Method for digital multiplexed detection and / or quantification of biomolecules and uses thereof - Patents.com

Abstract

The present invention relates to a digital multiplex method for detecting and / or quantifying multiple target biomolecules in a sample, wherein the biomolecules are selected from DNA, RNA, and proteins. The present invention further relates to various applications and kits of the digital multiplex method.

Description

[Technical field]

[0001] The present invention relates to a digital multiplex method for detecting and / or quantifying multiple target biomolecules such as DNA, RNA, and proteins in a sample based on isothermal amplification with specific molecular design. The present invention further relates to a method for diagnosis of diseases selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, skeletal muscle diseases, dental diseases, and prenatal diseases, as well as a method for agricultural diagnosis. The present invention also relates to a method for detecting biomarkers (biomolecules) in the food and agriculture food sector and in the environment. All such diagnostic and detection methods include using the digital multiplex method of the present invention. The present invention also relates to a kit for detecting and / or quantifying multiple target biomolecules, comprising particles functionalized with oligonucleotides, an enzyme, and a separation agent. [Background technology]

[0002] Several biomolecules, such as DNA, RNA, or proteins, are used as attractive diagnostic, prognostic, or predictive biomarkers. There is accumulating clinical evidence that such molecules, and especially nucleic acids, are closely related to various diseases (cancer, neurological or cardiovascular diseases, diabetes, etc.). Moreover, such molecules, and especially nucleic acids, are present in body fluids and therefore accessible via minimally invasive liquid biopsies (serum, plasma, urine). Circulating biomarkers can be repeatedly evaluated, allowing regular follow-up of treatment and recurrence, large population screening, or early diagnosis. However, the use of circulating nucleic acids, especially RNA, more specifically miRNA, as clinical markers remains challenging because it requires quantification of target molecules that are highly diluted in complex biological fluids. Moreover, in many cases, more than one target needs to be measured to establish a diagnosis. Therefore, precise measurement of a set of nucleic acid biomarkers has become a significant bottleneck, prompting the development of sensitive and quantitative multiplexed detection techniques. The most commonly used techniques for sensitive detection of some proteins are enzyme-linked immunosorbent assay (ELISA) and, for nucleic acids, polymerase chain reaction (PCR) for DNA detection and reverse transcription quantitative polymerase chain reaction (RT-qPCR) for RNA detection. Although all these techniques allow specific and sensitive detection, they have some drawbacks and need to be enhanced, especially for sensitive and quantitative multiplex detection.

[0003] For example, currently, quantification of ADN by PCR involves amplification of the target nucleic acid, which can introduce carryover contamination and reduce the specificity and sensitivity of the detection method. Classical methods for detecting RNA, especially miRNA, include DNA (or LNA-locked nucleic acid) microarrays ([1]-[5]) and PCR (polymerase chain reaction)-based techniques (RT qPCR-reverse transcription quantitative PCR or RT dPCR-RT digital PCR, [6]-[9]). Microarrays offer a large multiplex capacity (up to hundreds of targets). However, microarrays suffer from poor sensitivity and specificity. Moreover, these methods do not allow efficient quantification, since the results are highly dependent on the experimental conditions. Conversely, PCR-based methods show good sensitivity, but the simultaneously detected targets are limited to a few species at best. In addition, PCR-based methods require multi-step procedures, data normalization, and / or standard calibration, all of which introduce significant biases. Taken together, both techniques are not optimal for multiplexed and quantitative detection and / or measurement of highly diluted targets. The above mentioned drawbacks can be overcome using digital procedures. Digital techniques involve distributing the reaction sample into numerous small compartments, e.g. microscopic droplets, in which the presence of individual molecules is reported and direct counting is possible. Digital techniques have several advantages: in particular i) they are compatible with end-point assays, without the need for continuous monitoring of the reaction; ii) they provide absolute quantification without the use of calibration standards; iii) they provide ultimate sensitivity; iv) they also improve the sensitivity and specificity of the assay, especially for rare targets in a complex background.

[0004] For example, digital PCR (dPCR (

[19] ,

[20] )) is based on the isolation and amplification of individual targets in microcompartments, allowing absolute quantification by digital single-molecule counting. The digital readout provides absolute quantification without the need for assay calibration. It exploits the compartmentalization of single target molecules prior to signal amplification. As a result, only compartments receiving the target will be amplified and display a positive (1) signal, while other compartments remain negative (0) (hence the term "digital"). By applying Poisson's law, exact concentrations can be calculated with much higher accuracy than analog readouts based on standard calibration. However, digital PCR retains the drawbacks of PCR amplification chemistry with regard to laborious protocol optimization, sensitivity to inhibitors, and quantification bias due to the reverse transcription step required for nucleic acid detection, especially microRNA detection (

[10] -

[13] ). Moreover, multiplexed formats of digital assays have not been fully explored and are limited to combinations of three to five biomarkers due to crosstalk reactions between primer pairs and limited choice of spectrally resolved fluorescent probes, respectively (

[14] -

[16] ).

[0005] Sensitive detection of nucleic acids (DNA, RNA) is mainly based on target amplification by PCR-based techniques. Although its sensitivity is very high (down to single molecules), this technique requires careful primer and probe design, optimization of thermocycling protocols, and expensive equipment. In addition, the reverse transcription step required for RNA detection is known to introduce quantification bias

[13] . The detection of ribonucleic acids requires an additional reverse transcription step to convert the RNA sequence into a DNA strand that can be used for PCR amplification, thus biasing the quantification. Isothermal alternatives have been proposed, some of which do not rely on a reverse transcription step, but the exponential amplification mechanism is prone to leaky reactions that result in non-specific amplification

[17] . This inherent non-specific reactivity prevents these techniques from being used in routine analysis. State-of-the-art protein and small analyte detection relies on ELISA (enzyme-linked immunosorbent assay) techniques that use antigen / antibody recognition, which has sensitivity limitations and is only suitable for detecting high concentrations of analytes. It is noted that coupling antibody recognition with nucleic acid amplification improves the specificity and sensitivity of immunoassays (immuno-PCR

[18] ).

[0006] Multiplex PCR and multiplex ELISA assays have been described, which are generally based on orthogonal probes and / or amplification chemistries and limit the number of targets to fewer than 10 (typically 3-5). Another common problem with multiplex assay-based reactions such as PCR is that each target biomolecule to be detected requires an independent exponential reaction. For example, to measure three targets, three amplification reactions must be optimized to work without affecting each other under the same experimental conditions. The use of multiple orthogonal amplification systems leads to complex experimental designs and tedious optimization of reaction conditions. Microarrays, on the other hand, are based on spatially resolved capture of targets, allowing the detection of hundreds of markers at once. Despite the high multiplexing capacity of such arrays, the readout is analogue; the average concentration of the sample is calculated from the number of bound targets, which limits the sensitivity. In addition, data normalization is biased, resulting in poor quantification of the measurements.

[0007] Thus, although digital assays offer clear advantages in terms of ease of use and quantification, multiplexed approaches are needed to enable better diagnostics. Digital assays in multiplexed formats have not been well explored and are limited to very few combinations (in the best case, 3–6 biomarkers

[16] ). For non-DNA targets, digital PCR suffers from the same problems associated with qPCR (i.e., conversion steps, complex protocol design, thermocycling, enzyme inhibition). The aim of the present invention is to provide a digital multiplex detection and / or quantification method that allows absolute quantification and higher sensitivity in detecting and / or quantifying multiple types of biomolecules used as biomarkers, such as biopolymers, in particular nucleic acids, but does not have the above-mentioned drawbacks. It is also an aim to simplify the design of multiplex assays by using a single amplification mechanism for all targets. Summary of the Invention

[0008] The inventors of the present invention have previously developed an analog method for eliminating background amplification for detecting nucleic acids, which is also adapted to detect multiple nucleic acids (WO2017140815). The inventors of the present invention have surprisingly found that they can successfully digitize this analog multiplex method to obtain a digital multiplex method that allows increasing the detection sensitivity of multiple biomolecules, preferably used as biomarkers, and accurately quantifying such biomolecules in a single measurement without the need to perform several assays.

[0009] Thus, according to a first aspect, the present invention provides a digital multiplex method for detecting and / or quantifying a plurality of biomolecules in a sample, comprising: a) functionalizing a suspension of particles, preferably microparticles, with one or more oligonucleotides selected from a first oligonucleotide that is a conversion oligonucleotide (cT), a second oligonucleotide that is a report oligonucleotide (rT), a third oligonucleotide that is an amplification oligonucleotide (aT), and a fourth oligonucleotide that is a leak absorption oligonucleotide (pT); b) adding a barcode to the particles functionalized in step a) that allows for the identification of particles targeted to multiple biomolecules; c) contacting the particles obtained in step b) with a test sample to capture a plurality of target biomolecules; d) resuspending particles that have or have not captured target biomolecules in a common amplification mixture that includes a buffer, an enzyme, deoxynucleoside triphosphates (dNTPs), and optionally oligonucleotides; e) separating the particles in the suspension obtained in step d) from one another so that each particle can react independently; f) incubating the particles at a constant temperature so that each target biomolecule induces an amplification reaction that generates an amplified signal on the target-carrying particles; and g) detecting and / or measuring the signals of the particles, including the barcode signal and the amplified signal of each particle The present invention relates to a digital multiplexing method comprising the steps of:

[0010] The method of the present invention allows multiplex and digital detection of biomolecular targets, especially nucleic acid targets, thus allowing access to the absolute concentration of multiple targets in a single assay. Moreover, the method allows the particles to be washed after the capture step c), which allows the direct quantification of the biomolecular targets of interest from complex media such as biological samples. Indeed, in standard assays such as PCR or other in-solution isothermal amplification chemistries, the sample mixture and the amplification mixture are usually contaminated by impurities present in the sample. Therefore, the nucleic acid molecules must be purified before quantification. In the digital multiplex method of the present invention, since the particles irreversibly capture the target biomolecules, no additional extraction / purification steps are required before the particles are contacted with the test sample.

[0011] Another advantage of the digital multiplex detection and / or quantification method of the present invention is that the dynamic range can be adjusted according to the abundance of target biomolecules in the sample.In fact, the sensitivity of the method mainly depends on the particle number / target number ratio (used to calculate concentration from Poisson's law).Therefore, the method of the present invention allows the particle number to be adjusted according to the (expected) abundance of target biomolecules, which allows more accurate detection and control of the amount of substance used. Furthermore, advantages of the method of the present invention compared to RT-PCR analogue methods, especially when detecting and / or quantifying RNA, are that 1) it does not require a reverse transcription step that may hinder the quantitative nature of the assay, 2) it does not require the high temperatures and temperature cycles required for the PCR step, and 3) isothermal amplification is robust against the numerous chemicals that may be found in crude samples and that have been shown to inhibit PCR reactions (

[30] and [4]), avoiding cross-contamination issues since the target biomolecules are not amplified.

[0012] Yet another advantage of the digital multiplex method of the present invention is that its design is simplified by the use of a single amplification mechanism for any multiple species of biomolecules. The inventors have also demonstrated that the methods of the present invention can be used to directly detect biomolecules from any type of sample that contains them, including blood samples and other biological fluids. The specificity, sensitivity, simplicity and rapidity of the digital multiplex method of the present invention allows the method to be used in medical diagnostic methods, in particular in the diagnosis of diseases such as cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases and prenatal diseases. Also, according to a second aspect, the present invention relates to an in vitro method for the diagnosis of a disease comprising or selected from the group consisting of cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases and prenatal diseases, said diagnostic method comprising using the digital multiplex method of the present invention.

[0013] The specificity, sensitivity, simplicity and rapidity of the digital multiplex method of the present invention also allows the method to be used in agricultural diagnostic methods, especially for the diagnosis of diseases caused by biotic stresses, such as infectious and parasitic diseases, or diseases caused by abiotic stresses, such as nutritional deficiencies or adverse environments. According to a third aspect, the present invention also provides a method for producing ... composition comprising the steps of: - diseases caused by biotic stress, preferably of infectious and / or parasitic origin, or - Diseases caused by abiotic stress, preferably nutritional deficiencies and / or adverse environments An in vitro method for the agricultural diagnosis of a disease selected from the group comprising: The present invention also relates to in vitro methods comprising using the digital multiplexing method of the present invention. Kits for carrying out the methods of the invention are also provided.

