mg mL -1 , containing 50 wt% of R18 / F5 relative to the polymer) was rapidly added to 450 μL of 20 mM phosphate buffer, pH 7.4, 50 mM NaCl at 21 °C under shaking conditions (Thermomixer comfort, Eppendorf, 1100 rpm). While continuing to mix, 500 μL of 20 mM phosphate buffer, pH 7.4, 50 mM NaCl was added. Then, the acetonitrile residue was evaporated.

[0174] General protocol for DNA-functionalized nanoparticles

[0175] The freeze-dried single-stranded DNA sequences were purchased from Biomers or Eurogentec, dissolved in Milli-Q water, aliquoted and stored at -20 °C for further experiments. Aliquots of the corresponding nucleic acid fragments (SEQ ID N°1, SEQ ID N°8, SEQ ID N°14, SEQ ID N°19, SEQ ID N°20 or SEQ ID N°21) conjugated with dibenzocyclooctyne (DNA-DBCO) were added to 200 μL of the corresponding nanoparticles at a concentration of 20 μM in the reaction mixture. The reactants were mixed and left to stand overnight at 40 °C without shaking and protected from light. The reactants were then cooled to room temperature. The mixture was diluted to 4 mL with 20 mM phosphate buffer and the NPs were purified by centrifugation at 1000 g for 5 minutes at 20 °C using a centrifugal filter (Amicon, 0.5 mL, 100 kD, Sigma-Aldrich). The centrifugation step was repeated 6 times with the corresponding buffer to remove unreacted oligonucleotides. After each centrifugation, the overflow was discarded and the filtrate was resuspended in 4 mL of 20 mM phosphate buffer. This high level of purification is very important for achieving high sensitivity of the method. The obtained functionalized DNA-NPs were stored at 4 °C protected from light.

[0176] Nanoparticle characterization

[0177] Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZSP (Malvern Instruments S.A.). The nanoparticles were characterized by DLS using the standard cumulants and size distribution provided by volume analysis with the Zetasizer software. For data analysis, the following parameters were used: solvent (water) - temperature 25 °C, refractive index RI 1.33, viscosity 0.8872 cP. All nanoparticles were assumed to be homogeneous and spherical. Absorption spectra were recorded on a Cary 5000 scanning UV-visible spectrophotometer (Varian). Excitation spectra, emission spectra and anisotropy were recorded on an FS5 fluorescence spectrometer (Edinburg Instruments). For standard recording of fluorescence spectra, the excitation wavelength was set at 530 nm. The fluorescence spectra were corrected for detector response and light source fluctuations.

[0178] Transmission electron microscopy (TEM)

[0179] A 300-mesh carbon-coated copper-rhodium electron microscope grid (Euromedex, France) was surface-treated in a pentylamine atmosphere (0.45 mbar, 5 mA to 5.3 mA, 25 s) using an Elmo glow discharge system (Cordouan Technologies, France). Then, 5 μL of the NP solution was deposited onto the grid and left to stand for 2 minutes. The grid was then treated with a 2% uranyl acetate solution for 1 minute for staining. Observation was carried out at 200 keV using a Tecnai F20 electron microscope equipped with an FEG. The covered area of the nanoparticles of interest was recorded at a magnification of 29,000x on a GATAN CCD 2K*2K "US10001" camera. Image analysis was performed using Fiji software.

[0180] Nucleic acid detection on glass surface

[0181] The LabTek chamber (borosilicate coverslip, eight-well, ThermoFisher Scientific) was pretreated with 1 M KOH for 30 minutes, washed 3 times with PBS, and then incubated with 100 μL of 0.5 mg mL -1 of BSA-biotin (Sigma-Aldrich) in PBS for 20 minutes. Then, the BSA-biotin solution was removed, and the chamber was washed 3 times with 500 μL of PBS. Next, the chamber was incubated with 100 μL of a neutravidin (ThermoFisher Scientific) solution (0.5 mg mL -1 , in PBS) for 15 minutes and washed 3 times with 500 μL of PBS. Then, the chamber was incubated with 100 μL of a 1 μM P1 biotin capture sequence (SEQ ID N°2) solution in PBS for 20 minutes and washed 3 times with PBS. Finally, 100 μL of a detection solution containing 200 pM of probe P2 and different concentrations of synthetic targets in PBS dissolved in 0.01 g / L Tween 80 was added and incubated in the dark at 40 °C for 1 hour. Before measurement, the chamber was washed 3 times and covered with 200 μL of the same buffer.

