WO2018197725A1 - Electrochemical biosensor for the detection of nucleic acids - Google Patents

Abstract

The present invention can be included in the medical field. The present invention provides a method for detecting and/or quantifying target miRNA, RNA or DNA molecules in an isolated sample. The method involves the steps of (a) contacting the isolated samples with a reagent mixture, (b) adding hydrogen peroxide (H2O2) and an electrochemical mediator molecule, (c) using a magnet to capture the mixture obtained in step (b) onto an electrochemical sensor and (d) detecting and/or quantifying an electrochemical signal produced by the complex of step (c). The present invention also provides a device/kit and a combination product which comprises components which are useful for detecting and/or quantifying target miRNA, RNA or DNA molecules. Accordingly, the present invention also provides the use of the aforementioned device/kit and combination product for the detection and/or quantification of miRNA, RNA or DNA molecules in an isolated sample.

Description

ELECTROCHEMICAL BIOSENSOR FOR THE DETECTION OF NUCLEIC ACIDS

Technical field

The present invention can be included in the medical field. Specifically, the present application provides a method, device/kit and combination product for the detection and/or quantification of miRNA, RNA or DNA molecules.

Background art

MicroRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood, serum or plasma. These types of samples represent a challenge in terms of microRNA quantification.

Previously described electrochemical biosensor based on the use of AbS9.6 antibodies for selective capture of biotinylated DNA-miRNA hetero-duplexes and their further labeling using a commercial conjugate of Strep-HRP (Torrente-Rodriguez et al. ACS Sensors. 1 (2016) 896-903) demonstrated a LOD of 2.4 pM and a sensitivity of 9,548 nA nM^"1.

The newly developed method disclosed herein enables accurate and reproducible quantification of microRNAs in clinical samples, preferably in scarce clinical samples, and solves the problem of providing for a methodology capable of detecting and/or quantifying miRNA, RNA or DNA molecules of interest in at least one isolated complex clinical sample of different types of specimens, reliably, rapidly, reproducibly and with high specificity and sensitivity. Figures

Figure 1: Schematic illustration of the fundamentals involved in the preparation of the magnetic particle- based amperometric biosensor for the determination of miRNAs.

Figure 2: Effect of the number of incubation steps used to perform the determination of the target miRNA-21 on the resulting S/N current ratio using the developed methodology. Error bars estimated as triple of the standard deviation (n=3).

Figure 3: Effect of the pre-incubation time of AbS9.6 + ProtA-HRP40 mixture solution on the S/N current ratio. Error bars estimated as triple of the standard deviation (n=3).

Figure 4: Effect of the incubation time of target miRNA+AbS9.6-ProtA-HRP40 conjugate mixture on the S/N current ratio. Error bars estimated as triple of the standard deviation (n=3). Figure 5: Calibration plot constructed for synthetic target miRNA-21 determination with the developed electrochemical magnetosensor. Error bars estimated as triple of the standard deviation (n=3).

Figure 6: Calibration plots constructed for synthetic target miRNA-21 with the electrochemical biosensor developed by performing the labeling of the AbS9.6 using the ProtA-HRP40 (in gray) and ProtA-HRP (in black). Error bars estimated as triple of the standard deviation (n=3).

Figure 7: Selectivity of the magneto biosensor for the determination of miRNA-21. Current values were measured in the absence or in the presence of 0.025 nM target miRNA (miRNA-21), l-m(c), l-m(t) and NC sequences (miRNA-155 and miRNA-223). Error bars estimated as triple of the standard deviation (n=3).

Figure 8. Amperometric responses measured with the developed biosensor for the determination of the endogenous content of miRNA-21 in 250 ng raw RNAt extracted from cells and human breast tissues. Amperograms obtained for T and NT samples extracted from a breast cancer patient are also shown. Error bars estimated as triple of the standard deviation of three replicates.

Figure 9. Dependence of the S/B ratio obtained by comparing the amperometric responses obtained with the methodology of the present invention for 0 (B) and 0.01 nM (S) of target RNA by varying the length of the heterohybrid formed and by labelling it with AbRNA / DNA and ProtA-poli-HRP40.

Summary of the invention

The present invention provides a method for detecting and/or quantifying target miRNA, RNA or DNA molecules in an isolated sample. The method involves the steps of (a) contacting the isolated samples with a reagent mixture, (b) adding hydrogen peroxide (H₂O₂) and an electrochemical mediator molecule, (c) using a magnet to capture the mixture obtained in step (b) onto an electrochemical sensor and (d) detecting and/or quantifying an electrochemical signal produced by the complex of step (c). The present invention also provides a device/kit and a combination product which comprises components which are useful for detecting and/or quantifying target miRNA, RNA or DNA molecules. Accordingly, the present invention also provides the use of the aforementioned device/kit and combination product for the detection and/or quantification of miRNA, RNA or DNA molecules in an isolated sample.

Detailed description of the invention

Definitions

The terms "target miRNA", "target RNA" and "target DNA" refers to any miRNA, RNA or DNA which is of interest and which a skilled person might want to quantify or detect using the method, device/kit or combination product disclosed herein. The term "miRNA" ("microRNA") refers to a small non-coding RNA molecule (usually containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post- transcriptional regulation of gene expression. While the majority of miRNAs are located within the cell, some miRNAs, commonly known as circulating miRNAs or extracellular miRNAs, have also been found in extracellular environment, including various biological fluids and cell culture media.