[0014] Thus, according to a fourth aspect, the present invention provides a kit for detecting and / or quantifying a plurality of target biomolecules, comprising: a) a suspension of particles, preferably microparticles, functionalized with one or more oligonucleotides selected from a first oligonucleotide that is a conversion oligonucleotide (cT), a second oligonucleotide that is a report oligonucleotide (rT), a third oligonucleotide that is an amplification oligonucleotide (aT), and a fourth oligonucleotide that is a leak-absorbing oligonucleotide (pT), and having attached thereto a barcode that allows for the identification of particles that target multiple biomolecules; b) a mixture of enzymes, preferably selected from the group comprising polymerases, nicking or restriction enzymes, exonucleases, and deoxynucleoside triphosphates (dNTPs), and optionally oligonucleotides, and c) Separation agent The present invention relates to a kit comprising: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] As indicated above, the present invention relates to adapting recent background-free amplification chemistry to a supported format (i.e., at least a part of the chemical reaction is carried out on a support) and digital readout, thus providing absolute quantification of multiple target biomolecules. Said digitization allows the detection and / or quantification of multiple biomolecules, preferably those used as biomarkers, with increased sensitivity and accuracy without the need to perform several assays.

[0016] Digital multiplex method for detecting and / or quantifying multiple biomolecules - Patent Application 20070123633 Thus, according to a first aspect, the present invention provides a digital multiplex method for detecting and / or quantifying a plurality of biomolecules in a sample, comprising: a) functionalizing a suspension of particles, preferably microparticles, with one or more oligonucleotides selected from a first oligonucleotide that is a conversion oligonucleotide (cT), a second oligonucleotide that is a report oligonucleotide (rT), a third oligonucleotide that is an amplification oligonucleotide (aT), and a fourth oligonucleotide that is a leak-absorbing oligonucleotide (pT); b) adding a barcode to the particles functionalized in step a) that allows for the identification of particles targeted to multiple biomolecules; c) contacting the particles obtained in step b) with a test sample to capture a plurality of target biomolecules; d) resuspending particles that have or have not captured target biomolecules in a common amplification mixture that includes a buffer, an enzyme, deoxynucleoside triphosphates (dNTPs), and optionally oligonucleotides; e) separating the particles in the suspension obtained in step d) from one another so that each particle can react independently; f) incubating the particles at a constant temperature so that each target biomolecule induces an amplification reaction that generates an amplified signal on the target-carrying particles; and g) detecting and / or measuring the signals of the particles, including the barcode signal and the amplified signal of each particle The present invention relates to a digital multiplexing method comprising the steps of: The digital method according to the invention allows the simultaneous detection and / or quantification of multiple biomolecules, said biomolecules having the same or different structures and functions, and therefore the method of the invention is called a "multiplex" method.

[0017] In the context of the present invention, the term "biomolecule" relates to large macromolecules or biopolymers such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products. Biomolecules in the present invention are usually endogenous, but may also be exogenous (e.g., biopharmaceutical drugs). According to one preferred embodiment of the digital multiplex method of the invention, the biomolecules are selected from the group comprising proteins and nucleic acids, said proteins being preferably enzymes. In the context of the present invention, the term "enzyme" refers to a protein that has a catalytic function and is capable of catalyzing the conversion of a molecular substrate. The group of enzymes that can be detected and / or quantified by the digital multiplex method of the present invention is selected from nicking enzymes, restriction endonucleases, ADN N-glycosylases, AP-endonucleases, exonucleases ARN / ADN polymerases, ligases, and methylases. More specifically, the digital multiplex method of the present invention can be implemented to identify and / or quantify nicking enzymes and restriction endonucleases.

[0018] More preferably, the biomolecular targets of the present invention are nucleic acids, such as DNA, cDNA, RNA, mRNA, microRNA, and even more preferably, they are ribonucleic acids (RNA). In the context of the present invention, the term "nucleic acid" relates to a biopolymer or small biomolecule composed of nucleotides, which are monomers made of three components: a five-carbon sugar, a phosphate group, and a nitrogenous base. If the sugar is the compound ribose, the polymer is RNA (ribonucleic acid), and if the sugar is deoxyribose, which is derived from ribose, the polymer is DNA (deoxyribonucleic acid). As mentioned above, the digital multiplex methods of the present invention can be used to detect and / or quantify DNA molecules or complementary DNA (cDNA) encoding molecules. Even more preferably, the digital multiplex method of the present invention is used to detect and / or quantify ribonucleic acid (RNA) molecules selected from messenger RNA (mRNA), small interfering RNA (siRNA), and microRNA (miRNA).

[0019] According to a most preferred embodiment, the digital multiplex method of the present invention can be used to detect and / or quantify microRNA molecules. In the context of the present invention, the term "microRNA" refers to endogenous short non-coding RNA strands having a length of about 22 nucleotides that are involved in the post-transcriptional regulation of gene expression. To carry out the method of the invention, in a first step (step a)), it is necessary to functionalize a suspension of particles, preferably microparticles, with one or more oligonucleotides selected from a first oligonucleotide which is a conversion oligonucleotide (cT), a second oligonucleotide which is a report oligonucleotide (rT), a third oligonucleotide which is an amplification oligonucleotide (aT) and a fourth oligonucleotide which is a leak-absorbing oligonucleotide (pT). According to one embodiment, the particles may be functionalized with two oligonucleotides: a conversion oligonucleotide (cT) and a report oligonucleotide (rT). According to another embodiment, the particles are functionalized with three types of oligonucleotides: a conversion oligonucleotide (cT), an amplification oligonucleotide (aT), and a leak-absorbing oligonucleotide (pT); a conversion oligonucleotide (cT), a report oligonucleotide (rT), and an amplification oligonucleotide (aT); a conversion oligonucleotide (cT), a report oligonucleotide (rT), and a leak-absorbing oligonucleotide (pT). According to yet another embodiment, the particles are functionalized with four types of oligonucleotides: a conversion oligonucleotide (cT), a report oligonucleotide (rT), an amplification oligonucleotide (aT), and a leak-absorbing oligonucleotide (pT).

[0020] In the context of the present invention, the term "conversion oligonucleotide" or "target-specific conversion oligonucleotide" or "conversion template" or "cT" refers to an oligonucleotide that converts a target biomolecule into a universal short oligonucleotide (also referred to herein as "signal" or "signal sequence"). The conversion template may or may not be protected from degradation. According to one preferred embodiment of the method of the present invention, the conversion template can be designed in a specific manner depending on the biomolecule to be detected. For example, when detecting a given microRNA, the most 3' part of the conversion template is complementary or partially complementary to this microRNA target. The 5' part corresponds to the complementary sequence of the output (signal) sequence. Between these two elements, a nicking recognition / cleavage site is introduced so that the strand polymerized along the template using the microRNA as a primer is nicked to release the output sequence. In the case of the nicking enzyme Nt.BstNBI (used in the example described below), this sequence can be 5'-NNNNGACTC-3' (where N is one of the four standard DNA bases).

[0021] Optionally, when designing the conversion template, a spacer sequence may be included between the biomolecule binding site and the nicking recognition site. The spacer sequence comprises an oligodeoxyribonucleotide sequence of 1 to 20 nucleotides, preferably 4 to 12 nucleotides, more preferably 5 to 10 nucleotides. In one embodiment, the spacer sequence is composed of a sequence lacking at least one of four types of nucleotides (e.g., T, A, C, but not G). In particular, the spacer is a poly(T) spacer. In the context of the present invention, the term "poly(T) spacer" refers to a poly(T) homopolymer spacer having a length comprised between 1 and 20 nucleotides, preferably between 4 and 12 nucleotides, more preferably between 5 and 10 nucleotides. According to a particularly preferred embodiment, the conversion template contains a poly(T) spacer at the target biomolecule binding sequence (e.g., miRNA binding sequence) and at the nicking enzyme (e.g., Nt.BstNBI) recognition site, thus increasing the number of nucleotide DNA on the primer obtained after extension and nicking. This modified design of the conversion template allows for a significant reduction or even elimination of non-specific amplification (also called background amplification), thus increasing the sensitivity and reliability of the method of the invention.

[0022] Various examples of conversion template designs are given below. [Table 1]

[0023] In the context of the present invention, the term "report oligonucleotide" or "report probe" or "report template" or "rT" refers to an oligonucleotide that converts the presence of a trigger (signal sequence) sequence into a detectable signal. Such detectable signals are, for example, fluorescent signals, electrochemical signals, particle agglutination, colorimetric signals, preferably fluorescent signals. Thus, the report probe is preferably a fluorescent probe. In one embodiment, the report probe is a self-complementary structure modified at both ends by a fluorophore and / or a quencher. As used herein, the term "self-complementary" means that two different parts of the same molecule can hybridize with each other because they are base-complementary (AT and GC). In the present invention, the two ends of the single-stranded probe (a few nucleotides at 3' and 5' portions) can hybridize with each other and induce the quenching of the fluorophore by the quencher.

[0024] The reporting probe may also include a loop that contains a nicking recognition site. Compared to PCR probes (used for detection) used in the prior art that must be designed (sequence) and optimized (length) for each target, the reporting probes used in the digital multiplexing method of the present invention are modular, meaning that the reporting probes can be used for any target with signal transduction by the appropriate bistable module (autocatalytic template + pseudotemplate as described below). In the context of the present invention, the term "amplification oligonucleotide" or "autocatalytic template" or "aT" refers to an oligonucleotide that can exponentially amplify a trigger (signal sequence). Examples of oligonucleotides that can function as amplification oligonucleotides are shown in Table 1 below.

[0025] According to one embodiment of the digital method of the present invention, the amplification oligonucleotides contain a partially repeated structure that includes a nicking enzyme recognition site, and the leak-absorbing oligonucleotides can bind to the products of polymerization along the amplification oligonucleotides, extend them, inactivate them, and slowly release them, thereby inducing a threshold. According to one embodiment of the digital multiplex method of the present invention, the 3' ends of the amplification oligonucleotides exhibit reduced affinity for the amplified sequence. In the context of the present invention, the term "leak-absorbing oligonucleotide" or "pseudo-template" or "pT" refers to an oligonucleotide that binds to the amplified sequence more strongly than the autocatalytic template and drives the addition of a few nucleotides at the 3' of the amplified sequence, thus inactivating the amplified sequence with respect to further priming against the autocatalytic template (since the 3' end of the inactivated amplified sequence is no longer a mismatch to the autocatalytic template). The leak-absorbing oligonucleotide drives the inactivation of the trigger (signal sequence) synthesized by the leaky reaction, thus allowing the avoidance of non-specific amplification (i.e., amplification occurring in the absence of the amplified signal sequence), also referred to herein as background amplification. The pseudo-template needs to be protected from exonuclease degradation. This is done using a few phosphorothioate modifications at the 5' end (or other exonuclease blocking capabilities such as biotin-streptavidin modifications or modified bases). Examples of leak-absorbing oligonucleotides are shown in Table 1 below.

[0026] In yet another embodiment, the 3' end of the leak-absorbing oligonucleotide is complementary to the sequence amplified by the amplification oligonucleotide, and the 5' end of the leak-absorbing oligonucleotide serves as a template for adding an inactivation tail to the amplified sequence. In one embodiment for carrying out the present digital multiplex method, the concentrations of the amplification and leak-absorber oligonucleotides are selected such that the reaction of the amplification oligonucleotides is faster than the reaction of the leak-absorber oligonucleotides with high concentrations of amplified sequences, but the reaction to the leak-absorber oligonucleotides is faster than the reaction of the amplification oligonucleotides with low concentrations of amplified sequences, thereby effectively eliminating amplification unless the stimulation threshold is exceeded.

[0027] Each oligonucleotide used in the digital multiplex method of the present invention ultimately has a specific function that allows the conversion of a target biomolecule and the amplification of the resulting signal without simultaneously inducing background amplification (i.e., amplification that occurs in the absence of an amplified signal sequence). Examples of various oligonucleotides (templates) for use in the digital multiplexing methods of the present invention are shown in Table 1 below and in the Examples section of this application.

[0028] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0029] In Table 1 above, biotin and bioteg refer to biotinylated synthons using the aminoethoxy-ethoxyethanol linker and the longer triethylene glycol linker, respectively. * " indicates a phosphorothioate backbone modification and "p" indicates a 3' phosphate modification. In SEQ ID NOs: 16-23 and 30, thymidine (T) is replaced with deoxyuridine (U) to avoid nicking of the polymerization product of the signal on the template. Atto633, FAM, Cy5, HEC, BMN3, Cy3.5 are fluorophores and BHQ2, BHQ1, and BMNQ530 are quenchers. Based on the sequences listed above and the examples described herein, various combinations of templates can be used to carry out the digital multiplex method of the present invention. Starting from this specification and general knowledge of nucleotide design, a person skilled in the art will be able to determine other templates and other combinations to carry out the method of the present invention on any target biomolecule of interest.