[0182] Single-particle measurements were carried out in epi-fluorescence mode using a Nikon Ti-E inverted microscope (Apo TIRF, air, NA 1.4, Nikon) with a 20x objective lens. Excitation was provided by a light-emitting diode at 550 nm (SpectraX, Lumencor). The exposure time was set to 500 ms per frame image. The fluorescence signal was recorded using a Hamamatsu Orca Flash 4 camera.

[0183] Single-particle analysis was performed using Fiji software. The positions of the particles were detected by applying Fiji routines to the projections (maximum intensity) of all frames obtained for each experiment. After automatic background subtraction, the number of particles of interest with a diameter of 10 pixels was measured. At least 10 image sequences (1024 pixels × 1024 pixels) were analyzed for each condition.

[0184] Magnetic bead nucleic acid detection

[0185] Synthetic target solutions at different concentrations (SEQ ID N°6 in Figures 3, 5, and 6) were dissolved in PBS with 0.01 g / L Tween 80 and, when indicated, co-incubated with 50 nM of the P1 biotin capture sequence and 10 nM of the sticky-probe (P3) capture sequence. Cell lysates and RNA extracts were diluted in RNA Secure (Sigma) with 0.01 g / L Tween 80 and co-incubated as indicated with 20 nM of each biotin capture sequence P1 and 10 nM of each sticky-probe (P3) capture sequence P3. The solutions were heated at 95 °C for 30 seconds, cooled on ice for 5 minutes, and then incubated at 40 °C for 20 minutes without stirring. Then, 0.1 mg of streptavidin magnetic beads pre-washed twice with PBS were added. The suspension was incubated with gentle stirring at 40 °C for 1 hour. The complex composed of magnetic beads, target sequences, and capture sequences was separated from the unreacted nucleic acids by a magnetic field. The beads were washed three times with the same incubation buffer. 200 pM of the nanoprobe (the probe contains SEQ ID N°19, SEQ ID N°20, or SEQ ID N°21) was added and incubated with gentle stirring at 40 °C for 1 hour. The complex composed of magnetic beads, target sequences, capture sequences, and nanoprobes was separated from the unreacted nanoparticles by a magnetic field. The beads were washed three times with the same incubation buffer and the complex was resuspended in 50 μL of the same buffer and then transferred to a 96-well black plate. Direct detection of nucleic acids was performed on the bead surface by measuring the fluorescence intensity in a microplate (SPARK, TECAN).

[0186] The oligonucleotides used in the examples and their respective nucleic acid sequences are shown in Table 1 and the sequence listing attached to this patent application. N1, SPIKE, and ORF1 respectively represent regions from the SARS-CoV-2 virus used as targets, and these regions are used to refer to the nucleotide sequences of probes P1, P2, and P3. B, C, and NS respectively designate the non-coding sequences of probe P2 that will bind to NP. NP designates the super-bright nanoparticles. When using P3, the NP for detecting all target sequences is the same, P2 is not complementary to the target, and the P3 probe varies according to the target to be detected.

[0187]

[0188]

[0189]

[0190] Table 1

[0191] 1.2. Results

[0192] This example discloses an embodiment of the present invention, in which a single recognition event is combined with the binding of super-bright NPs to a solid support. The dye-loaded polymeric NPs were selected because of their high brightness, which is due to a large number of encapsulated dyes R18 with a bulky counterion F5-TPB, which minimizes self-quenching. To formulate the NPs, a polymer PEMA-AspN3 carrying azide and carboxylate functional groups was used (Melnychuk et al., 2020). The hydrophobic PEMA block in this polymer provides excellent optical properties for the encapsulated dyes, while the carboxylate ensures the formation of small NPs and the exposure of azide reactive groups on the NP surface. The NPs were formulated by nanoprecipitation of an acetonitrile solution of PEMA-AspN3 into phosphate buffer (pH 7.4). The R18 / F5-TPB dye was loaded into the NPs at 50 wt% relative to the polymer (i.e., 33 wt% relative to the total particle mass) by premixing it with the polymer in acetonitrile prior to nanoprecipitation, following a previously developed method (Melnychuk et al., 2020). The obtained NPs had a size of 24.5 nm and good polydispersity. Then, DNA (SEQ ID N°1) was grafted onto the NP surface using a click reaction between the DBCO group of the DNA oligonucleotide and the azide of the polymeric NPs. According to DLS, the size of the obtained NPs after purification by ultrafiltration was 31.5 nm. The increase of about 7 nm corresponds to twice the thickness of the DNA shell on the NP surface. The absorption and fluorescence spectra of the obtained DNA-NPs corresponded well to the encapsulated R18 / F5-TPB dye. The fluorescence quantum yield was 28%, consistent with earlier work on similar NPs. Given the 50 wt% dye loading of the polymer and an NP size of 24.5 nm, the estimated number of dyes per NP was about 1100. Thus, the brightness of the NPs corresponded to about 1000 dyes, with a high quantum yield.