As used herein the term "magnetic particles^'" refers to nano- to micro-magnetic particles (Φ = 10 nm to 10 μιη) with magnetic properties which may or may not be superparamagnetic. As used herein the term "RNA/DNA hetero-duplex" or "DNA/DNA homo-duplex" is understood as a duplex formed by the hybridization of two complementary sequences. The terms "RNA/DNA hetero- duplex" and "DNA/RNA hetero-duplex" are used interchangeably.

As used herein, the term "specifically binds" is understood as any antibody or a fragment thereof capable of recognizing said duplexes with affinities constants in the range of 10⁵-10¹² mol^"1.

The term "poly-HRP" refers to an enzymatic label comprising horseradish peroxidase polymer.

The term "adaptor molecule" refers to any molecule which allows for a covalent and/or non-covalent interaction between poly-HRP and the antibody or fragment thereof. For example, the adaptor molecule may be protein A which is conjugated to poly-HRP.

The term "electrochemical sensor" refers to any rigid or flexible support which can function as a working electrode in an electrochemical transduction.

The term "combination product" can refer to (i) a product comprised of two or more regulated components that are physically, chemically, or otherwise combined or mixed and produced as a single entity; (ii) two or more separate products packaged together in a single package or as a unit and comprised of drug and device products, device and biological products, or biological and drug products; (iii) a drug, device, or biological product packaged separately that according to its investigational plan or proposed labeling is intended for use only with an approved individually specified drug, device, or biological product where both are required to achieve the intended use, indication, or effect and where upon approval of the proposed product the labeling of the approved product would need to be changed, e.g., to reflect a change in intended use, dosage form, strength, route of administration, or significant change in dose; or (iv) any investigational drug, device, or biological product packaged separately that according to its proposed labeling is for use only with another individually specified investigational drug, device, or biological product where both are required to achieve the intended use, indication, or effect. Method for detecting and or quantifying target miRNA, RNA or DNA

The strategy for miRNA determination we report here relies on the efficient hybridization of the target miRNA with a, preferably biotinylated, complementary DNA probe (b-DNACp) immobilized onto Streptavidin-functionalized magnetic microcarriers (Strep-MBs), recognition of the perfectly matched DNA-miRNA heterohybrids with an antibody specific for DNA-RNA heteroduplexes, labeled with Protein A conjugated with an HRP homopolymer, (ProtA-PolyHRP40), and amperometric detection at SPCEs. This approach is generally schematized for illustrations purposes only in Figure 1 and involves two main steps: (i) selective capture of the target miRNA at the b-DNACp-modified MBs and simultaneous labeling of the b-DNACp-miRNA heteroduplexes with the anti DNA-RNA hybrid antibody and ProtA-PolyHRP (preferably with PolyHRP40 or PolyHRP80), and (ii) amperometric detection of the cathodic current produced upon addition of H202 using HQ as a redox mediator in solution after magnetic capture of the modified MBs on the working electrode surface of the SPCE. The measured magnitude of the cathodic current is related to the amount of HRP immobilized on the surface of MBs, this being in turn related to the number of hetero- duplexes formed and, therefore, proportional to the concentration of the target miRNA in the sample.

Therefore, in a first aspect, the present invention provides a method for detecting and/or quantifying target miRNA, RNA or DNA molecules in an isolated sample comprising the steps of: (a) contacting the isolated sample with a mixture comprising: (i) a magnetic particle coated with a complementary DNA molecule, wherein the complementary DNA molecule is complementary to the target miRNA, RNA or DNA, (ii) an antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo- /hetero-duplexes, conjugated to (iii) a poly-HRP, through (iv) one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody or fragment thereof covalently or non-covalently; (b) adding hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor; (c) using a magnet to capture the mixture obtained from step (b) onto an electrochemical sensor; and (d) detecting and/or quantifying an electrochemical signal produced by the complex of step (c), thereby detecting and/or quantifying the target molecule; wherein the antibody fragment is selected from the group consisting of "Fab" fragments, "F(ab')2 fragments, "Fv" fragments, single chain Fv fragments or "scFv", "Diabodies" and "bispecific antibodies" (Bab). In a preferred embodiment, the present invention provides a method for detecting and/or quantifying target miRNA molecules in an isolated sample.

As stated above, preferably the magnetic particle is first coated with a complementary DNA molecule before being incorporated into the mixture. In this sense, the magnetic particle should be functionalized in a manner which will allow the magnetic particle to bind to a modified or non-modified DNA or RNA molecule, wherein the DNA or RNA molecule has been designed to bind to a specific target sequence. The binding of the DNA or RNA molecule to the surface of the magnetic particle can be covalent or non- covalent. For example, the DNA or RNA molecule may be conjugated to a short peptide sequence capable of forming a covalent or non-covalent interaction with a protein molecule which has been covalently bound to the magnetic particle through a sulfhydryl- or amine-reactive cross-linker, or the DNA or RNA molecule may be conjugated to an amino acid and covalently attached to an NHS-activated magnetic particle. It is further noted, that the or each magnetic particle can be coated with just one complementary DNA or RNA molecule designed to bind to a specific target sequence, or may be coated, in order to significantly enhanced the sensitivity of the present methodology, with one or more polynucleotide sequences each comprising more than one, preferably at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100, complementary DNA or RNA molecules each designed to bind to a specific target sequence before being incorporated into the mixture. In this sense, each one or more polynucleotide sequences shall contain repeated sequences of the complementary DNA or RNA molecules designed to bind to the specific target sequence. In this regard, it is important to note that, as shown below, by varying the length of the heterohybrid formed (i.e. in bp (base pairs)) and by incubating it with an antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo-/hetero-duplexes, conjugated to (iii) a poly-HRP, through (iv) one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody or fragment thereof covalently or non-covalently (preferably forming the conjugates by using ProtA-poli-HRP40 or more preferably ProtA- poli-HRP80), there is a significant increase in the sensitivity of the methodology as shown in the table below and in figure 9.