[0030] In particular, the selection of the template sequence depends on experimental parameters such as the operating temperature, the rate and specificity of the enzyme used, especially the nickase. Preferably, an amplification template containing a Nb.BsmI nickase site and / or a template operating with Nt.BstNBI or Nb.BssSI can be used. However, other nicking enzymes can be used, including, among others, Nt.BspQI, Nt.CviPII, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, and Nt.BsmAI. More than one nickase and more than one nickase recognition site can be used in the same mixture. In the context of the present invention, functionalization of a particle means immobilizing or attaching any one of the oligonucleotides to the particle. Therefore, the oligonucleotide used in the digital method of the present invention can be modified to be fixed to particle surface.For example, in the method of the present invention, oligonucleotide can be attached to particle using biotin-avidin linkage, but oligonucleotide can be attached to particle using many other grafting chemistries, including but not limited to amino coupling, disulfide bond, carboxyl coupling, self-assembled monolayer, other thiol reactive chemistry, click chemistry, double biotin-avidin linkage, nuclear linker-mediated hybridization, and any covalent ligation and non-covalent immobilization chemistry.

[0031] In one preferred embodiment of the method of the present invention, the modified oligonucleotides immobilized on the particle surface are conversion oligonucleotides (cT) and / or report oligonucleotides (rT). According to one embodiment, instead of being directly attached to the surface of the particle, some oligonucleotides can be attached to the free end of the attachment means (or linker) using a nucleic acid sequence or a non-polymerizable linker (sometimes also called a "spacer"), such as a PEG linker or an aliphatic linker. Such a spacer can be, for example, a nucleic acid spacer (modified or unmodified polynucleotide), or a non-polymerizable spacer, such as a polyethylene glycol spacer or an aliphatic spacer. In one embodiment of the digital multiplex method of the invention, the functionalization of the suspension of particles, preferably microparticles, in step a) is carried out exclusively with converter oligonucleotides (cT) and report oligonucleotides (rT).

[0032] In this embodiment, only the conversion oligonucleotide (cT) and the report oligonucleotide (rT) are immobilized on the particle before the particle is contacted with the barcode and the test sample. The third oligonucleotide (aT) and the fourth oligonucleotide (pT) are added later to the amplification mixture of step d). In this embodiment, the amplification mixture of step d) further comprises an oligonucleotide. In a preferred embodiment, the first and second oligonucleotides are attached to the particle using a biotin-(strept)avidin linkage, although as shown above, numerous other grafting chemistries may be used to attach such oligonucleotides. Additionally, spacers may be used. The particles used in the digital multiplexing methods of the present invention are preferably microparticles. According to one embodiment, such particles are porous, i.e., such particles are made of a porous material. According to another embodiment, the particles are made of hydrogels. Such hydrogel particles can be prepared, for example, according to the protocol described in Xu et al. (2005). According to yet another embodiment, the particles are non-porous solid particles.

[0033] The size of the particles used in the method of the present invention is comprised between 10 nm and 500 μm, preferably between 100 nm and 100 μm, more preferably between 500 nm and 10 μm. In one preferred embodiment, the particles are 1 μm streptavidin-coated particles. Such particles can be purchased, for example, from Invitrogen. According to another embodiment of the digital multiplex method of the present invention, the converter oligonucleotides and the report oligonucleotides may be biotinylated for attachment to avidin or streptavidin modified particles. According to one embodiment, the digital multiplexing method of the invention may comprise, prior to step a) of functionalizing the particles, a step of washing the particles with a buffer and then resuspending the washed particles in the same buffer, e.g. to remove preservatives contained in the storage buffer. Washing may be performed after the functionalization of step a).However, the advantage of the multiplex method of the present invention is that such washing is not necessary.By avoiding the washing step, it is possible to detect and / or quantify target biomolecules more quickly and accurately. In step b) of the digital method of the invention, a barcode is added to the particles functionalized in step a) that allows the identification of particles targeted to multiple biomolecules, so that each particle batch is barcoded according to the biomolecule that targets it.

[0034] Thus, in the context of the present invention, the term "barcode" relates to a label attached or associated with a particle that allows discrimination between different particle populations used to detect different target biomolecules of interest. A number of strategies have been developed to barcode particles, for example as described in references

[21] -

[25] . According to one embodiment, fluorescent dyes can be grafted (covalently or not) or directly incorporated into the particles by mixing them with a monomer mixture, as can be done with quantum dots. According to another embodiment, Raman spectroscopy labels, photonic crystals, and / or rare earth ion incorporation can be used to barcode the particles. Other means for distinguishing particle populations in the digital multiplexing methods of the present invention may include using particles having different shapes, sizes, or size distributions. In one preferred embodiment, the barcode is a fluorescent barcode. Preferably, said fluorescent barcode is a combination of fluorescent molecules of different grafting densities that gives each particle type a distinguishable identity when excited with a fluorescence recording device.

[0035] In particular, each particle population is barcoded with a fluorescent dye by co-grafting biotin-labeled fluorescent oligonucleotides and can therefore be distinguished using its fluorescent properties. In one preferred embodiment of the digital method of the invention, the particle functionalization in step a) is performed simultaneously with the barcoding in step b), i.e. the barcode is added simultaneously with one or more of the four oligonucleotides. After the particles are functionalized and barcoded in steps a) and b), respectively or simultaneously, the particles are contacted with a test sample to capture various target biomolecules in step c) of the digital multiplex method of the present invention. As used herein, the term "target biomolecule" or "biomolecular target" relates to a biomolecule, as defined above, which is detected and / or quantified by the method of the present invention. These target biomolecules are present in a sample. The sample may be of any type. For example, the sample may be obtained from a test subject, the subject being an animal, preferably a mammal, more preferably a human. Such a sample may also be called a biological sample.

[0036] In the context of the present invention, the term "biological sample" relates to solid or fluid biological material obtained from a living organism. A solid biological sample may be cells, tissue parts (biopsies), whole tissues, or organs, etc. Preferably, the sample used in the method of the present invention is a fluid sample. In the context of the present invention, the term "sample of a biological fluid" or "fluid sample" relates to any sample obtained from a bio-organic fluid produced by a living organism. The biological fluid is selected from the group comprising extracellular fluid, intravascular fluid, interstitial fluid, lymphatic fluid and transcellular fluid. In particular, the sample of biological fluid is selected from the group comprising blood and blood components, urine, saliva, tears, sweat, and the like. In a more preferred embodiment, the sample of biological fluid is a blood sample or a blood component sample. By "blood sample" or "blood component sample" is meant whole blood or one of its components, in particular selected from the red blood cell fraction, the white blood cell fraction, platelets, plasma, or serum. In another embodiment of the invention, the sample may be obtained from a non-living organism. For example, the sample may be obtained from air, water, soil, digestive products, etc. The type of sample depends on the application of the digital multiplex method of the invention. According to the invention, the sample contains or is likely to contain biomolecules.

[0037] In step c), the target biomolecules are randomly captured by cognate particles according to a Poisson distribution by hybridization with conversion oligonucleotides (cT). Therefore, the number of particles must be such that the number of target biomolecules of Poisson capture results in a proportion of particles that have captured at least one target that is less than 100% of the total number of particles. Preferably, this number is less than 95%. In one embodiment, the particles are mixed with the test sample in a reaction buffer. After incubation, the particles are pelleted and eluted for 10 min. 6 ~10 12 particles / mL, preferably 10 7 ~10 11 particles / mL, more preferably 10 8 ~10 10 The particles are resuspended in storage buffer at a concentration comprised between 10 particles / mL. In the most preferred embodiment, the concentration of the particles is between 10 9 According to one embodiment of the method of the invention, after step c), the particles that have or have not captured target biomolecules may be washed with a washing buffer before the next step d), in which the particles are resuspended in an amplification mixture. According to a preferred embodiment, after capturing the target biomolecules on the particles, the particles may be washed, especially if a polymerase such as Klenow polymerase is used in step d). In this last case, the particles are resuspended several times in the washing buffer (e.g. 2-10 times, preferably 3-7 times, more preferably 4-5 times). In step d) of the digital multiplex method of the present invention, the particles with captured target biomolecules are resuspended in an amplification mixture containing buffer, enzymes, deoxynucleoside triphosphates (dNTPs), and optionally oligonucleotides.

[0038] In the context of the present invention, the term "amplification mixture" relates to a reactive mixture comprising agents that allow the amplification of a target sequence. In particular, such agents are selected from buffers, enzymes, deoxynucleoside triphosphates (dNTPs) and, optionally, oligonucleotides, as defined below. According to one embodiment of the digital multiplex method of the invention, the enzymes used in step d) are selected from the group comprising a polymerase, a nicking or restriction enzyme, and an exonuclease, where the polymerase, the nicking enzyme, and the restriction enzyme drive the isothermal amplification, and the exonuclease allows to avoid saturation of the system. In particular, the polymerases used in the digital multiplex method of the present invention include Bst2.0 DNA polymerase, Bst large fragment DNA polymerase, Klenow fragment (3'->5' exon - ) (also referred to herein as Klenow polymerase), Phi29 DNA polymerase, Vent (exo - ) DNA polymerase, more particularly the polymerase is selected from the group consisting of Vent(exo) - ) DNA polymerase (purchased from New England Biolabs (NEB)). A mixture of two or more polymerases can also be used.

[0039] According to a preferred embodiment, the polymerase Vent(exo - ) DNA polymerase and Klenow fragment (3'->5' exo - ) are used together. In particular, in this case, the Klenow fragment (3'->5' exo) can improve the capture of target biomolecules to the surface of the particles before encapsulating the particles. Then, Vent (exo - ) DNA polymerase is used in the amplification reaction. The nicking enzyme is selected from the group including Nb.BbvCI, Nb.BstI, Nb.BssSI, Nb.BsrDI, in particular the nicking enzyme is Nb.BsmI and / or Nt.BstNBI (purchased from New England Biolabs (NEB)). A mixture of two or more nickases can also be used. According to one embodiment of the method of the present invention, the nicking enzyme may be replaced by a restriction enzyme. In contrast to a nicking enzyme, which only cuts a single strand, a restriction enzyme cuts two strands. Therefore, when a restriction enzyme is used instead of a nicking enzyme, it is necessary to protect the oligonucleotides used in the method of the present invention. This protection can be performed, for example, by carrying out a chemical modification of the oligonucleotide. Such modifications can be phosphorothioate backbone modifications, locked nucleic acid sugar modifications, peptide nucleic acid modifications, or the replacement of one nucleic acid base with another, for example methylation of the nucleic acid base (e.g., guanine to O). 6 -methylguanine or cytosine with methylcytosine). Examples of such modifications that can also be used in the methods of the invention are disclosed in Loenen et al., (2014).

[0040] The exonuclease used in the digital method of the invention is for example selected from RecJf, exonuclease I, exonuclease VII, in particular the exonuclease is ttRecJ exonuclease obtained according to the protocol described by Yamagata (Yamagata et al., 2001). The reaction buffer used for the mixture of enzyme and oligonucleotide is adapted to the selected oligonucleotide template. A person skilled in the art will be able to adapt conventional buffers to specific molecular designs. Examples of such buffers are also given in the experimental part of this application. For example, in a preferred embodiment of steps c) and d) of the method of the present invention, the reaction buffer comprises 20 mM Tris HCl pH 7.9, 10 mM (NH4)2SO4, 40 mM KCl, 10 mM NaCl, 10 mM MgSO4, 25 μM each dNTP, 0.1% (weight / volume) Synperonic F104, 2 μM netropsin, and 200 μg / mL BSA.

[0041] In one embodiment, capture step c) and resuspension step d) can be performed simultaneously, i.e. particles and the sample containing target biomolecules are directly injected into the amplification mixture.In this case, washing steps can be avoided.Generally, this embodiment is performed when the test sample is not toxic or does not interfere with the amplification reaction.For example, it is performed when the sample contains a purified extract of microRNA. After step d) of resuspending particles that have or have not captured target biomolecules, step e) is performed of separating the particles in suspension from each other so that each particle can react independently. According to one embodiment, particle separation can be obtained by various means using microchamber arrays, droplet microfluidics, or particle dispersion in immiscible fluids. For example, the separation step is performed in microdroplets (e.g., Biorad's QX200 system or Stilla Technologies' Naica system). Separation is performed in millions of water-in-oil droplets fabricated by combining two immiscible liquids: a sample aqueous phase; and a continuous hydrophobic phase such as undecane-1-ol, silicone oil, mineral oil, and more preferably, perfluorinated oil, because it is highly immiscible with water, biocompatible, low viscosity, transparent, and compatible with PDMS devices.