[0193] To detect the target, two approaches based on the principle of similar hybridization were considered, in which the target DNA links the NPs to the solid support.

[0194] In the first approach, the glass surface and the NP surface were functionalized with two different capture DNA sequences (SEQ ID N°2 and SEQ ID N°1, respectively) Figure 2A)。To functionalize the glass surface, an established pathway based on the adsorption of BSA-biotin, further conjugation with streptavidin, and finally addition of the corresponding DNA capture carrying biotin units was used ( Figure 2A )。When the target DNA / RNA is present, the capture sequence hybridizes with the target, thereby immobilizing the NPs on the glass surface. After the washing step, the latter can be further observed by fluorescence microscopy. Given the high brightness of these NPs, visualization can be directly performed by using the LED excitation light source of a simple epi-fluorescence microscope. The N1 region of SARS-COV-2 RNA was selected as the target. When no short target was present, almost no NPs were detected on the NP surface (data not shown). Then, addition of the short target (SEQ ID N°6) resulted in the appearance of fluorescent spots on the surface, and the signal increased significantly with the amount of the target ( Figure 2B )。Therefore, the DNA-NPs do not bind non-specifically to the DNA-functionalized glass surface, while the target oligonucleotides immobilize the NPs on the glass surface. Importantly, the number of immobilized NPs is closely related to the concentration of the target oligonucleotides ( Figure 2B )。The detection limit of these measurements is extremely low, ranging from 1 fM to 10 fM. Experiments using non-coding DNA targets yielded lower signals, indicating sequence-specific detection (data not shown). These experiments provide a proof-of-concept for nucleic acid detection by a target-driven DNA-NP immobilization strategy.

[0195] Inspired by the results obtained on glass surfaces, and in order to simplify the detection protocol, where a fluorescence microscope is not required for DNA / RNA detection, the glass surface was replaced by streptavidin-functionalized magnetic beads. Using magnetic beads has two important features. First, magnetic beads have been well used for RNA extraction. Second, magnetic beads are easily processed by magnetic steps, allowing for efficient washing and further use in simple detection protocols, such as with a plate reader. Here, the target oligonucleotide (SEQ ID N°6) hybridizes with the capture DNA-biotin (as in the case of the glass-based approach) and is further immobilized on the surface of the magnetic beads (Figure 3A). After washing the beads with the help of a magnetic rack, the resuspended beads are mixed with DNA-NP (as in the case of the glass-based approach). In this case, the presence of the target is expected to immobilize the DNA-NP on the bead surface, while the excess unreacted DNA-NP can be washed away by magnetic separation. As a result, magnetic beads hybridized with the target and fluorescent NP were obtained. The complex can be resuspended and further detected by a fluorescence plate reader, avoiding the use of an expensive microscope. The results showed that an increase in the target concentration led to an increase in the fluorescence signal recorded by the plate reader (Figure 3B). Without the target, the signal was very low, close to the signal of only the magnetic beads. Thus, without the target, the DNA-NP does not interact with the magnetic beads, while the presence of the target triggers the hybridization of DNA-NP on the magnetic bead surface. This signal was clearly observed at 1 fM, while the estimated limit of detection was 0.3 fM. The results obtained were consistent with those obtained with immobilization on glass, indicating that target-triggered immobilization of DNA-NP is a robust nucleic acid detection method. However, in the case of using magnetic beads, comparable or even better sensitivity can be obtained using a simpler and cheaper fluorescence instrument. To verify the sequence specificity of oligonucleotide detection, the same bead-based experiment was performed in the presence of total RNA lysates from cells containing a large number of RNA sequences. Importantly, a similar dose-dependent response was observed in the presence of total RNA lysates from cells, so the large number of RNA sequences present in the lysate did not interfere with the hybridization process. These results confirmed that the detection of the target nucleic acid is sequence-specific. The limit of detection obtained was 1 fM, close to the limit of detection without lysate.