In this embodiment, the magnetic particle is thus functionalized in a manner which will allow the magnetic particles to bind to a polynucleotide comprising more than one modified or non-modified DNA or RNA molecules, wherein each, preferably all, of the DNA or RNA molecules have been designed to bind to a specific target sequence. In this manner, the binding of the polynucleotides comprising the DNA or RNA molecules to the surface of the magnetic particle can be covalent or non-covalent and more than one antibody will bind to each of the polynucleotides functionalizing the magnetic particles thus significantly increasing the sensitivity of the present methodology. It is noted that the possibility of using magnetic particles coated with one or more polynucleotide sequences which in turn comprise more than one, preferably at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, complementary DNA or RNA molecules designed to bind to a specific target sequence, is understood herein as directly applicable to all aspects of the present invention including the device or the kit described in the present specification as well as to the combination product or any uses of the same. In this sense, in the context of the present invention, the term "polynucleotide" should be generally understood as one or more polynucleotide sequences each comprising more than one, preferably at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100, complementary DNA or RNA molecules each designed to bind to the specific target sequence,

In addition, in another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo- /hetero-duplexes, can be optionally conjugated to one or more secondary antibodies directed to the antibody portion of the said antibody or fragment to provide further amplification of a signal. In this embodiment, each of the secondary antibodies is in turn labelled or conjugated to a HRP, preferably a poly-HRP, through one or more adaptor molecule(s) which bind(s) the HRP or poly-HRP and the antibody or fragment thereof covalently or non-covalently (preferably forming the conjugates by using ProtA-poli-HRP40 or more preferably ProtA-poli-HRP80), thereby localizing more signal-generating moieties at the site of the molecule of interest. Other types of assays in which the disclosed conjugates can be used are readily apparent to those skilled in the art. It is noted, that the possibility of using one or more secondary antibodies directed to the antibody portion of the said antibody or fragment thus providing a further amplification of the signal, is understood herein as directly applicable to all aspects of the present invention including the device or the kit described in the present specification as well as to the combination product or any uses of the same.

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the magnetic particle is coated with a biotin- or biotin analogue-binding protein and the DNA, RNA or polynucleotide has been modified with a biotin or biotin analogue molecule. In this embodiment, the DNA, RNA or polynucleotide has been conjugated to the biotin or biotin analogue molecule. Examples of biotin-analogues include: digoxigenin, biotin-propanolol and fluorescently labeled biotin molecules. Examples of biotin- or biotin analogue-binding proteins include: streptavidin, avidin, spyavidin, neutravidin, traptavidin and anti-biotin antibodies (such as ab53494 from abeam). Preferably, the biotin- or biotin analogue-binding protein is streptavidin (SA) and the DNA, RNA or polynucleotide is conjugated to a biotin molecule. In a preferred embodiment, the SA-magnetic particle and biotin- DNA, RNA or polynucleotide molecule are pre- incubated for at least 10, 15, 20, 25 or 30 minutes before being incorporated into the mixture.

The present invention may comprise one or more adaptor molecules. For example, the binding of poly- HRP to the antibody or fragment thereof relies on the biotinylation of the antibody and the conjugation of the poly-HRP to a streptavidin molecule or the biotinylation of the antibody and the poly-HRP and the use of a divalent streptavidin (Fairhead et al. Journal of Molecular Biology. 426(1) (2014) 896-903 199- 214). In the aforementioned example, the term "adaptor molecule" refers to both the biotin molecule(s) and the streptavidin. In a preferred embodiment, the adaptor molecule is protein A which is conjugated to poly-HRP, other adaptor molecule could be IgG which is conjugated to HRP.

The antibody or fragment thereof may be any antibody or fragment thereof capable of specifically binding a DNA/DNA homo-duplex and/or a DNA/RNA hetero-duplex. In a preferred embodiment, the antibody or fragment thereof is antibody S9.6. Antibody S9.6 is a mouse monoclonal antibody, commercialized by Kerafast and generated against a ΦΧ174 bacteriophage-derived synthetic DNA-RNA antigen which recognizes selectively RNA-DNA hybrids of various lengths. The ATCC reference for this monoclonal antibody is S9.6 (ATCC® HB-8730™). Other potentially useful antibody is Covalab (clone D5H6).

The term "electrochemical mediator" refers to any molecule which can be oxidized in the presence of hydrogen peroxide and HRP and detected and/or quantified by an electrochemical sensor. The oxidized electrochemical mediator can accept electrons from the electrochemical sensor and the change in current can be correlated with the amount of oxidized electrochemical mediator. In a preferred embodiment, the electrochemical mediator molecule is selected from the group consisting of 3,3',5,5'- tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid), o-phenylenediamine dihydrochloride, hydroquinone, hydroxymethyl ferrocene, ferrocene, tetrathiafulvalene and osmium complex. Preferably, the electrochemical mediator molecule is hydroquinone. The term "isolated sample" refers to any in vitro sample. In a preferred embodiment, the isolated sample refers to an in vitro sample which has been isolated from a human. Preferably, the isolated sample is a tissue sample or biological fluid such as blood (preferably whole blood), serum, plasma, cerebrospinal fluid, saliva or urine. In a preferred embodiment, the method includes a pre-incubation step where the antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA hetero/homo-duplexes is incubated with one or more adaptor molecule(s) and poly-HRP before being incorporated into the mixture. Preferably, the method includes a pre-incubation step where the S9.6 antibody is incubated with a protein A-polyHRP conjugate (ProtA-poly-HRP). In a preferred embodiment, the S9.6 antibody is pre-incubated with ProtA- poly-HRP for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes.