[0042] According to one preferred embodiment, separation is carried out by creating a water-in-oil emulsion having aqueous droplets containing the particles suspended in the reaction mixture. According to one embodiment, the droplets used in the digital method of the invention have a size comprised between 0.001 pL and 100 pL, preferably between 0.1 pL and 10 pL, more preferably between 0.5 and 5 pL. In another embodiment, the separation of step e) may be carried out by using a microfluidic chip allowing a Poisson distribution of the particles in the droplets of the emulsion used. If separation is performed in microfluidic droplets, the particle concentration in the amplification mixture prior to separation in step e) is selected such that on average only a small number of particles are encapsulated in each droplet. Preferably, this number is smaller than 1. Other microfluidic methods that make it possible to obtain a Poisson's law distribution, a small number of particles or a single particle per droplet of emulsion, and to minimize empty droplets, are selected from acoustic focusing, inertial focusing, "close-packed ordering". These methods are known to those skilled in the art, who will be able to adapt the parameters for their implementation to the digital multiplexing method of the invention.

[0043] Furthermore, to separate the particles in step e), specific devices such as microchamber devices (e.g., SlipChip or QuantStudio 3D from Thermofisher Scientific) can be used, where the particles are distributed within microchambers that separate them from each other. According to another embodiment, the particles are isolated either physically by dispersing them in an immiscible fluid, or chemically by closing the pores of the particles (e.g., using layer-by-layer deposition or polymer cross-linking or medium gelling). In this embodiment, the amplification reaction is carried out within the pores of the particles. In another embodiment, the separation step can be carried out using non-porous particles, in which case the amplification reaction is carried out on particles surrounded by a liquid microlayer that allows separating said particles from other particles, making it possible to limit signal diffusion between the particles. As used herein, the term "digital multiplex method" refers to a detection and / or quantification method in which multiple targets are randomly captured by a population of functionalized and barcoded particles, each particle type being specific for a target type, and each target type being captured by particles that follow a Poisson distribution. A signal amplification reaction is then performed within or around each particle (in the liquid microlayer), and only particles that have captured at least one target show a positive signal. This allows for the direct counting of discrete events in each particle population, thus allowing absolute quantification of the associated target biomolecules.

[0044] The main advantage of the digital multiplex method of the present invention compared with the PCR digital multiplex method (where primers and probes are specific to each target biomolecule), for example, is that multiple target biomolecules captured by particles are not directly detected, but are converted into a common signal sequence, and then converted into a common measurable signal, which makes it possible to avoid the sensitivity and specificity problems caused by direct detection of target biomolecules, and the complicated design and implementation of orthogonal amplification systems. The digital multiplex method of the present invention also includes a step f) of incubating the particles at a constant temperature so that each target biomolecule induces an amplification reaction and a report signal. As used herein, the term "signal" or "signal sequence" relates to a nucleic acid sequence, preferably a single-stranded DNA, obtained by converting the sequence of a target biomolecule, preferably a nucleic acid molecule, more preferably a microRNA, into a sequence that can be amplified.

[0045] The converted signal sequence is therefore amplified in step d) of the digital multiplex method of the present invention. According to one embodiment, the amplification in step f) is carried out at a constant working temperature in the range of 35-60°C, more preferably 37-55°C, even more preferably 45-50°C. In one embodiment, the digital multiplex method of the present invention comprises a step f1) of recovering the particles. In one preferred embodiment, the recovery is performed by disrupting the compartments containing the particles. In this way, the particles are recovered in a solution, preferably in an aqueous medium. The recovery of the particles is performed when the particles are separated from each other by emulsification, i.e. in an unmissable liquid, preferably in a water-in-oil emulsion. The disruption of the compartments in step f1) can be performed by a means selected from perfluorooctanol treatment, surfactant dilution with a surfactant-free continuous phase, or electrostatic pulse, preferably by using an electrostatic pulse gun (Zerostat 3, Milty, UK).

[0046] In another embodiment of the digital multiplex method of the present invention, if the particles are separated from each other in solution, e.g. in an aqueous solution, there is no need to perform the recovery step f1) and the detection and / or quantification of step g) can be performed directly. In step g) of the digital multiplex method of the present invention, the particles need to be barcoded (or labeled) in order to detect and / or measure the signals of the particles, including the barcode signal and the amplified signal of each particle. As indicated above, barcoding is performed in step b) of the digital multiplex method of the present invention or is carried out simultaneously with the functionalization of the particles in step a). For this purpose, many different barcodes can be used. As indicated above, not a few methods have been developed in the art for barcoding particles. Such methods are disclosed in references

[28] -

[32] . For example, such methods can be selected from fluorescent dyes that may be grafted (covalently or non-covalently) or directly incorporated into the particles by mixing them with a monomer mixture, as can be done with quantum dots; flow lithography, photonic crystals, and rare earth ion incorporation. The skilled person will be able to adapt conventional barcoding means to the digital method of the present invention.

[0047] According to one preferred embodiment of the digital multiplex method of the present invention, the barcode used is a fluorescent signal. During the incubation for amplifying the signal sequence in step f), the particle-bound target biomolecules trigger an amplification reaction, which in turn induces the activation of the report template, preferably of the fluorescent probe. Thus, according to one embodiment of the digital multiplex method of the present invention, the step g) of detecting and / or measuring the barcode signal comprises detecting and / or measuring the barcode signal of each particle associated with the target biomolecule and at the same time the signal of the report template associated with the amplified signal, said signal being preferably a fluorescent signal. In one embodiment, when the digital multiplex method of the present invention is used to measure the absolute concentration of a target biomolecule in a tested biological sample, the fluorescent particles and non-fluorescent particles that have captured the target biomolecule are counted and their ratio is calculated.

[0048] Preferably, flow cytometric analysis allows for simultaneous measurement of barcode signals and counting of positive (target present) / negative (target absent) particles, thus allowing calculation of accurate concentrations of multiple target biomolecules in the initial sample. The positive population corresponds to particles that have captured at least one target biomolecule. Considering the Poisson random capture of targets by each particle population, it is possible to calculate the concentration of associated target biomolecule by first calculating the Poisson distribution parameter λ.

number

[0049] The measured concentration of the target biomolecule can then be determined by the following formula: [Let7a]=[part].λ [Let7a]=[part].ln(1-F pos ) where [parts] is the initial concentration of particles. For example, counting and analysis in the digital multiplex method of the present invention can be performed by flow cytometry on an Attune NxT (Thermo Fisher Scientific, Massachusetts, USA). According to another embodiment, other conventional methods, such as fluorescence microscopy, may be used to count positive / negative particles.

[0050] According to a preferred embodiment, the digital multiplexing method of the present invention comprises: a) functionalizing a suspension of particles, preferably microparticles, with a first oligonucleotide, which is a conversion oligonucleotide (cT), and a second oligonucleotide, which is a report oligonucleotide (rT); b) adding a barcode to the particles functionalized in step a) that allows for the identification of particles targeted to multiple biomolecules; c) contacting the particles obtained in step b) with a test sample to capture a plurality of target biomolecules; d) resuspending the particles that have captured or have not captured the target biomolecule in an amplification mixture that includes a buffer, an enzyme, a third oligonucleotide that is an amplification oligonucleotide (aT), a fourth oligonucleotide that is a leak-absorbing oligonucleotide (pT), and deoxynucleoside triphosphates (dNTPs); e) separating the particles in the suspension obtained in step d) from one another so that each particle can react independently; f) incubating the isolated particles at a constant temperature so that each target biomolecule induces an amplification reaction and a barcode signal; f1) recovering the particles; and g) detecting and / or measuring the signals of the particles, including the barcode signal and the amplified signal of each particle Includes.

[0051] A schematic diagram illustrating the principle of this preferred embodiment of the digital multiplexing method of the present invention is shown in FIG. The specific technical features of the digital multiplexing method according to this preferred embodiment are detailed above. Use of the digital multiplex detection and / or quantification method of the invention for diagnostic purposes The biomolecules mentioned above can be present in all types of samples.For example, the biomolecules mentioned above can be present in samples obtained from non-living organisms (for example, soil samples, water samples, air samples, food samples, etc.) or samples obtained from living organisms, such as cells, body fluids, or tissues.The biomolecules mentioned above can be present, for example, in all body fluids, and therefore can be accessed through minimally invasive liquid biopsy (serum, plasma, urine).

[0052] According to one embodiment of the present invention, the target biomolecules detected and / or measured by the digital multiplex method of the present invention are used as biomarkers. In the context of the present invention, the term "biomarker" relates to a naturally occurring molecule, preferably a biomolecule, protein, enzyme, gene, nucleic acid, or characteristic by which a particular pathological or physiological process, disease, etc. can be identified. Such biomarkers can be used to detect disease in organisms such as plants, animals, preferably mammals, and more preferably humans. Furthermore, such biomarkers can be used for the detection of one or several abnormalities as well as in the food and agri-food industries or in the environment. Biomarkers, for example, may be present in all bodily fluids and therefore accessible via minimally invasive liquid biopsies (serum, plasma, urine, tears, saliva, sweat, etc.).

[0053] Preferably, they can be used as biomarkers to detect diseases selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases, and prenatal diseases. In the context of the present invention, the term "detection" is used in a general manner to define the detection of a target biomolecule in a sample as defined above. Also, as used herein, the term "detection" refers to the diagnosis or prognosis of one or several of the above-listed diseases or their symptoms. Detection also includes predicting one or several of said diseases or predicting the risk that a subject will develop one or several of these diseases. Moreover, the term "detection" further relates to agronomic diagnosis, i.e. the diagnosis of plant pathologies, in particular plant pathologies having a biotic or abiotic origin as defined in the present invention.

[0054] In the context of the present invention, the term "cancer" refers to a malignant neoplasm characterized by deregulated or uncontrolled cell proliferation. In particular, a "cancer cell" refers to a cell that exhibits deregulated or uncontrolled cell proliferation. The term "cancer" includes primary malignant tumors (e.g., those in which cells have not migrated to sites within the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., metastases, resulting from the migration of tumor cells to secondary sites different from the site of the original tumor). Such cancers may be selected, in particular, from the group of solid tumors and / or the group of hematopoietic cancers.

[0055] In one embodiment of the invention, the cancer is selected from the following: osteolysis, sarcoma of bone (osteosarcoma, Ewing's sarcoma, giant cell tumor of bone), bone metastasis, glioblastoma and brain cancer, lung cancer, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic, and promyelocytic), acute T-cell leukemia, basal cell carcinoma, cholangiocarcinoma, bladder cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic ... Myeloblastic leukemia, chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngeal carcinoma, cystadenocarcinoma, diffuse large B-cell lymphoma, proliferative abnormal changes (dysplasia and metaplasia), embryonal carcinoma, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, erythroleukemia, esophageal cancer, estrogen receptor positive breast cancer, essential thrombocythemia, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, hepatocellular carcinoma, hormonal The following conditions are considered to be related to the following: lymphoma-insensitive prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoblastic leukemia, lymphoma (Hodgkin and non-Hodgkin), malignant tumors and hyperproliferative disorders of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin, and uterus, lymphoid malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, melanoma, myel ... Lung cancer, myxosarcoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, solid tumors (carcinoma and sarcoma), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovium, sweat gland carcinoma, thyroid cancer, Waldenstrom's macroglobulinemia, testicular tumor, uterine cancer, and Wilms' tumor.

[0056] In the context of the present invention, the term "neurological disease" refers to diseases of the central and peripheral nervous system, including the brain, spinal cord, cranial nerves, peripheral nerves, nerve roots, autonomic nervous system, neuromuscular junction, and muscles. Neurological diseases are selected from neurodevelopmental diseases, neurodegenerative diseases, or psychiatric diseases. Neurological diseases include epilepsy, Alzheimer's disease and other dementias, cerebrovascular diseases including stroke, migraine, and other headache disorders, multiple sclerosis, Parkinson's disease, neuroinfections, brain tumors, traumatic disorders of the nervous system due to head trauma, and the like. As used herein, "cardiovascular disease" refers to heart or vascular failure, including coronary heart disease: disease of the blood vessels that supply the heart muscle; cerebrovascular disease: disease of the blood vessels that supply the brain; peripheral artery disease: disease of the blood vessels that supply the arms and legs; rheumatic heart disease: damage to the heart muscle and heart valves due to rheumatic fever caused by streptococcus; congenital heart disease: malformations of the heart structure present at birth, and deep vein thrombosis, and pulmonary embolism: blood clots in the leg veins that can break off and travel to the heart and lungs. As used herein, "inflammatory disease" preferably refers to acute pancreatitis; ALS; Alzheimer's disease; cachexia / anorexia; asthma; atherosclerosis; chronic fatigue syndrome, fever; diabetes (e.g., insulin-dependent diabetes); glomerulonephritis; graft-versus-host rejection; hemorrhagic shock; hyperalgesia, inflammatory bowel disease; inflammatory conditions of the joints, including osteoarthritis, psoriatic arthritis, and rheumatoid arthritis; ischemic injury, including cerebral ischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhage, or stroke, these each of which may be linked to neurodegeneration); pulmonary disease (e.g., ARDS); multiple myeloma; multiple sclerosis; myeloid (e.g., AML and CML) and other leukemias; myopathy (e.g., muscle protein metabolism, especially in sepsis); osteoporosis; Parkinson's disease; pain; preterm labor; psoriasis; reperfusion injury; septic shock; side effects of radiation therapy, temporomandibular joint disease, tumor metastasis; or inflammatory conditions resulting from strains, sprains, cartilage injuries, trauma, orthopedic surgery, infection, or other disease processes.