[0196] Example 2: Detection of target nucleic acids of different lengths optionally in the presence of a third probe

[0197] Viral RNA is a long and folded molecule, presenting regions that are difficult to access. Even in biological or clinical samples, viral RNA is usually present in the form of fragments generated by the breakdown of relatively unstable RNA molecules. Therefore, longer RNA sequences were studied. The first target (SEQ ID N°23) is a 100-mer (100 nt), in which the sequence complementary to the biotin-capturing DNA of SEQ ID N°2 is located within this sequence, while the sequence complementary to the DNA-NP of SEQ ID N°1 is located at its end. The second target (SEQ ID N°24) is 1000 nt, in which both target sequences are located within the entire sequence. They were tested using a glass surface and a bead-based method. The results showed that both methods could easily detect the 100-nt RNA target (Figures 4A and 4B), showing a clear dose-dependence in terms of the number of particles (glass surface method) or fluorescence intensity (bead method). However, it was noted that the number of particles and fluorescence intensity decreased in both cases compared to 48 nt. In fact, the values obtained when detecting the 48-nucleotide target were 10 to 100 times higher than those obtained when detecting the same concentration of 100-nucleotide long nucleic acids. Given that the target lengths were not very different, this significant decrease in the performance of the two techniques may be related to the fact that the complementary sequence captured by biotin is located within the entire 100-nt sequence, causing a steric problem for the interaction between the magnetic beads and streptavidin. On the other hand, the 1000-nt RNA target did not show an obvious response, although an increase in the signal of the sample with the target was observed compared to the control (Figures 4A and 4B). Thus, the combination of long targets with internal sequences complementary to the DNA-NP reduces the performance of our hybridization method, regardless of the nature of the immobilization surface (glass or bead). It is speculated that it is difficult for the DNA-NP to hybridize spatially with such targets because it requires an unfavorable steric turn in the RNA strand. Therefore, in the next step, a series of optimizations were carried out on the hybridization mechanism and the particles themselves, while focusing on the simpler bead-based method.

[0198] To improve the hybridization of the NP with the target sequence located within the entire sequence, the inventors designed a third probe, P3, named the "Post-it note" sequence, which is complementary to the target at one end and to the DNA-NP at the other end, connected by an A20 linker ( Figure 5A ). In this case, the steric problem of the DNA-NP was completely solved. To verify this method, this approach was tested on a 48-nucleotide target nucleotide sequence (SEQ ID N°6) from the N1 region of the SARS-CoV-2 virus, and it was found that the signal showed an obvious dose-dependent response, as Figure 5B shown. The application of this method to long RNA sequences is shown below.

[0199] Example 3: Influence of NP properties on sandwich hybridization sensitivity

[0200] To increase the signal obtained in this assay, the number of encapsulated dyes per particle was varied. Initially, the NP size increased. This was achieved by nanoprecipitating the same polymer at a higher salt concentration. In the presence of 50 mM NaCl in phosphate buffer, NPs with a polymer core of 52.8 nm were obtained according to DLS. TEM images showed a particle size of 35 ± 8 nm, corresponding to approximately 3200 dyes per particle. The absorption and fluorescence spectra of these NPs were similar to those of the parent smaller analogues. Given that these NPs exhibited a good fluorescence quantum yield (41%), they were expected to be approximately 3 times brighter than the original formulation. Their fluorescence brightness corresponded to 3200×0.41×125000 = 1.6×10 8 M -1 cm -1 . These brighter NPs were tested using a hybridization method with a sticky note-like (probe P3) DNA, and a significant enhancement in the signal intensity was found for all tested 48 nt target concentrations ( Figure 6A ). Thus, larger particles did provide a stronger signal. Next, using the larger NPs, the importance of dye loading was verified ( Figure 6B ). Using the same DNA sticky note method, it was observed that at target concentrations below 10 pM, NPs with a low dye loading of as low as 0.1 wt% and 1 wt% had poor signals. In contrast, at a loading of 10 wt%, especially 50 wt%, the signal was clearly observable at all tested 48 nt target concentrations, including 0.1 pM. It can be concluded that for the magnetic bead method, the high brightness of DNA-NP is of utmost importance: approximately 64 dyes per NP (1 wt% dye loading) was insufficient to obtain good assay performance, while good results were observed with approximately 640 dyes per NP (10 wt% loading). This may be because at low dye loading, the scattering signal of the magnetic beads was comparable to the signal of the bound NPs. However, for ultra-bright NPs, only a few hybridized DNA-NPs were sufficient to detect the signal, and thus a very low concentration of the target was required. Therefore, in subsequent experiments, the focus was on large NPs with a 50 wt% dye loading.