In a preferred embodiment, the S9.6-ProtA-poly-HRP complex is incubated with the magnetic particle coated with a complementary DNA molecule and the isolated sample. In this embodiment, the magnetic particle has been pre-incubated with the complementary DNA molecule and the S9.6 antibody has been pre-incubated with ProtA-polyHRP. Preferably, the S9.6-ProtA-poly-HRP complex is incubated with the magnetic particle coated with a complementary DNA molecule and the isolated sample for at least 5, 10, 15, 20, 25, 30, 40, 50, or 60 minutes. a preferred embodiment, the method of the present invention comprises the steps of: a) contacting the isolated sample with a mixture comprising a magnetic particle coated with a complementary DNA, RNA or polynucleotide molecule, wherein the complementary DNA, RNA or polynucleotide molecule is complementary to the target miRNA, RNA or DNA,

b) adding an antibody or fragment thereof complexed with poly-HRP through the use of one or more adaptor molecule(s) to the mixture,

c) adding hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor,

d) using a magnet to capture the mixture obtained from step (c) onto an electrochemical sensor; and e) detecting and/or quantifying an electrochemical signal produced by the complex of step (d), thereby detecting and/or quantifying the target molecule. a preferred embodiment, the method of the present invention comprises the steps of:

a) contacting the isolated sample with a mixture comprising a magnetic particle coated with a complementary DNA, RNA or polynucleotide molecule, wherein the complementary DNA molecule is complementary to the target miRNA, RNA or DNA,

b) adding an antibody or fragment thereof complexed with ProtA-poly-HRP to the mixture, preferably a S9.6-ProtA-poly-HRP complex,

c) adding hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor,

d) using a magnet to capture the mixture obtained from step (c) onto an electrochemical sensor; and e) detecting and/or quantifying an electrochemical signal produced by the complex of step (d), thereby detecting and/or quantifying the target molecule. a preferred embodiment, the method of the present invention comprises the steps of:

a) contacting the isolated sample with a mixture comprising a magnetic particle coated with a complementary DNA, RNA or polynucleotide molecule (complementary for the target nucleic acid molecule) and an antibody or fragment thereof complexed with poly-HRP through the use of one or more adaptor molecule(s),

b) adding hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor,

c) using a magnet to capture the mixture obtained from step (c) onto an electrochemical sensor; and d) detecting and/or quantifying an electrochemical signal produced by the complex of step (d), thereby detecting and/or quantifying the target molecule. In a preferred embodiment, the method of the present invention comprises the steps of:

a) contacting the isolated sample with a mixture comprising a magnetic particle coated with a complementary DNA, RNA or polynucleotide molecule (complementary for the target nucleic acid molecule) and an antibody or fragment thereof complexed with ProtA-poly-HRP, preferably a S9.6-ProtA-poly-HRP complex,

b) adding hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor,

c) using a magnet to capture the mixture obtained from step (c) onto an electrochemical sensor; and d) detecting and/or quantifying an electrochemical signal produced by the complex of step (d), thereby detecting and/or quantifying the target molecule.

Furthermore, results demonstrate also that the use of ProtA conjugated with HRP homopolymers is an interesting strategy for signal amplification. In fact, the use of poly-HRP40 instead of HRP increases the sensitivity of the method 120-fold. Therefore, there is a clear advantage in using polymeric forms of HRP over a monomeric HRP molecule. Hence, the present method may use HRP polymers since HRP polymerization range is important for detection enhancement. In this regard, it is noted that SA-PolyHRP conjugates are made of 5 (five) identical covalent HRP homopolymer blocks that may be, also covalently, coupled to multiple streptavidin molecules. Three different homopolymers are currently used in our normal production process. These are PolyHRP20, PolyHRP40 and PolyHRP80. Therefore, in a preferred embodiment, the poly-HRP is selected from the group consisting of poly-HRP20, poly-HRP40 and/or poly-HRP80. In a preferred embodiment, the method of the present invention only comprises one or two incubation steps (excludes pre- incubation steps). In the first incubation step, the complementary DNA molecule is incubated with the magnetic particle for at least 5, 10, 15, 20, 25 or 30 minutes, however such incubation step could be very considered as a pre-incubation step. In this sense a first or second incubation step (depends on whether the first incubation step is considered or not a pre-incubation step), the isolated sample is incubated with the coated magnetic particles and an antibody or fragment thereof complexed with poly-HRP through the use of one or more adaptor molecule(s). Figure 1 provides an example for the two incubation steps of this embodiment.

In a preferred embodiment, the electrochemical sensor comprises (i) a solid electrode made of a material selected from the list consisting of: gold, carbon, platinum, CD-trode, screen-printed electrodes, silver, mercury, graphite, glassy carbon, carbon nanotubes, gold nanowires, gold nanoparticles, metallic oxide nanoparticles, carbon paste, boron-doped diamond and composites, and (ii) a magnet, preferably a neodymium magnet. Preferably, the electrochemical sensor comprises screen-printed electrodes and a neodymium magnet.