[0057] In the context of the present invention, an "autoimmune disease" is defined as a condition in which the subject's body produces antibodies against the subject's own tissues and cells. Examples of autoimmune diseases are type I diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, etc. Viral or bacterial diseases are caused by pathogenic viruses or bacterial strains. Examples of such diseases are AIDS, ascariasis, athlete's foot, dysentery, chickenpox, cholera, cold, dengue fever, diarrhea, diphtheria, filariasis, gonorrhea, herpes, hookworm, influenza, leprosy, measles, mumps, oriental cyst, pinworm disease, plague, pneumonia, poliomyelitis, rabies, ringworm, septicemic sore throat, sleeping sickness, smallpox, syphilis, tetanus, typhoid, vaginitis, viral encephalitis, whooping cough, etc.

[0058] "Skin diseases" include, for example, acne, alopecia, basal cell carcinoma, Bowen's disease, congenital erythropoietic porphyria, contact dermatitis, disseminated superficial actinic porokeratosis, dystrophic epidermolysis bullosa, eczema (atopic eczema), extramammary Paget's disease, epidermolysis bullosa simplex, erythropoietic protoporphyria, fungal infection of the nails, Hailey-Hailey disease, herpes simplex, hidradenitis suppurativa, Conditions affecting the skin such as hypertrichosis, hyperhidrosis, ichthyosis, impetigo, keloids, keratosis pilaris, lichen planus, lichen sclerosus, melanoma, melanosis, mucous membrane pemphigoid, pemphigoid, pemphigus vulgaris, pityriasis lichenoides, pityriasis rubra pilaris, plantar warts (verrucae), polymorphous photoeruption, psoriatic pyoderma gangrenosum, rosacea, scabies, shingles, squamous cell carcinoma, Sweet's syndrome, urticaria, and angioedema, vitiligo. As used herein, "muscle skeletal disease" refers to diseases of bone, muscle (myopathy) and musculoskeletal junction. For example, such diseases are selected from back pain, bursitis, fibromyalgia, fibrous dysplasia, growth plate injury, hereditary connective tissue disorder, Marfan syndrome, osteogenesis imperfecta, osteonecrosis, osteoporosis, Paget's disease of bone, scoliosis, spinal stenosis, tendonitis, etc.

[0059] As used herein, "dental disease" refers to problems with the teeth and mouth. Examples of such diseases are dental cavities, periodontal (gum) diseases, oral cancer, oral infectious diseases, traumatic injuries, and genetic disorders. As used herein, "prenatal disease" relates to diseases that may affect pregnancy or fetal development, such as: AIDS, amniotic fluid, bleeding during pregnancy, cervical disorders, gestational diabetes, disseminated intravascular coagulation (DIC), ectopic pregnancy, erythroblastosis fetalis, fetal growth problems, high blood pressure during pregnancy, HELLP syndrome, hydatidiform mole, hyperemesis gravidarum, intrauterine growth retardation, large for gestational age (LGA), miscarriage, placental abruption, placenta previa, placental insufficiency, polyhydramnios, prenatal testing, pregnancy loss, preterm labor and delivery, rubella, small for gestational age (SGA), systemic lupus erythematosus, toxoplasmosis, twin-to-twin transfusion syndrome, twins, triplets, multiple births, vaginal bleeding during pregnancy. Therefore, the digital multiplex method of the present invention can be used in an in vitro diagnostic method for diagnosing a disease selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases, and prenatal diseases. Thus, in a second aspect, the present invention relates to an in vitro method for the diagnosis of a disease selected from the group comprising cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases due to viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases and prenatal diseases, comprising the use of a digital multiplex method according to the present invention.

[0060] According to one embodiment, the diagnostic method comprises: - providing a sample from a subject; and - detecting the presence or absence of one or more of said diseases by using the digital multiplex method of the invention. Includes The specificity, sensitivity, simplicity and rapidity of the digital multiplex method of the present invention allows it to be used in agricultural diagnostic methods, in particular for the diagnosis of diseases caused by biotic stresses, such as infectious and parasitic diseases, or abiotic stresses, such as nutritional deficiencies or adverse environments. According to a third aspect, the present invention also provides a method for producing ... composition comprising the steps of: - diseases caused by biotic stress, preferably of infectious and / or parasitic origin, or - Diseases caused by abiotic stress, preferably nutritional deficiencies and / or adverse environments 1. An in vitro method for the agricultural diagnosis of a disease selected from the group consisting of: The present invention also relates to in vitro methods, including using the multiplexed digital methods of the present invention.

[0061] According to one embodiment, the diagnostic method comprises: - preparing a sample obtained from any one of the plant parts, and - detecting the presence or absence of one or more of said diseases by using the digital multiplex method of the invention. Includes. In the context of the present invention, the term "agronomic diagnosis" relates to the diagnosis of plant pathologies, said diagnosis comprising carrying out analyses of plant samples to identify fungi, viruses, bacteria, nematodes and any other organisms causing biotic stress and / or to identify biomolecules whose presence is due to abiotic stress. In the context of the present invention, the term "plant pathology" or "plant disease" relates to a plant abnormality manifested by alterations in the morphological, physiological characteristics or behavior of the plant due to biotic or abiotic stress. As used herein, the term "biotic stress" also relates to stress or disease caused by a living organism. The biotic stress may be caused by any living organism, but preferably by organisms of infectious and / or parasitic origin and selected from fungi, viruses, bacteria and nematodes.

[0062] According to one embodiment, the agronomic diagnostic method of the present invention can be used to diagnose diseases caused by fungi, said diseases being selected from anthracnose, black knot disease, blight, chestnut blight (such as leaf blight), canker, clubroot, wilt, elm wilt, ergot, Fusarium wilt, Panama disease, leaf blister, mildew (such as downy mildew and powdery mildew), oak wilt, rot (such as base rot, gray mold rot, heart rot), rust (such as blister rust, cedar apple rust, and coffee rust), scab (such as apple scab), ear smut, wheat smut, corn smut, snow rot, sooty mildew, and verticillium wilt. According to another embodiment, the agronomic diagnostic method of the present invention can be used to diagnose a disease caused by a virus, said disease being selected from dwarf disease, mosaic disease, sorosis, and spotted wilt disease. According to yet another embodiment, the agronomic diagnostic method of the present invention may be used to diagnose a disease caused by bacteria, said disease being selected from aster yellows, bacterial wilt, blight (such as fire blight and rice bacterial spot), canker, crown gall, rot, base rot, and scab.

[0063] In addition, the agricultural diagnostic method of the present invention can be used to detect root-knot nematodes (Meloidogyne spp., etc.), cyst nematodes (Heterodera spp. and Globodera spp., etc.), root lesion nematodes (Pratylenchus spp., etc.), borer nematodes (Radopholus similis, etc.), Ditylenchus dipsaci, pinewood nematodes (Bursaphelenchus xylophilus, etc.), reniform nematodes (Rotylenchulus reniformis, etc.), grape spur nematode (Xiphinema index), false root-knot nematode (Nacobbus The present invention can be used for the diagnosis of diseases caused by nematodes selected from the group consisting of Rice root nematode, Rice root nematode, Rice root nematode, and Rice root nematode, Aphelenchoides besseyi. According to one embodiment, the agronomic diagnostic method of the present invention is used to diagnose diseases caused by "abiotic stress", the term "abiotic stress" defines diseases that are not caused by living organisms. Such diseases are preferably caused by nutritional deficiencies and / or adverse environments. For example, abiotic stress can be caused by inappropriate pH, water availability (drought stress), temperature (heat stress and cold stress), oxygen and / or gas availability, mineral deficiencies (salt stress), and toxic compounds (e.g., pollutants).

[0064] The specificity, sensitivity, simplicity and rapidity of the digital method of the invention also make it possible to use it in methods for detecting biomolecules in the field of food, agri-food industry and in the environment. In particular, the digital method of the invention is used to detect abnormalities in food, agri-food and the environment. This detection is carried out by using the digital method of the invention to detect biomolecules that can be considered as biomarkers of said abnormalities. Such biomolecules may, for example, be part of a living organism, or may be produced by the activity of a living organism, or may be artificial biomolecules. Such biomolecules (or biomarkers) are selected from the group comprising biopolymers, in particular DNA, RNA, proteins, and enzymes. Said biomolecules are present in the original and / or transformed products of agri-foods and foods.

[0065] Biomolecules may also be present in the environment, for example, in the air, water, and / or soil. Thus, according to one aspect, the present invention also relates to an in vitro method for detecting biological molecules (biomarkers) in agri-food, the food industry and / or the environment, comprising using the digital method of the present invention. In the context of the present invention, the term "food" relates to any food product, basic or transformed, produced without the use of industrial processes or by using such processes. In the context of the present invention, the term "agri-food" relates to the agri-food industry, i.e. the commercial production of food by agricultural enterprises. The term "environment" or "environmental" relates to the natural environment, i.e. an ecological unit functioning as a natural system without extensive civilized human intervention, including all plants, microorganisms, soil, rocks, atmosphere, and natural phenomena occurring within their boundaries and natural conditions. Such terms also relate to non-natural or artificial environments that can be created by humans. According to one aspect, the present invention also relates to an in vitro method for detecting anomalies in the food and agri-food industry and / or the environment, using the digital method of the present invention. Preferably, the method comprises the steps of: - preparing samples obtained from food, agri-food or the environment; - detecting a test biomolecule (biomarker) in said sample by the digital method of the invention; Includes.

[0066] Kit for carrying out the digital multiplex method of the present invention The present invention also relates to kits that can be used to detect and / or quantitate multiple target biomolecules according to the methods of the present invention. Thus, according to a fourth aspect, the present invention provides a kit for detecting and / or quantifying a plurality of target biomolecules, comprising: a) a suspension of particles, preferably microparticles, functionalized with one or more oligonucleotides selected from a first oligonucleotide which is a conversion oligonucleotide (cT), a second oligonucleotide which is a report oligonucleotide (rT), a third oligonucleotide which is an amplification oligonucleotide (aT), and a fourth oligonucleotide which is a leak-absorbing oligonucleotide (pT), and carrying different barcodes that allow for the identification of particles targeting different biomolecules; b) a mixture of enzymes, preferably selected from the group comprising a polymerase, a nicking or restriction enzyme, and an exonuclease, and optionally an oligonucleotide, and c) Separation agent The present invention relates to a kit comprising:

[0067] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: a) a suspension of particles, preferably microparticles, functionalized with a first oligonucleotide, which is a conversion oligonucleotide (cT), and a second oligonucleotide, which is a report oligonucleotide (rT), and barcodes are attached to allow for the identification of particles targeted to different biomolecules; b) an enzyme, preferably selected from the group comprising a polymerase, a nicking or restriction enzyme, and an exonuclease, and a mixture of oligonucleotides comprising a third oligonucleotide which is an amplification oligonucleotide (aT) and a fourth oligonucleotide which is a leak-absorbing oligonucleotide (pT); c) a separating agent, preferably a water-in-oil emulsion The present invention relates to a kit comprising:

[0068] According to yet another embodiment, the components of item b) above are provided separately, i.e. the oligonucleotides are provided separately from the enzyme and are subsequently mixed. The components described in items a), b), and c) of the kit correspond to the same as those used in the digital multiplex method of the present invention as described above. The present invention also relates to the use of said kit for carrying out the digital multiplex method of the present invention. The kit of the present invention may also include instructions for use. Specific embodiments of the present invention will be clearly understood from the following examples and drawings. [Brief description of the drawings]