[0201] Example 4: Optimized conditions

[0202] By testing the A10 linker and the A20 linker, the importance of the linker between biotin and the capture sequence was determined (Haider et al., 2016). Longer linkers showed the advantage of lower background noise in the no-target control, thus improving the sensitivity of the method.

[0203] Finally, the optimized conditions included the following improvements: (1) increasing the particle size to 50 nm while maintaining a 50 wt% dye loading; (2) DNA sticky note (probe P3); (3) A20 linker for biotin-capturing DNA. Using this optimized method ( Figure 7A ), 100 nt RNA targets and 1000 nt RNA targets spiked into RNA lysates were tested.

[0204] In addition, the importance of the tetraethylene glycol (TEG) linker between the oligonucleotide and biotin was verified, which was reported to improve the capture of biotin by magnetic beads. In the case of the 100 nt target, excellent responses of the optimized assay were observed independent of the presence of the TEG linker ( Figure 7B ). The response was better compared to the original method: a detectable signal was clearly observed for a 0.01 pM target, while it was 1 pM for the original method. Most importantly, the new method was able to clearly detect 0.01 pM in the 1000 nt target, and the dose response for biotin capture using TEG was more pronounced. These results provide strong proof-of-concept for the detection of viral RNA by a new method combining ultrasbright DNA-NPs with magnetic bead capture.

[0205] Example 5: Testing of clinical samples

[0206] Clinical samples were then tested using the optimized RNA sensing assay. The RNA of SARS-CoV-2 is long and contains many potentially detectable target sequences. Three pairs of target sequences were identified in different parts of the viral genome. These SARS-CoV-2 sequences were: N1 (SEQ ID N°6, SEQ ID N°7, SEQ ID N°23, SEQ ID N°24), spike (SEQ ID N°13), and ORF1a (SEQ ID N°22). Three biotin-capturing (SEQ ID N°2, SEQ ID N°9, SEQ ID N°15) and three DNA sticky note probes (SEQ ID N°3, SEQ ID N°4, SEQ ID N°5, SEQ ID N°10, SEQ ID N°11, SEQ ID N°12, SEQ ID N°16, SEQ ID N°17, SEQ ID N°18) (including one of the above) were designed. It was expected that using 3 pairs of primers would significantly increase the probability of viral RNA capture and DNA-NP hybridization.

[0207] The clinical samples used were collected from the Microbiology Department of the University Hospital Center (CHU) of Strasbourg or purchased from CliniSciences. A total of 10 nasopharyngeal swab samples from qRT-PCR-positive SARS-CoV-2 patients and 10 nasopharyngeal swab samples from qRT-PCR-negative patients were included. All samples were received in inactivated transport medium. RNA extracts were prepared from all 20 samples using a minispin column kit.

[0208] First, the optimized assay was tested for its response to SARS-CoV-2 RNA in patient RNA extracts. The target concentration was varied by diluting the SARS-CoV-2 positive sample RNA extract into the SARS-CoV-2 negative sample while keeping the total RNA concentration constant. It was clearly observed that the SARS-CoV-2 positive RNA extract showed a stronger fluorescence signal relative to the SARS-CoV-2 negative extract ( Figure 8 ). Dilution of the positive sample caused the signal to gradually decrease, indicating that the response was dose-dependent. Notably, a significant response was still observed even after diluting the positive sample 10,000-fold (CT = 18.75), indicating that in theory, this method could detect SARS-CoV-2 RNA in the RNA extract with a sensitivity equivalent to approximately 30 PCR cycles.

[0209] Finally, an attempt was made to directly detect SARS-CoV-2 RNA from clinical samples without RNA extraction. The hypothesis was that magnetic beads commonly used for RNA extraction could effectively capture viral RNA with the help of biotinylated capture DNA. Additionally, the protocol included magnetic separation and washing prior to application of the DNA-NP to ensure that the buffer used in the original clinical sample did not damage the DNA-NP. Two series of clinical samples from two different sources were used: a commercial source (CliniSciences, CS series) and CHU from the university hospital (CH series). Direct detection of these samples showed that for the CS series, all three tested SARS-CoV-2 positive samples showed stronger signals compared to the three negative samples ( Figure 9A ). For the CH series, the responses of all five tested positive samples systematically showed higher signals compared to four out of five controls ( Figure 9B ), and thus only 1 false positive result was shown among the 10 tested CH samples. Then, dilutions of the samples were prepared in buffer, and it was found that in the case of SARS-CoV-2, the signal decreased with dilution, which was not the case for the controls ( Figure 9C ). These results confirmed the ability of the DNA-Np-based assay to directly detect SARS-CoV-2 in clinical samples without the need for a dedicated RNA extraction step.