In a preferred embodiment, the limit of detection of the present method is below 2.4, 2.0, 1.5 or 1.0 pM. In a preferred embodiment the sensitivity of the present method is above 10,000, 20,000, 30,000, 40,000 or 50,000 nA nM ¹.

Therefore, in brief, the present invention describes a novel amperometric, preferably disposable, biosensor for the rapid, facile and sensitive detection of target nucleotides, preferably miRNAs. The method, illustrated for miRNAs such as miRNA-21 determination, is based on the selective capture of the target miRNA by specific DNA capture probe-modified MBs, recognition of the resulting DNA-miRNA heteroduplexes by a specific antibody further labeled with a bacterial protein conjugated with an homopolymer containing multiple HRP molecules, and amperometric detection upon magnetic capture of the modified MBs onto a SPCE. As illustrated in the examples, the biosensor exhibits very interesting analytical performance using a simple approach, providing a LOD of 0.4 pM within 30 min, without requiring previous reverse transcription of RNA to cDNA, complex amplification protocols or the use of an internal reference. In addition the biosensor shows successful applicability in the analysis of raw RNAt samples extracted from cancer cells and human tumor specimens. The short assay time, simplicity and feasibility to tailor the final sensitivity, to be applied for the determination of any target RNA and to perform multiple analyses in a single experiment, position this versatile methodology as a promising tool for high-throughput and simple miRNAs/RNAs bioanalysis applicable to a broad range of settings.

Device or kit

In a second aspect, the present application provides a device or kit. The device or kit comprises: (a) a magnetic particle which can be coated with a modified DNA, RNA or polynucleotide molecule, (b) a modified DNA, RNA or polynucleotide molecule which is complementary to a target miRNA, RNA or DNA molecule, (c) an antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo-/hetero-duplexes, wherein, the antibody fragment is selected from the group consisting of "Fab" fragments, "F(ab')2 fragments, "Fv" fragments, single chain Fv fragments or "scFv", "Diabodies" and "bispecific antibodies" (Bab), (d) a poly-HRP, (e) one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody covalently or non-covalently, (f) hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor, (g) an electrochemical sensor, wherein the electrochemical sensor is any rigid or flexible support which can function as a working electrode in an electrochemical transduction, and (h) a magnet, preferably a neodymium magnet.

The magnetic particle is functionalized in a manner which will allow the magnetic particle to bind to a modified or non-modified DNA, RNA or polynucleotide molecule. The binding of the DNA, RNA or polynucleotide molecule to the surface of the magnetic particle can be covalent or non-covalent. For example, the DNA molecule may be conjugated to a short peptide sequence capable of forming a covalent or non-covalent interaction with a protein molecule which has been covalently bound to the magnetic particle through a sulfhydryl- or amine-reactive cross-linker, or the DNA molecule may be conjugated to an amino acid and covalently attached to an NHS-activated magnetic particle.

In a preferred embodiment, the magnetic particle is coated with a biotin- or biotin analogue-binding protein and the DNA, RNA or polynucleotide has been modified with a biotin or biotin analogue molecule. Preferably, the magnetic particle has been coated with streptavidin and the DNA, RNA or polynucleotide has been conjugated to a biotin molecule.

The antibody or fragment thereof may be any antibody or fragment thereof capable of specifically binding a DNA/DNA homo-duplex and/or a DNA/RNA hetero-duplex. In a preferred embodiment, the antibody or fragment thereof is antibody S9.6 or clone D5H6 provided by Cobalab.

In a preferred embodiment, the poly-HRP is selected from the group consisting of poly-HRP20, poly- HRP40 and poly-HRP80. In a preferred embodiment, the adaptor molecule is protein A which is conjugated to the poly-HRP. In a preferred embodiment, the electrochemical mediator molecule is selected from the group consisting of 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), o- phenylenediamine dihydrochloride, hydroquinone, hydroxymethyl ferrocene, ferrocene, tetrathiafulvalene and osmium complex. Preferably, the electrochemical mediator molecule is hydroquinone. In a preferred embodiment, the electrochemical sensor comprises a solid electrode made of a material selected from the list consisting of: gold, carbon, platinum, CD-trode, screen-printed electrodes, silver, mercury, graphite, glassy carbon, carbon nanotubes, gold nanowires, gold nanoparticles, metallic oxide nanoparticles, carbon paste, boron-doped diamond and composites. Preferably the electrochemical sensor comprises screen-printed electrodes.

Combination product

In a third aspect, the present application provides a combination product which comprises (a) an antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo-/hetero-duplexes, wherein, the antibody fragment is selected from the group consisting of "Fab" fragments, "F(ab')2 fragments, "Fv" fragments, single chain Fv fragments or "scFv", "Diabodies" and "bispecific antibodies" (Bab), (b) a poly-HRP, and (c) one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody covalently or non-covalently. The antibody or fragment thereof may be any antibody or fragment thereof capable of specifically binding a DNA/DNA homo-duplex and/or a DNA/RNA hetero-duplex. In a preferred embodiment, the antibody or fragment thereof is antibody S9.6. In a preferred embodiment, the poly-HRP is selected from the group consisting of poly-HRP20, poly- HRP40 and poly-HRP80. In a preferred embodiment, the adaptor molecule is protein A which is conjugated to the poly-HRP.