[0069] [Figure 1] Principle of multiplex and digital detection of microRNA. A population of barcoded particles is functionalized with target-specific conversion and reporting oligonucleotides. The particles are mixed with the sample, resulting in random capture of the target biomolecules by the particles. After washing, the particles are compartmentalized (e.g., water-in-oil droplets) with amplification and leak-absorbing oligonucleotides and an enzyme mix. After incubation at a constant temperature (e.g., 50°C), the particles are analyzed by flow cytometry to access the absolute concentration of each target, calculated from the ratio of positive / negative particles in each population. [Figure 2-1] Results of flow cytometry analysis of Let7a detection: a. Particles BLet7a before incubation. b. Particles BLet7a after 4 h incubation at 50°C (NC = no target). [Figure 2-2] Results of flow cytometry analysis of Let7a detection. c. Particles BLet7a after 4 h incubation at 50° C. in the presence of 1 pM target. d. Measured concentration calculated from the ratio of positive particles Fpos: [Let7a]=[parts].ln(1-Fpos). [Figure 3-1]Microscopy readout vs. flow cytometry readout. a. Fluorescence images of encapsulated particles after incubation. White dots correspond to particles with FAM-modified rT (positive and negative), positive droplets are shown in grey, corresponding to the fluorescence of the Atto633-modified probe in solution. MicroRNA was detected in grey droplets but not in dark grey droplets. [Figure 3-2] Microscopy readout vs. flow cytometry readout. b. Histogram of flow cytometry readout. MicroRNA was detected in 79% of the bead-containing droplets. c. Comparative analysis of microscopy readout (statistics calculated from 250 bead-containing droplets) and flow cytometry readout. [Figure 4] Detection concentration as a function of particle concentration in the inclusion mix. [Diagram 5] Enzyme capture by oligonucleotides on particles. The enzyme mixture (polymerase, nickase, exonuclease) is incubated with or without particles grafted with oligonucleotides. After 30 min incubation at 30°C, the particles are pelleted and the supernatant is mixed with the molecular program (amplification and pseudotemplate oligonucleotides) in solution and spiked with 1 pM Let7a. The samples are incubated at 50°C and the fluorescence of rT is monitored in real time. a. Real-time fluorescence curve showing the amplification reaction. b. Extraction start time of the fluorescence curve. [Figure 6-1] Effect of neutral particles on false positive and true positive rates. a. Cytometric fluorescence histograms of the negative control (no target, top row) and the positive control (1 pM Let7a, bottom row) as a function of particle concentration. [Figure 6-2] Effect of neutral particles on false positive and true positive rates. b. Percentage of positive particles as a function of overall particle concentration. [Figure 6-3] Effect of neutral particles on false positive and true positive rates. c. Target biomolecule concentration in positive particles. [Figure 7]Let7a range detection using neutral particles. 105 BLet7a complemented with 2 × 105 BN were used to quantify 0, 0.2, and 1 pM Let7a. a. Cytometry fluorescence histograms. b. Comparison of measured Let7a concentrations according to theoretical spike-in concentrations. [Figure 8-1] Concentrations of Let7a and miR92a detected in a two-target assay. a, Cytometric fluorescence histograms of three particle populations (BLet7a, B92a, BN) with fluorescent barcodes (Atto633). [Figure 8-2] Concentrations of Let7a and miR92a detected by the two-target assay. b. Cytometric fluorescence results of B92a and BLet7a (rT fluorescence). [Figure 8-3] Concentrations of Let7a and miR92a detected in the dual-target assay. c. Measured concentrations of the two targets. [Figure 9-1] Triplex assay for simultaneous quantification of let7a, mir92a, and mir203a. a. Fluorescence intensity of barcode (Atto633) of each particle population (measured by flow cytometry). [Figure 9-2] Triplex assay for simultaneous quantification of Let7a, mir92a, and mir203a. Particle populations are, from left to right: a. Neutral particles, miR92a particles, miR203a particles, and Let7a particles, and b. Cytometric fluorescent signals of miR92a, miR203a, Let7a particles depending on the sample. [Figure 9-3] Triplex assay for simultaneous quantification of let7a, mir92a, and mir203a. c. Measured concentrations of each microRNA target. [Figure 10] Flow cytometry discrimination of ten particle populations using two-dimensional fluorescent barcoding. 1 μm streptavidin-coated particles are functionalized with various ratios of biot-TTTT-FAM (5 levels) and biot-TTTTT-DyXL510 (2 levels). [Figure 11]Let7a detection from human colon total RNA extracts. a. Cytometric fluorescent signal of Let7a particles. b. Measured concentrations of Let7a in negative controls and in samples containing 10 ng / μL of colon total RNA. [Figure 12] Dynamic range adjustment as a function of particle number. The graph shows the evolution of the dynamic range for 20 μL samples with different target concentrations. The dynamic range is composed between the limit of detection (LoD) and the lower limit of quantification (hLoQ). For simplicity, we assume that the LoD is equal to the limit of blank (LoB = average percentage of false positive events), which is set provisionally to 5%. The hLoQ is set provisionally to 95% of the positive events (i.e., above this value the quantification is considered unreliable). The bottom bar represents the appropriate number of particles to be used at each target concentration to fall within the dynamic range. As a result, the dynamic range can be adjusted. [Figure 13] Detected Let7a concentrations as a function of the presence / absence of Klenow DNA polymerase (3'->5'exo-) during capture onto particles. [Figure 14] Effect of an enhanced "hard" wash procedure on reducing background amplification. [Figure 15] Design of poly(T) conversion template. Expected concentrations are 0M (negative control) and 1.00E-12M (1 pM sample). [Figure 16] Comparison of expected and measured patterns in 6-plexed miRNA detection. [Figure 17] Adjustable dynamic range. [Figure 18] Detection of three microRNAs from human total RNA. EXAMPLES

[0070] Methods and Materials Oligonucleotides All oligonucleotides (templates and synthetic microRNAs) used in this study were purchased from Biomers (Germany). Oligonucleotide sequences were purified by HPLC. Template sequences were protected from exonuclease degradation by 5' phosphorothioate modification. cT (conversion template) and rT (report template) were modified with polythymidylic acid linkers followed by 3' and 5' modification of biotin moieties, respectively. The oligonucleotides are shown in Table 2 below.

[0071] [Table 3]

[0072] Table 2. Oligonucleotide sequences used in the present invention. * " indicates phosphorothioate backbone modification. "p" indicates 3' phosphate modification. "Biotin" and "bioteg" refer to biotinylated synthons using aminoethoxy-ethoxyethanol linkers and the longer triethylene glycol linker, respectively. Uppercase and lowercase letters represent deoxyribonucleotides and ribonucleotides, respectively. "aT" corresponds to the autocatalytic template, "pT" corresponds to the pseudotemplate, "rT" corresponds to the reporting template, and "cT" corresponds to the conversion template. Atto633, FAM, and DyXL510 are fluorophores. dTFAM is a deoxythymidine nucleoside derivatized with 6-FAM (6-carboxyfluorescein) via a spacer arm.

[0073] particle functionalization Streptavidin-coated 1 μm particles (Dynabeads C1) were obtained from Invitrogen. Before functionalization, the particles were washed three times with washing buffer (20 mM Tris-HCl pH 7.5, 1 M NaCl, 1 mM EDTA, 0.2% Tween 20 (Sigma-Aldrich)) and then resuspended in the same buffer. Biotinylated oligonucleotides are added to the particle suspension, mixed well by vortexing and incubated for 15 min at room temperature. After functionalization, the particles were washed once with washing buffer, once with storage buffer (5 mM Tris-HCl pH 7.5, 50 mM NaCl, 500 μM EDTA, 5 mM MgSO4) and finally resuspended in storage buffer. The grafted particles are stored at 4 °C until use.

[0074] MicroRNA capture All capture mixes were assembled in 200 μL PCR tubes at 4 °C. Mix particles into reaction buffer (10 for each bead population). 9 beads / mL, 20 mM Tris HCl pH 8.9, 10 mM (NH4)2SO4, 40 mM KCl, 10 mM NaCl, 10 mM MgSO4, 25 μM each dNTP, 0.1% (w / v) Synperonic F104, 2 μM netropsin). Samples were spiked with synthetic microRNA targets (serialized diluted in 1× Tris-EDTA buffer using low DNA retention chips) or target-containing fluids (plasma, urine, cell extracts, tissue extracts, etc.). Samples were incubated for 1 h at 30° C. with agitation at 2000 rpm in a ThermoMixer (Eppendorf). Particles were then pelleted and 10 9 The particles were resuspended in storage buffer at a concentration of 10 ...

[0075] Reaction mixture assembly All reaction mixtures were assembled in 200 μL PCR tubes at 4 °C. Templates (aT and pT) and particles (3 × 10 8particles / mL, including neutral particles and detection particles), enzymes (200u / mL Nb.BsmI, 10u / mL Nt.BstNBI, 80u / mL Vent(exo - ), and 23 nM ttRecJ) were mixed with reaction buffer (20 mM Tris HCl pH 8.9; 10 mM (NH4)2SO4, 40 mM KCl, 10 mM NaCl, 10 mM MgSO4, 25 μM each dNTP, 0.1% (wt / vol) Synperonic F104, 2 μM netropsin) and BSA (200 μg / mL).

[0076] Droplet generation and incubation A two-inlet (one for oil and one for aqueous samples) flow focusing microfluidic mold was prepared by patterning a 4-inch silicon wafer by standard soft lithography techniques using SU-8 photoresist (MicroChem Corp., MA, USA). A 10:1 mixture of Sylgard 184 PDMS resin (40 g) / crosslinker (4 g) (Dow Corning, MI, USA) was poured into the mold, degassed under vacuum, and baked at 70 °C for 2 h. After curing, the PDMS was peeled off from the wafer, and 1.5 mm diameter inlet and outlet holes were punched with a biopsy punch (Integra Miltex, PA, USA). Immediately after oxygen plasma treatment, the PDMS layer was fixed onto a 1 mm thick glass slide (Paul Marienfeld GmbH&Co.KG, Germany). Finally, the chip was subjected to a second bake at 200 °C for 5 h to make the channels hydrophobic. The aqueous sample phase (amplification mix + particles) and the continuous phase (fluorinated oil Novec-7500, 3M (Emulseo, France) containing 1% (w / w) fluorosurfactant) were mixed on-chip using a pressure controller MFCS-EZ (Fluigent, France) and 200 μm diameter tubes (CIL, France) to generate 0.5 pL droplets by hydrodynamic flow focusing. The droplets were transferred to PCR tubes and incubated at 50 °C for the amplification reaction to occur.

[0077] particle analysis After incubation, the droplets were mixed (1:5 vol / vol) with surfactant-free fluorinated oil (Novec 7500). The emulsion was broken using an electrostatic pulse gun (Zerostat 3, Milty, UK). Once all the water droplets had merged into one water droplet, the oil phase was discarded and the water droplet was resuspended in sheath fluid (Attune NxT focusing fluid, Thermo Fisher Scientific, MA, USA). Samples were analyzed by flow cytometry in an Attune NxT (Thermo Fisher Scientific, MA, USA).

[0078] Example 1 DNA-grafted particles for digital detection of microRNAs The digital multiplex strategy of the present invention relies on the use of DNA-grafted particles that can capture single nucleic acid targets, trigger exponential amplification reactions, and report positive signals. The ratio of positive particles to negative particles provides access to the absolute concentration of the target in the initial sample, which is calculated from Poisson's law. We first investigated the possibility of using this digital approach to detect one microRNA target.

[0079] To that end, 1 μm streptavidin-coated magnetic particles were functionalized with a conversion template (cT, SEQ ID NO: 65) and a report template (rT, SEQ ID NO: 64) targeting the microRNA Let7a. The particles are mixed with a sample containing 0 or 1 pM of synthetic Let7a RNA sequence (SEQ ID NO: 69) along with an amplification mechanism (aT, SEQ ID NO: 61 pT, SEQ ID NO: 62, enzyme, buffer, and dNTPs). The suspension is then encapsulated using a microfluidic flow focusing intersection, allowing the compartmentalization of individual particles into water-in-oil droplets. Upon incubation at 50° C., particles that have captured at least one microRNA begin to produce multiple copies of short DNA sequences (called signals) through polymerization / nicking cycles, which ultimately trigger an amplification reaction. The output strands of this reaction in turn hybridize with the supported probes, leading to an increase in the fluorescence of the particles. The particles are finally recovered by breaking the emulsion and analyzed by flow cytometry. The results are shown in FIG. 2. It can be noted that without incubation (and therefore no amplification, FIG. 2a), a single population of weakly fluorescent particles is observed, corresponding to background amplification of the supported probe. After incubation in the presence of 1 pM Let7a, a population of highly fluorescent particles (31%) is distinguished from a negative population (69%). This positive population corresponds to particles that have captured at least one target biomolecule. Assuming that the target is captured by the particles in Poisson random fashion, it is possible to calculate the concentration of Let7a measured in the assay by first calculating the parameter λ of the Poisson distribution.