[0210] In summary, the kit and method according to the present invention thus represent a highly sensitive nucleic acid sensing method. The target-driven immobilization of DNA-NPs on a solid surface allows the detection of RNA / DNA targets. The kit and method of the present invention are capable of achieving simple and automated high-throughput nucleic acid, especially RNA, detection in biological samples. Even at low RNA concentrations, allowing the LOD to be in the sub-fM range, the brightness of the NPs also plays a crucial role in obtaining excellent fluorescence signals.

[0211] For the detection of long nucleic acids, the presence of a third probe designated as an intermediate between the long target nucleic acid and the super-bright particles and an adapter of sufficient length for biotin-captured DNA are important, probably due to the slower diffusion kinetics and RNA folding blocking the accessibility of the target sequence from within the entire sequence. The optimized assay allows the detection of 1000 nt SARS-CoV-2 RNA fragments with fM sensitivity. In addition, the ability to detect SARS-CoV-2 in clinical samples was demonstrated, with a sensitivity equivalent to 19 to 30 RT-PCR cycles.

[0212] Finally, thanks to the combination of DNA-NPs with magnetic beads, the method is capable of directly detecting SARS-CoV-2 in clinical samples without a dedicated RNA extraction step. This work presents a new method for simple and rapid RNA detection, where super-bright DNA-NPs provide the highest sensitivity, and the use of magnetic beads allows bypassing RNA extraction and allows for a simple detection mode using a plate reader.

[0213] In summary, the inventors designed a highly sensitive RNA sensing method based on ultra-bright DNA-functionalized dye-loaded polymeric NPs, magnetic beads, and corresponding primers. Target-driven immobilization of DNA-NPs on a solid support allows detection of RNA / DNA targets on a glass surface by fluorescence microscopy and directly on magnetic beads by a fluorescence plate reader. The latter method is of particular interest as it enables simple and automated high-throughput RNA detection in biological samples. Even at low RNA concentrations, allowing LODs in the fM range and even sub-fM range, the brightness of the NPs plays a crucial role in obtaining excellent fluorescence signals. Additionally, detection of long RNAs is considered challenging, probably due to slower diffusion kinetics and RNA folding that may hinder accessibility of the target sequence from within the entire sequence. Therefore, a DNA sticky note-like (probe P3) capture sequence was employed as an intermediate between the long RNA and our DNA-NPs to ensure sufficient linker length for biotinylated capture DNA. The optimized assay allows detection of 1000 nt SARS-CoV-2 RNA fragments with fM sensitivity. Additionally, the ability to detect SARS-CoV-2 in clinical samples was demonstrated, with sensitivity equivalent to 19 to 30 RT-PCR cycles. Finally, thanks to the binding of DNA-NPs to magnetic beads, the method enables direct detection of SARS-CoV-2 in clinical samples without a dedicated RNA extraction step. This work presents a new method for simple and rapid RNA detection, where ultra-bright DNA-NPs provide the highest sensitivity, while the use of magnetic beads allows bypassing RNA extraction and allows a simple detection mode using a plate reader. The method can be easily extended to other types of RNAs, enabling rapid molecular diagnosis of various diseases, particularly viral infections and cancer.

[0214] Example 6: Detection of microRNA targets

[0215] To detect microRNA (miRNA) targets that are 20 - 22 bases in length, the inventors designed a simple system based on hybridization between complementary sequences. The system consists of a capture sequence (10 - 12 bases) complementary to one end of the miRNA target on a glass surface and NPs (ULPs) coated with a nucleic acid (10 to 12 bases) complementary to the other end of the miRNA target as a detection probe. Locked nucleic acid (LNA) was incorporated into the capture sequence on the glass surface to enhance the stability of the short duplex. LNA was not incorporated into the complementary oligonucleotide on the NP surface as the formed duplex was already stable enough, which may be attributed to the cooperative effect of adjacent DNAs grafted to the NPs.