In a preferred embodiment, the combination product further comprises a magnetic particle which can be coated with a modified DNA, RNA or polynucleotide molecule and/or a modified DNA, RNA or polynucleotide molecule which is complementary to a target miRNA, RNA or DNA molecule. Preferably, the combination product further comprises a magnetic particle which can be coated with a modified DNA molecule. The magnetic particle is functionalized in a manner which will allow the magnetic particle to bind to a modified or non-modified DNA molecule. The binding of the DNA, RNA or polynucleotide molecule to the surface of the magnetic particle can be covalent or non-covalent. For example, the DNA molecule may be conjugated to a short peptide sequence capable of forming a covalent or non-covalent interaction with a protein molecule which has been covalently bound to the magnetic particle through a sulfhydryl- or amine-reactive cross-linker, or the DNA, RNA or polynucleotide molecule may be conjugated to an amino acid and covalently attached to an NHS-activated magnetic particle.

In a preferred embodiment, the magnetic particle is coated with a biotin- or biotin analogue-binding protein and/or the DNA, RNA or polynucleotide has been modified with a biotin or biotin analogue molecule. Preferably, the magnetic particle has been coated with streptavidin and/or the DNA, RNA or polynucleotide has been conjugated to a biotin molecule.

In a preferred embodiment, the combination product further comprises hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly- HRP and detected and/or quantified by an electrochemical sensor.

In a preferred embodiment, the electrochemical mediator molecule is selected from the group consisting of 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), o- phenylenediamine dihydrochloride, hydroquinone, hydroxymethyl ferrocene, ferrocene, tetrathiafulvalene and osmium complex. Preferably, the electrochemical mediator molecule is hydroquinone.

The components of the combination product may be sold together or separately. If sold together, the components can be present in one container as a mixture or in multiple separate containers packaged as a single product. Some components may be lyophilized or in solution. If sold separately, the components should be labeled to make it clear that the components are to be used in combination with the other components of the combination product disclosed in this document. Uses of the device/kit and combination product

In a fourth aspect, the present invention provides the use of any of the devices, kits or combination products disclosed herein for any of the methods for detecting and/or quantifying target miRNA, RNA or DNA molecules in an isolated sample which have been disclosed in this document. In a preferred embodiment, the device, kit or combination product of the present invention further comprises instructions which outline the use of the device, kit or combination product in a method for detecting and/or quantifying target miRNA, RNA or DNA molecules in an isolated sample according to any one of the methods disclosed in the present document.

Examples

Apparatus and electrodes

Amperometric measurements were made with a CHI812B potentiostat (CH Instruments) controlled by CHI812B software. All measurements were carried out at room temperature. Screen-printed carbon electrodes (SPCEs) (DRP-110) consisting of 4-mm diameter carbon working electrodes were purchased from Drop-Sens. Furthermore, a specific cable connector (DRPCAC) acts as interface between the SPCEs and the potentiostat. A Bunsen AGT-9Vortex was used for the homogenization of the solutions. A magnetic separator (DynaMag2) was purchased from Invitrogen Dynal, and a constant temperature incubator shaker from Ivymen-Comecta was also used. A Raypa steam sterilizer, biological safety cabinet Telstar Biostar, a temperature freezer New Brunswick Scientific, a refrigerated centrifuge Sigma 1-15 K, Forma Scientific Infrared CO₂ Incubator and Stuart SBH130 Analogue Block Heater were also used.

Reagents and solution

All the reagents were of the highest available grade. Sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Scharlab; Prot A-HRP conjugate, hydroquinone (HQ), and H₂0₂ (30%, w/v) were purchased from Sigma-Aldrich; an anti-DNA-RNA Hybrid [S9.6] Antibody (AbS9.6) from Kerafast (USA), and a high sensitivity Protein A protein Poly-horseradish peroxidase conjugate (ProtA-HRP40) from Antibodies-Online were also used. Streptavidin-modified MBs (Strep- MBs, 2.8 μιη, 10 mg mL^"1, Dynabeads M-280 Streptavidin, 11206D) were purchased from Dynal Biotech ASA.

Buffer solutions were prepared with Milli-Q water (18 ΜΩ cm at 25 °C). Binding and Washing buffer (B&W) consisting of 10 mM Tris-HCl solution containing 1 mM EDTA and 2 M NaCl, pH 7.5 (sterilized after their preparation) and phosphate buffer 0.05 M, pH 6.0, were used. A commercial blocker casein solution (a ready-to-use, PBS solution of 1% w/v purified casein) was purchased from Thermo Scientific.

The sequences of DNA and RNA synthetic oligonucleotides used are described in Table 1 and were purchased from Sigma- Aldrich.

Table 1. Oligonucleotides used in this work.

These oligonucleotides were dissolved in nuclease free water at a final concentration of 100 μΜ, divided into small aliquots and stored at -80°C.

Magnetic bead modification

5.0 μΐ. of Strep-MBs suspension was transferred into a microcentrifuge tube and washed twice with 50 μΐ. B&W. Between washings, the particles were placed in the magnetic concentrator and, after 3 min, the supernatant was discarded. Washed MBs were incubated for 30 min at 30 °C under continuous stirring (950 rpm) with 25 μΐ. of 0.1 μΜ of the biotinylated antiDNA-21 capture probe solution (prepared in B&W). After two washing steps with 50 μΐ. of blocker casein solution, b-antiDNA-21 Cp-coated MBs were resuspended in 25 μΐ. of the previously prepared recognition and labeling mixture solution and incubated during 30 min (950 rpm, 30 °C). For the preparation of such mixture solution, the anti-DNA- RNA Hybrid Antibody (AbS9.6) and the ProtA-HRP40 were mixed, at a concentration of 2 μg mL^"1 for both reagents (which corresponds with 1 : 1,000 and 1 :25 dilutions, respectively) in a microcentrifuge tube containing blocker casein solution and pre-incubated during 60 min at room temperature. Subsequently, this AbS9.6-ProtA-HRP40 conjugate solution was supplemented with the appropriate amount of the synthetic target or the RNA_t extracted from the biological samples. Control experiments without target miRNA were performed for each hybridization and enzymatic labeling process in order to evaluate the blank signal. The ProtA-HRP40-AbS9.6-miRNA/b-DNACp-MBs assembly was then washed twice again with 50 μΐ. of blocker casein solution. Finally, the modified-MBs were resuspended in 50 μΐ. of 0.05 M sodium phosphate buffer solution (pH 6.0) to perform the amperometric detection.