[0080]

number

[0081] Thus, the measured concentration in the initial sample is 620 fM, which is consistent with the theoretical concentration (1 pM) when considering the uncertainty in the target dilution (dilution from a 100 µM stock solution). The negative control reports 2.6% false positive particles. Furthermore, we analyzed samples containing 1 pM Let7a target by flow cytometry and compare the results obtained by flow cytometry (using rT on beads modified with FAM fluorophore, SEQ ID NO: 64) and fluorescence microscopy (using rT in solution modified with Atto633 dye, SEQ ID NO: 63). Microscopic image analysis (Figure 3a) shows that 79% of particle-containing droplets (white dots) detected the target (indicated by grey fluorescent droplets), a result in good agreement with the flow cytometry readout where 85% of positive events were detected (Figure 3b).

[0082] Example 2 Enzyme capture effect We investigated the effect of particle concentration on the false positive rate. 7 The particles were incubated in the presence of 0 or 1 pM Let7a target (20 μL). 5 or 10 6 10 μL of particles were resuspended in the master mix, then emulsified in droplet microfluidics, incubated at 50 °C for 4 h, and analyzed by flow cytometry (Figure 4). 5 or 10 6At particles / μL, the negative controls recorded 16.4% and 2.6% false positive events, respectively, indicating that particle concentration has a significant effect on assay sensitivity. We show that pre-incubating particles in a master mix before dispensing results in the concentration of enzymes (one or several) on the DNA-grafted particles. As a result, overconcentration of enzymes (e.g., DNA polymerase) accelerates non-specific amplification reactions and ultimately increases the false positive rate.

[0083] To further demonstrate this effect, we designed the experiment shown in Figure 5. The enzyme mixture (polymerase, nickase, exonuclease) used for microRNA detection is incubated for 30 minutes: i) without particles, ii) with unfunctionalized particles, and iii) with oligonucleotide-functionalized particles. The particles are then pelleted and the supernatant is used as an enzyme mix to detect Let7a in solution, adding 10 pM Let7a and a molecular program (a set of oligonucleotides including aT, pT, cT, rT, and SEQ ID NO: 63), and incubating the mix at 50°C while monitoring the fluorescence of the report template in real time (Figure 5a). Figure 5b compares the onset times (Cq) of the three samples. When using the enzyme mixture preincubated without particles or with unfunctionalized particles, the Cq is very similar (around 100 minutes), demonstrating that the particles themselves do not affect the reaction. Mixing the enzyme and oligo-loaded particles can slow down the reaction and induce false negatives.

[0084] To counter this effect and accelerate the reaction rate and increase the sensitivity, reactions are performed at a constant particle concentration. Furthermore, particle dilution is compensated for by adding a population of "neutral particles" while keeping the particle concentration constant. These neutral particles carry the same amount of reporter template as the detection particles, but no conversion template. Thus, although they capture the same amount of enzyme as the detection particles, they are unable to capture microRNA and trigger amplification. In particular, the concentration of detection particles for Let7a (105 B of beads / µl Let7a ) from 0 to 2×10 5 The positive BN of samples incubated with 0 or 1 pM of Let7a target was used. Let7a The percentage of positive results is shown in Table 1. We demonstrated that the addition of neutral beads reduces the percentage of false positives compared to the negative control, but does not affect microRNA quantification. 5 At 3 x 10 particles / μL, the reaction is complete after 4 hours. 5 At particles / µL, the reaction takes approximately 10 h and 10 6 At 1000 particles / μL, the reaction is not complete even after 24 hours. Finally, we determined that the optimal particle concentration is approximately 3 × 10 5 It was concluded that the ELISA could be performed at 100 μg / μL.

[0085] To verify these experimental conditions, we performed digital detection of Let7a at different concentrations. Figure 7 shows that the false positive rate is only 0.7%, which proves the effect of adding neutral particles. In positive samples, the detected concentration is consistent with the expected concentration. Overall, these results indicate that DNA-functionalized particles are suitable for digital detection of one microRNA target. Multiplexed and Digital Detection of MicroRNAs Using a system that was well characterized for the detection of one target, we proceeded to the detection of several targets in a single assay. Figure 8 shows particle B targeting the microRNAs Let7a and mir92a, respectively. Let7a and B. 92a Figure 1 shows a duplex assay performed with two populations of 1000 ng / ml ...

[0086] As with single target assays, particle populations are significantly turned on only in the presence of their specific target. The false positive percentage of the negative control is very low (B Let7a 0.14%, B92 a Note that the false positive percentage is higher when other targets are present. For example, in the Let7a only sample, B 92a 7% of the miRNAs are turned on. This is probably due to the co-encapsulated particles and can be mathematically compensated for. The detected concentration is consistent with the expected concentration. Therefore, in this experiment, we have demonstrated that it is possible to effectively measure two miRNAs in a single assay. We then moved to a three-target assay (Let7a, mir203a, and mir92a) to further demonstrate the generalizability of the digital multiplex approach. Cognate particles, i.e., particles corresponding to each of the target biomolecules (i.e., modified with the corresponding conversion template (cT), SEQ ID NOs: 66-68) and neutral particle populations are spiked with 1 pM of one of the three targets. The results of the flow cytometry analysis are shown in Figure 9. The multiplexing capacity is limited only by the number of different particle populations, which can be separated, for example, by flow cytometry. The multiplexing capacity is limited only by the number of different particle populations, which can be separated, for example, by flow cytometry. FIG. 10 shows the discrimination of ten bead populations using a combination of two fluorescent barcodes (five and two levels of fluorescence, respectively).

[0087] MicroRNA detection in biological samples With this experiment, we demonstrated the suitability of the current digital detection method for detecting microRNA targets from biological samples (RNA extracts from human intestinal cells). In the proposed procedure, the target capture step is decoupled from the signal amplification. Thus, after capture, the particles can be washed and resuspended in a suitable buffer that is compatible with downstream biochemical reactions. Thus, the assay is compatible with biological samples made of complex media, and the washing steps allow the removal of initial matrix that may interfere with the amplification reaction. Before proceeding to the quantification of Let7a targets, human colon total RNA at a final concentration of 10 ng / μL was added to B Let7a FIG. 11 shows the detection concentration of Let7a, which is approximately 6×10 per ng of extract. 4 This demonstrates that the present invention allows for the detection of endogenous microRNAs in biological samples. Sensitivity and dynamic range adjustment Assay sensitivity is crucial when considering the quantification of low abundance targets. The sensitivity of a digital assay correlates closely with the ratio of positive / negative events, as determined by the average number of targets per particle (λ). In the present assay, this parameter was adjusted by changing the amount of detection particles. Thus, the dynamic range and sensitivity of the assay can be adjusted independently for each target. According to the diagram shown in Figure 12, for high abundance targets that are expected to saturate the particle population (for λ>5, the expected percentage of positive particles is >99%), the number of detection particles can be increased to adjust the parameter λ. Conversely, the amount of detection particles targeting poorly expressed targets can be decreased. Instead, if the target concentration is low, it is possible to increase the amount of sample used during the capture step. This results in an increase in the number of particles hybridizing to the target, and therefore an increase in the λ value.

[0088] Example 3 Use of two polymerases for nucleic acid detection. Enables sensitive quantification of DNA synthesis targets (Vent(exo -In addition to using exon 10 polymerase, we also used Klenow (exon 10) in the amplification reactions to quantify microRNAs. - We first performed this assay to detect Let7a. Acquisition Step miRNA capture was achieved by incubating detection particles, miRNA target, 25 μM dATP, dTTP, dCTP, and dGTP, and Klenow polymerase for 2 hours at 40° C. Concentrations are shown in Table 3 below.

[0089] [Table 4] Table 3: Concentrations of Klenow polymerase, particles, and microRNA Agitation was performed during incubation to avoid particle precipitation. After incubation, the particles were collected and washed twice with a storage buffer having the composition shown in Table 4 below. [Table 5] Table 4: Composition of storage buffer

[0090] Reaction mixtures A and B used in this assay are shown in Table 5 below. [Table 6] Table 5: Composition of reaction mixtures A and B

[0091] Enclosure Mix A and Mix B are encapsulated in 9 μm water-in-oil droplets in a 50% / 50% ratio using a double water inlet flow focusing microfluidic device. incubation The droplets are incubated at 50 °C for 8 h. particle analysis Droplets are disrupted using an electrostatic gun (Zerostat3, Milty, UK). Particles are resuspended in Attune NxT focusing fluid (ThermoFisher) and analyzed by flow cytometry (Attune NxT flow cytometer, ThermoFisher).

[0092] FIG. 13 shows that during the capture step (i.e., capture of miRNA(Let7a) on the particle surface), Klenow (exo - The detection of Let7a was clearly increased by introducing Klenow (exo - ) was detected, whereas Klenow (exo - When ) was not present, only 31 fM was detected. Klenow (exo - ) To optimize the action of the polymerase and to eliminate residual background amplification (due to non-specific amplification), we enhanced the experimental protocol described above by implementing additional post-capture wash steps in stringent buffers and a sonication step to remove captured polymerase molecules.

[0093] Acquisition Step miRNA capture was achieved by incubating detection particles, miRNA target, and Klenow polymerase for 2 hours at 40° C. with agitation (2000 rpm) to avoid particle precipitation. The concentrations are as follows: ·Particles: 10 9 particles / mL Klenow polymerase: 50 units / mL Post-capture wash: "Soft" cleaning procedure: Magnetically pool the particles and discard the remainder of the capture mix Resuspend in storage buffer Vortex for 30 seconds Discard the supernatant Resuspend in storage buffer Vortex for 30 seconds Discard the supernatant Resuspend in storage buffer Vortex for 30 seconds "Hard" cleaning instructions: Magnetically pool the particles and discard the remainder of the capture mix Resuspend in BW buffer Vortex for 30 seconds Sonicate in an ultrasonic bath for 30 seconds Discard the supernatant Resuspend in BW buffer Vortex for 30 seconds Sonicate in an ultrasonic bath for 30 seconds Discard the supernatant Resuspend in storage buffer Vortex for 30 seconds Discard the supernatant Resuspend in storage buffer Vortex for 30 seconds Discard the supernatant Resuspend in storage buffer Vortex for 30 seconds

[0094] The storage buffer has the same composition as shown above in Table 4. The composition of the BW buffer is shown below in Table 6. [Table 7] Table 6: Composition of BW buffer

[0095] Reaction mixtures A and B have the same compositions as those shown above in Table 5. Furthermore, encapsulation, incubation, and particle analysis are carried out in the same manner as described above. Figure 14 shows that the detection concentration from the negative control (sample without biomolecular target) was reduced by 80% due to the "hard" wash procedure, demonstrating that Klenow polymerase molecules are captured on the particles and that the "hard" wash procedure can eliminate non-specific amplification (background amplification).

[0096] Example 4 Design of conversion templates as poly(T) conversion templates As demonstrated above, the addition of Klenow polymerase to the capture step increased the detection of RNA targets. To further improve the sensitivity of our multiplex method, we designed a conversion template that contains a poly(T) spacer between the microRNA binding sequence and the Nt.BstNBI recognition site, thus increasing the number of DNA nucleotides on the primer. The experimental conditions (capture step, post-capture wash (hard wash protocol), encapsulation, incubation, and particle analysis) were as described above. Note, however, that only dATP (25 μM) was introduced during the capture step. I Figure 15 shows that the T5 and T15 (corresponding to the 5-15 nucleotide long poly(T) spacer corresponding to the sequences 21toBc T5 T7 biot (for T5) and 21toBc T15 T7 biot (for T15)); converter templates allow accurate quantification of miR21, with detection concentrations of 1.20 pM and 1.02 pM, respectively, compared to only 0.28 pM detected with the original T0cT. Furthermore, the T5 converter does not increase the background amplification.

[0097] Example 5 Six-plex detection of synthetic miRNA Using this set of optimized conditions (Klenow polymerase in capture, "hard" wash step, T5 converter template), the inventors evaluated the detection of synthetic miRNA spiked into water. Acquisition Step miRNA capture was performed by incubating detection particles, miRNA target, and Klenow polymerase at the following concentrations for 2 hours at 40°C: Particles: 2.5×10 in each of the 6 subpopulations 8 particles / mL (total 1.5×10 9particles / mL) Klenow polymerase: 50 units / mL

[0098] The concentrations of the six miRNAs are shown in Table 7 below. [Table 8] Table 7: Concentration of miRNA

[0099] Agitation was performed during incubation to avoid particle settling. Other experimental conditions (capture step, post-capture wash (hard wash protocol), encapsulation, incubation, particle analysis) were as described above. Note, however, that only dATP (25 μM) was introduced during the capture step. Figure 16 shows that the measured pattern is very close to the expected pattern (1.00E-12). The new experimental conditions allow for multiplexed quantification of RNA targets, which can be applied to the detection of disease-related molecular signatures.