[0216] The oligonucleotides used in the examples and their respective nucleic acid sequences are as Figure 10As shown, RNA or DNA, DNA target), biotin capture and recognition sequence (DBCO miR): See Table 2 and the sequence listing attached to this patent application.

[0217] In this patent application, the symbol "T" represents thymine in DNA, and the symbol "U" represents uracil in RNA. In accordance with the requirements of the ST26 standard, in the attached sequence listing, the symbol "T" will be interpreted as thymine in DNA and uracil in RNA.

[0218] In this patent application, in SEQ ID N°27 and SEQ ID N°31 of Table 2, locked nucleic acids are represented by bold and underlined characters. In accordance with the requirements of the ST26 standard, in the attached sequence listing, modified nucleotides (such as locked nucleic acids) are represented as the corresponding unmodified nucleotides in the sequence.

[0219]

[0220] Table 2

[0221] The target oligonucleotide hybridizes with the capture DNA-biotin and is further immobilized on the glass surface. After washing the glass surface, the particles are observed under a microscope.

[0222] When the target is present, it hybridizes with the surface capture sequence at one end and the complementary DNA of the NP at the other end, immobilizing the NP in this way and allowing detection by microscopy. In the absence of the target, the NPs remain in solution and are therefore washed away.

[0223] The LOD (limit of detection) of the target as shown in Figure 10 was calculated to be 4.5 fM for miR200a and 2 fM (DNA version) for miR21. The results for the RNA versions of the microRNAs were very similar.

[0224] These results indicate that relatively short oligonucleotides linked to NPs (10 to 12 bases) are sufficient to detect microRNAs in a sandwich assay.

[0225] References:

[0226] Cai H. et al., Optofluidic analysis system for amplification-free, direct detection of Ebola infection. Sci Rep 5, 14494 (2015).

[0227] Egloff S. et al., Enzyme-free amplified detection of cellular microRNA by light-harvesting fluorescent nanoparticle probes. Biosens Bioelectron 179, 113084 (2021)

[0228] Haider M. et al., A Double-Hybridization Approach for the Transcription-and Amplification-Free Detection of Specific mRNA on a Microarray, Microarrays, Mar. 5(1):5, 2016.

[0229] Lim S.H. et al., Quantitative Analysis of Nucleic Acid Hybridization on Magnetic Particles and Quantum Dot-Based Probes. Sensors 9, 5590–5599 (2009).

[0230] Melnychuk&Klymchenko, DNA-Functionalized Dye-Loaded Polymeric Nanoparticles: Ultrabright FRET Platform for Amplified Detection of Nucleic Acids. J. Am. Chem. Soc. 140, 10856–10865 (2018).

[0231] Melnychuk et al., Light-Harvesting Nanoparticle Probes for FRET-Based Detection of Oligonucleotides with Single-Molecule Sensitivity. Angewandte Chemie International Edition 59, 6811–6818 (2020).

[0232] Ngo H.T. et al., Direct Detection of Unamplified Pathogen RNA in Blood Lysate using an Integrated Lab-in-a-Stick Device and Ultrabright SERS Nanorattles. Sci Rep 8, 4075 (2018).

[0233] Reisch A. et al., Collective fluorescence switching of counterion-assembled dyes in polymer nanoparticles. Nat Commun 5, 4089 (2014).

[0234] Reisch A. et al., Tailoring Fluorescence Brightness and Switching of Nanoparticles through Dye Organization in the Polymer Matrix. ACS Appl. Mater. Interfaces 9, 43030 - 43042 (2017).

[0235] Reisch A. et al., Charge-Controlled Nanoprecipitation as a Modular Approach to Ultrasmall Polymer Nanocarriers: Making Bright and Stable Nanoparticles. ACS Nano 9, 5104 - 5116 (2015).

[0236] Severi C. et al., Smartphone-assisted detection of nucleic acids by light-harvesting FRET-based nanoprobe. Biosensors and Bioelectronics 168, 112515 (2020)

[0237] Zheng Z. et al., Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood. Clin Chem 52, 1294 - 1302 (2006).

Claims

1. A kit for detecting a target nucleic acid in a sample, wherein the kit at least comprises: i) Probe P1, which comprises or consists of a nucleic acid fragment NA1 linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, ii) A functional unit F2 bound to a solid surface, wherein F2 exhibits a high affinity for F1 or is covalently bound to F1, iii) Probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, said super-bright luminescent particle exhibiting a brightness of at least 10 7 M -1 cm -1 , preferably at least 2×10 7 M -1 cm -1 , more preferably at least 5×10 7 M -1 cm -1 , 10×10 7 M -1 cm -1 or at least 40×10 7 M -1 cm -1 in brightness.