Electrochemical measurements

To perform the amperometric measurements, the SPCE was positioned on a homemade casing of Teflon with an encapsulated neodymium magnet, and the 45 μΐ_^ of the modified MBs suspension were pipetted onto the SPCE where MBs were magnetically captured on the working carbon electrode. Then, the magnet holding block was immersed into an electrochemical cell containing 10 mL of 0.05 M phosphate buffer of pH 6.0 and 1.0 mM HQ (prepared just before performing the electrochemical measurement). Amperometric measurements in stirred solutions were made by applying a detection potential of -0.20 V vs. Ag pseudo-reference electrode upon addition of 50 μΕ of a 0.1 M H2O2 solution until the steady-state current was reached (approximately 100 s). The amperometric signals given through the manuscript corresponded to the difference between the steady-state and the background currents. Unless otherwise indicated, the presented data corresponded to the average of at least three replicates and the confidence intervals were calculated for a = 0.05.

Cell culture, human tissues and RNAt extraction

Protocols used for cell culture, collection of human tissues and RNAt extraction were as discussed below. Briefly, breast cancer cells MCF-7 were grown at 37 °C in a humidified atmosphere containing 5% C02 and maintained in high-glucose DMEM (Dulbecco's modified Eagle's medium), supplemented with fetal bovine serum (10%), penicillin (100 U mL-1), streptomycin (100 μg mL-1), and L-glutamine (2.5 mM) (GIBCO-Invitrogen, Carlsbad, CA, USA), whereas nontumorigenic epithelial MCF-IOA cells were cultured in high-glucose DMEM/Ham's Nutrient Mixture F12 (1 :1) with L-glutamine (2.5 mM), horse serum (5%, Gibco), human insulin (10 mg mL-1, Sigma), hydrocortisone (0.5 mg mL-1, Sigma), EGF (10 ng mL-1), and cholera toxin (100 ng mL-1, QuadraTech Ltd.).

With informed consent approved by Getafe University Hospital (Madrid, Spain) a 0.3 to 0.5 cm breast cancer tissue (T) and paired normal adjacent tissue (NT) from breast cancer patients were sectioned by the pathologist immediately after surgical excision of the tumor and placed in vials filled with RNAlater solution. This stabilization reagent was removed after 48 h of storage at 4 °C and the tissues kept frozen at -80 °C until their use. RNAt was isolated from all these samples using Tri Reagent (Molecular Research Center, Inc.). Briefly, PBS washed cells were scraped off and spun down. After homogenizing the pellet for 5 min in Tri Reagent at room temperature, the extraction with chloroform was carried out and the RNAt, in the upper aqueous phase, was precipitated with isopropylalcohol and washed twice in 70 % EtOH. Finally, the pellet was dried out during 10 min in a heating plate at 80 °C, dissolved in RNase-free water and stored at -80 °C [35]. RNAt quality and concentration were evaluated by measuring the absorbance at the appropriate wavelengths (260, 230 and 280 nm) with an ND-1000 spectrophotometer, obtaining in all cases ratio values confirming pure RNA.

Example 1 : Outline of method

We propose a novel strategy for miRNA determination based on efficient hybridization of the target miRNA with a biotinylated complementary DNA probe immobilized onto Streptavidin-functionalized magnetic microcarriers (Strep-MBs), recognition of the perfectly matched DNA/miRNA heterohybrids by a specific DNA-RNA antibody labeled in a final step with a commercial Protein A-Poly-HRP conjugate (ProtA-HRP40) through the Fc region of the DNA-RNA antibody and amperometric detection at SPCEs. The general approach is schematized in Figure 1 and involves two main steps: (i) selective capture of the target miRNA by efficient hybridization with the b-DNACp-modified MBs and simultaneous recognition of the resulting miRNA/b-DNA-MBs assembly by the conjugate AbS9.6-ProtA-HRP40 and (ii) amperometric detection of the catalytic current produced upon H2O2 addition using HQ as redox mediator in solution after magnetic capture of the resulting ProtA-HRP40-AbS9.6- miRNA/b-DNA-MBs on the working electrode surface of the SPCE. The measured catalytic current is related to the amount of HRP immobilized on the surface, which is related to the number of hetero-duplexes formed with the synthetic b-DNA capture probe and, therefore, proportional to the concentration of the target miRNA in the sample.

The feasibility of this approach, the optimization of all the operational characteristics involved, and its analytical performance were evaluated with miRNA-21 as target analyte because it is considered as a promising biomarker and therapeutic target for cancer. Example 2: Optimization of the experimental variables

All the experimental variables involved in this electrochemical biosensing approach were optimized by taking as the selection criterion the largest ratio between the current values measured at -0.20 V (vs the Ag pseudoreference electrode and potential value previously optimized for the HRP/HQ/H2O2 system (Conzuelo et al. Analytica Chimica Acta, 737 (2012) 29-36) in the absence (N) and in the presence of 0.25 nM (S) of the synthetic target miRNA-21. The tested variables, the checked range for each variable, and the values selected for further work are summarized in Table 2. Table 2. Optimization of the different experimental variables affecting the performance of amperometric magnetosensor developed for the determination of miRNA-21.