[0100] Example 6 Adjustable Dynamic Range The detection concentration of the microRNA target is calculated using the following formula: [microRNA] = -ln(1-F pos ).[particle] 捕捉 In the formula, F pos is the percentage of positive particles, ranging from 0% to 100%. pos When values ​​of are extreme (close to 0% or 100%), the variability of the measured miRNA concentrations is very high and such F pos Quantification at low values ​​is less reliable, and therefore the dynamic range over which miRNA targets can be reliably quantified is limited. However, it is possible to increase the dynamic range by adjusting the particle concentration during the capture step: lowering the concentration of detection particles allows detection of lower concentrations of targets.

[0101] To assess the dynamic range of the test and to verify that the dynamic range can be modified by varying the particle concentration, two different particle concentrations are used to quantify different amounts of Let7a. Acquisition Step miRNA capture was achieved by incubating detection particles, miRNA target, and Klenow polymerase at the following concentrations for 2 hours at 40°C: Klenow polymerase: 50 units / mL in all samples Buffer: miR buffer 1x dATP only

[0102] [Table 9] Table 8: Concentration of miRNA After the capture step, a hard wash was performed as described above. Reaction mixture:

[0103] [Table 10] Table 9: Reaction mixtures and concentrations

[0104] Enclosure Mix A and Mix B are encapsulated in 9 μm water-in-oil droplets in a 50% / 50% ratio using a dual water inlet flow focusing microfluidic device. incubation The droplets are incubated at 50 °C for 8 h. particle analysis The droplets are disrupted using an electrostatic "gun." Particles are resuspended in Attune NxT focusing fluid (ThermoFisher) and analyzed by flow cytometry (Attune NxT flow cytometer, ThermoFisher). Figure 17 shows that the concentration of detection particles in the capture mix is ​​2 × 10 9When the particle concentration is 2×10 8 When reduced to particles / mL, the dynamic range shifts by approximately one order of magnitude (1 pM to 10 fM).

[0105] Example 7 Multiplexed detection from human colon total RNA In this system, we work with synthetic miRNA targets spiked into water. In this experiment, we demonstrate the multiplex detection of three microRNA targets in human colon total RNA. In this assay, we detect the human miRNAs Let7a, miR21, with Lin4 from Caenorhabditis elegans as a control. Acquisition Step miRNA capture was achieved by incubating detection particles, miRNA target, and Klenow polymerase at the following concentrations for 2 hours at 40°C: Klenow polymerase: 50 units / mL in all samples Particles: 5×10 in each of the three subpopulations 8 particles / mL (total 1.5×10 9 particles / mL) ·Total RNA: 2.5μg / mL Lin4: 1 pM (exogenous spike-in control) Buffer: miR buffer 1x dATP only After the capture step, a hard wash was performed as described above.

[0106] Reaction mixture: Reaction mixtures A and B were the same (same composition and same concentrations) as shown in Table 9 above. Further, encapsulation, incubation and particle analysis were performed as described in Example 6. Figure 18 shows that the system successfully detected three microRNAs from a sample of human colon total RNA, including two endogenous targets (let7a and mir21) and one exogenous target (spike-in lin4). The expected concentration of the control microRNA lin4 was 1 pM. The measured concentration is 0.94 pM. Preferred aspects of the present invention are as follows. [1] A digital multiplex method for detecting and / or quantifying multiple types of target biomolecules in a sample, comprising the steps of: a) functionalizing a suspension of particles, preferably microparticles, with one or more oligonucleotides selected from a first oligonucleotide that is a conversion oligonucleotide (cT), a second oligonucleotide that is a report oligonucleotide (rT), a third oligonucleotide that is an amplification oligonucleotide (aT), and a fourth oligonucleotide that is a leak-absorbing oligonucleotide (pT); b) adding a barcode to the particles functionalized in step a) that allows for the identification of particles targeted to multiple biomolecules; c) contacting the particles obtained in step b) with a test sample to capture the plurality of target biomolecules; d) resuspending the particles, which may or may not have captured the target biomolecule, in a common amplification mixture containing a buffer, an enzyme, deoxynucleoside triphosphates (dNTPs), and optionally oligonucleotides; e) separating the particles in the suspension obtained in step d) from one another so that each particle can react independently; f) incubating the particles at a constant temperature so that each target biomolecule induces an amplification reaction that generates an amplified signal on the particles carrying the target; and g) detecting and / or measuring the signals of the particles, including the barcode signal and the amplified signal of each particle; A digital multiplexing method comprising: [2] The functionalization of the suspension of particles, preferably microparticles, in step a) is carried out with the first oligonucleotide and the second oligonucleotide, The third oligonucleotide and the fourth oligonucleotide are added to the amplification mixture in step d). The digital multiplex method described in [1] above. [3] The digital multiplex method according to [1] or [2], wherein steps a) and b) are carried out simultaneously. [4] The digital multiplex method according to any one of [1] to [3] above, further comprising a step e1) of recovering the particles. [5] The digital multiplex method according to any one of [1] to [4] above, wherein the enzyme used in step d) is selected from the group including a polymerase, a nicking enzyme or a restriction enzyme, and an exonuclease. [6] The digital multiplex method according to any one of [1] to [5] above, wherein the suspension obtained in step d) is separated in step e) into droplets, preferably water-in-oil emulsion droplets, preferably having a droplet size comprised between 0.001 and 100 pL, preferably 0.01 and 10 pL, more preferably 0.1 and 5 pL. [7] The digital multiplex method according to any one of [1] to [6] above, wherein the constant temperature in step f) is between 30 and 55°C, preferably between 37 and 52°C, and more preferably between 45 and 50°C. [8] The digital multiplex method according to any one of [1] to [7] above, wherein the functionalized particles are selected from porous or non-porous particles and hydrogel particles having a size comprised between 10 nm and 500 μm, preferably between 100 nm and 100 μm, and more preferably between 50 nm and 10 μm. [9] The digital multiplex method according to any one of [1] to [8], wherein the step g) of detecting and / or measuring the barcode signal includes detecting and / or measuring the barcode signal of each particle associated with the target biomolecule and a signal resulting from the amplification, and the barcode signal is preferably a fluorescent signal.

[10] The digital multiplex method according to any one of [1] to [9], wherein the target biomolecules are of the same type or different types, and the biomolecules are nucleic acids or proteins.

[11] The digital multiplex method described in

[10] , wherein the target biomolecule is a nucleic acid, preferably selected from the group including DNA, cDNA, RNA, mRNA, and microRNA, and more preferably, the nucleic acid is microRNA.

[12] The digital multiplex method according to any one of [1] to

[11] above, wherein the target biomolecule is used as a biomarker.

[13] An in vitro method for diagnosing a disease selected from the group consisting of cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases, and prenatal diseases, comprising using the digital multiplex method according to any one of [1] to

[12] above.

[14] - Diseases caused by biotic stress, preferably of infectious and / or parasitic origin, or - Diseases caused by abiotic stress, preferably nutritional deficiencies and / or adverse environments An in vitro method for the agricultural diagnosis of a disease selected from the group comprising: An in vitro method comprising using the multiplex digital method according to any one of [1] to

[12] above.

[15] A kit for detecting and / or quantifying multiple types of target biomolecules, comprising: a) a suspension of particles, preferably microparticles, functionalized with one or more oligonucleotides selected from a first oligonucleotide which is a conversion oligonucleotide (cT), a second oligonucleotide which is a report oligonucleotide (rT), a third oligonucleotide which is an amplification oligonucleotide (aT), and a fourth oligonucleotide which is a leak-absorbing oligonucleotide (pT), and carrying different barcodes that allow for the identification of particles targeting different biomolecules; b) a mixture of enzymes preferably selected from the group comprising polymerases, nicking or restriction enzymes, and exonucleases, and c) Separation agent Including the kit.

[0107] Bibliographic references TIFF0007623961000016.tif218167 TIFF0007623961000017.tif249164 TIFF0007623961000018.tif197170

Claims

1. 1. A digital multiplex method for detecting and / or quantifying multiple target biomolecules in a sample, comprising the steps of: a) functionalizing a suspension of particles with a first oligonucleotide, a converter oligonucleotide (cT), that converts said target biomolecule into a signal sequence, and a second oligonucleotide, a reporter oligonucleotide (rT), that converts the presence of said signal sequence into a detectable signal; b) adding a barcode to the particles functionalized in step a) that allows for the identification of particles targeted to multiple biomolecules; c) contacting the particles obtained in step b) with a test sample to capture the plurality of target biomolecules, the number of particles being sufficient to randomly capture the plurality of targets by the particles according to a Poisson distribution, and the proportion of particles capturing at least one target being less than 100% of the total number of particles; d) resuspending the particles that have captured or have not captured the target biomolecule in an amplification mixture containing a buffer, an enzyme, a third oligonucleotide that is an amplification oligonucleotide (aT) that exponentially amplifies the signal sequence, a fourth oligonucleotide that is a leaky absorption oligonucleotide (pT) that drives the inactivation of the signal sequence synthesized by a leaky reaction, thereby allowing the avoidance of non-specific amplification, and deoxynucleoside triphosphates (dNTPs); e) separating the particles in the suspension obtained in step d) from one another so that each particle can react independently, the separation of the particles being obtained by microchamber arrays, water-in-oil droplets or particle dispersion in an immiscible fluid, f) incubating the particles at a constant temperature so that each target biomolecule induces an amplification reaction that generates an amplified signal on the particles carrying the target; and g) detecting and / or measuring the signals of the particles, including the barcode signal and the amplified signal of each particle; thereby enabling absolute quantification of associated target biomolecules in the initial sample by enabling direct counting of discrete events for each particle population.

2. 2. The digital multiplexing method of claim 1, wherein steps a) and b) are performed simultaneously.

3. 3. The digital multiplexing method according to claim 1 or 2, further comprising a step e1) of recovering the particles.

4. The digital multiplex method according to any one of claims 1 to 3, wherein the enzymes used in step d) are selected from the group comprising polymerases, nicking or restriction enzymes, and exonucleases.

5. 5. The digital multiplexing method according to any one of claims 1 to 4, wherein the suspension obtained in step d) is separated into droplets in step e).

6. Digital multiplexing method according to any one of claims 1 to 5, wherein the constant temperature in step f) is comprised between 30 and 55°C.

7. 7. The digital multiplexing method according to any one of claims 1 to 6, wherein the functionalized particles are chosen from porous or non-porous particles and hydrogel particles having a size comprised between 10 nm and 500 μm.

8. The digital multiplex method according to any one of claims 1 to 7, wherein the step g) of detecting and / or measuring the barcode signals comprises detecting and / or measuring the barcode signal of each particle associated with the target biomolecule and the signal resulting from the amplification.

9. The digital multiplex method according to any one of claims 1 to 8, wherein the target biomolecules are of the same type or different types, and the biomolecules are nucleic acids or proteins.

10. The digital multiplexing method of claim 9 , wherein the target biomolecule is a nucleic acid.

11. The digital multiplex method according to any one of claims 1 to 10, wherein the target biomolecule is used as a biomarker.

12. - diseases caused by biotic stress, or -Diseases caused by abiotic stress An in vitro method for the agricultural diagnosis of a disease selected from the group comprising: An in vitro method comprising using the digital multiplex method according to any one of claims 1 to 11.

13. 1. A kit for detecting and / or quantifying multiple target biomolecules, comprising: a) a suspension of particles functionalized with a first oligonucleotide, a converter oligonucleotide (cT), that converts the target biomolecule into a signal sequence, and a second oligonucleotide, a reporter oligonucleotide (rT), that converts the presence of the signal sequence into a detectable signal, and carrying different barcodes that allow for the identification of particles targeted to different biomolecules; b) a mixture comprising a third oligonucleotide, which is an amplification oligonucleotide (aT) that exponentially amplifies the signal sequence, a fourth oligonucleotide, which is a leaky absorption oligonucleotide (pT) that drives the inactivation of a signal sequence synthesized by a leaky reaction, thereby allowing the avoidance of non-specific amplification, and an enzyme; and c) A separating agent selected from the group consisting of water-in-oil emulsions, non-porous particles, and microchamber devices. Including the kit.

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