2. The kit according to claim 1, wherein the solid surface is selected from: an immobilized surface and the surface of solid particles, and the solid particles are preferably magnetic beads or glass beads.

3. The kit according to any one of the preceding claims, wherein the super-bright luminescent particles are fluorescent polymeric nanoparticles loaded with super-bright dyes.

4. The kit according to any one of the preceding claims, wherein the nucleic acid fragment NA2 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid.

5. The kit according to any one of claims 1 to 3, which comprises at least one probe P3, which comprises or consists of: a first part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein T3 is different from T1, and a second part, which comprises or consists of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, wherein the first part and the second part are joined by a nucleotide linker.

6. The kit according to claim 5, which comprises at least two probes P1, each of the probes P1 comprising or consisting of: - A nucleic acid fragment NA1, which is at least respectively designated as NA1-1 or NA1-2, and is respectively complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid.

7. The kit according to claim 5 or 6, which comprises: i) At least two probes P3, which comprise or consist of: a first part, which comprises or consists of a nucleic acid fragment having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are joined by a nucleotide linker, and ii) At least one probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to super-bright luminescent particles.

8. A method for detecting a target nucleic acid molecule in a sample, wherein the method at least comprises the following steps: a) Under conditions suitable for hybridization of complementary nucleic acid sequences and suitable for formation of non-covalent complexes of nucleic acids, bringing into contact at least the following substances: - Probe P1, which comprises or consists of a nucleic acid fragment NA1 linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, - A functional unit F2 bound to a solid surface, wherein F2 exhibits a high affinity for F1 or is covalently bound to F1, and - Probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle, said particle exhibiting a brightness of at least 10 7 M -1 cm -1 to form a mixture b) Applying a physical external force to the mixture of step a) to separate the non-covalent complexes of the nucleic acids, and c) Measuring the luminescence emission intensity associated with the non-covalent complex of the nucleic acid.

9. The method according to claim 8, wherein the solid surface is the surface of solid particles, preferably magnetic beads or glass beads.

10. The method according to any one of claims 8 or 9, wherein the super-bright luminescent particles are fluorescent polymeric nanoparticles loaded with super-bright dyes.

11. The method according to any one of claims 8 to 10, wherein the nucleic acid fragment NA2 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T2 of the target nucleic acid.

12. The method according to any one of claims 8 to 11, wherein step a) comprises contacting at least the following substances: - At least one probe P1, which comprises or consists of a nucleic acid fragment NA1 linked to a functional unit F1, wherein NA1 comprises or consists of a nucleotide sequence complementary to the nucleotide sequence of region T1 of the target nucleic acid, - A functional unit F2 bound to the solid surface, wherein F2 exhibits a high affinity for F1 or is covalently bound to F1, and - At least one probe P2, which comprises or consists of a nucleic acid fragment NA2 covalently linked to a super-bright fluorescent particle, wherein the nucleic acid fragment NA2 comprises or consists of a nucleotide sequence complementary to probe P3, and - At least one probe P3, which comprises or consists of: a first part, which comprises or consists of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are joined by a nucleotide linker.

13. The method according to claim 12, wherein step a) comprises contacting at least the following substances Two probes P1, wherein each of the probes P1 comprises or consists of: a nucleic acid fragment NA1, wherein each of the NA1 is designated as NA1-1 or NA1-2 and is respectively complementary to the nucleotide sequence T1-1 or T1-2 of the target nucleic acid.

14. The method according to claim 12 or 13, wherein step a) comprises contacting the following substances i) At least two probes P3, which comprises or consists of: a first part, which comprises or consists of a nucleic acid fragment NA4 having a nucleotide sequence complementary to the nucleotide sequence of NA2, and a second part, which comprises or consists of a nucleic acid fragment NA3 having a nucleotide sequence complementary to the nucleotide sequence of region T3 of the target nucleic acid, wherein the first part and the second part are joined by a nucleotide linker, and ii) At least one probe P2, which comprises or consists of a nucleic acid fragment NA2 linked to a super-bright luminescent particle.

15. Use of the kit according to any one of claims 1 to 7 or the method according to any one of claims 8 to 14 for detecting a target nucleic acid.

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