Experimental variable Tested range Selected value

[b-DNACp], μΜ 0 - 1 0.1 t b-DNACp incubation, mm 0 - 60 30

Strep-MBs, μΐ. 2.5 - 10 5.0

Number of incubation steps 2 - 4 2

AbS9.6 dilution 1 : 100 - 1 : 10,000 1 : 1,000

ProtA-HRP40 dilution 1 :5 - 1 :250 1 :25 t ProtA-HRP40 + AbS9.6 pre-incubation, mm 30 - overnight 60

t mixture solution incubation? mm 15 - 60 30

Example 3 : Optimization of the number of incubation steps

In order to simplify as much as possible the assay protocol, the effect of the number of steps used in the fabrication of this magnetobiosensor was investigated. The procedures tested, using in all cases 30 min- incubation steps, are summarized in Table 3. Figure 2 compares the amperometric responses measured for 0 and 0.1 nM target miRNA-21 as well as the corresponding signal-to-noise (S/N) ratios. As can be observed, the method involving only two incubation steps provided the highest S/N current ratio which, moreover, reduced drastically the total assay time from 120 min to 60 min. At this point, it is important to highlight the fact that, given the possibility to prepare and store in advance the b-DNACp-MBs, this bioelectrochemical strategy would enable the determination of miRNAs in only 30 min, accordingly, this simplified protocol was employed for the implementation of the electrochemical biosensing strategy for miRNA-21.

Table 3. Different protocols evaluated to perform the determination using the new electrochemical biosensing strategy.

Example 4: Optimization of the pre-incubation times

As can be deduced from the study of the number of incubation steps, a better recognition of the AbS9.6 is performed by the ProtA-HRP40 when both reagents are free in solution. Thus, the pre-incubation time of the AbS9.6 + ProtA-HRP40 mixture solution was studied in order to optimize the recognition of the AbS9.6's Fc region by the ProtA-HRP40 and improve the sensitivity of the final approach. Accordingly with the results shown in Figure 3, 60 min was selected as optimal pre-incubation time. Additionally, the effect of the incubation time of the mixture solution containing the target miRNA and the ProtA-HRP40- labeled AbS9.6 was also evaluated and displayed in Figure 4. As can be seen, the largest S/N current ratio was obtained between using incubation times between 15 and 30 min, while no significant improvement was observed using longer times which demonstrated that 30 min is enough time to carry out efficient hybridization and labeling processes. Accordingly, 30 min was selected to carry out this step.

Example 5: Analytical characteristics

The reproducibility of the amperometric responses obtained with different biosensors prepared in the same manner was evaluated by measuring the current values for 0.025 nM of synthetic target miRNA-21. A relative standard deviation (RSD) value of 3.1% was calculated from the measurements made with ten different sensors, demonstrating a great reproducibility of the whole MBs-based sensor fabrication and the signal transduction protocols used. The calibration curve constructed for the synthetic target miRNA is displayed in Figure 5 and the corresponding analytical characteristics are summarized in Table 4.

Table 4. Analytical characteristics obtained for the determination of synthetic target miRNA-21 using the developed electrochemical magnetosensor.

r 0.9995

Slope, nA nM^"1 55,314 ± 921

Intercept, nA 228 ± 45

Linear range, pM 1.0 - 100

Limit of detection (LOD), pM 0.4

Limit of quantification (LOQ), pM 1.0

A limit of detection (LOD) as low as 0.4 pM (10 attomoles in 25 μΐ. sample) was calculated according to the 3><Sb/m criterion, where Sb was estimated as the standard deviation for 10 blank signal measurements and m is the slope value of the calibration plot.

The comparison of the analytical performance of this approach with the previously described electrochemical biosensor based on the use of AbS9.6 antibodies for selective capture of biotinylated DNA-miRNA hetero- duplexes and their further labeling using a commercial conjugate of Strep-HRP (Torrente-Rodriguez et al. ACS Sensors. 1 (2016) 896-903) demonstrated a LOD 6 times lower (0.4 vs 2.4 pM) and a sensitivity 6 times higher (55,314 vs 9,548 nA nM^"1) which can be attributed to the use of AbS9.6 as detector antibody instead of as capture one and the small size of the S9.6 binding epitope. According to Qavi et al. (Qavi et al. Analytical. Chemistry. 83 (2011) 5949-5956) the S9.6 binding epitope is on the order of 6 base pairs in size and ~3 S9.6 antibodies can bind to a single DNA/miRNA duplex. Furthermore, the poly-HRP conjugate-based enzymatic labeling strategy used in this work could result an interesting approach for signal amplification.

Example 6: Storage stability of b-DNACp-MBs

The storage stability of the b-DNACp-MBs was evaluated by storing the modified magnetic microcarriers at 4 °C in microcentrifuge tubes containing 50 μL· of filtered PBS. Each working day, the amperometric responses obtained with sensors prepared using them for 0.0 and 0.025 nM synthetic miRNA-21 solution following the protocols described in the experimental section, were measured. Results achieved demonstrated that no significant decrease in the resultant S/N ratio was observed during at least 20 days (no longer times have still been assayed), suggesting the possibility of preparing sets of b-DNACp-MBs conjugates and storing them under the above-mentioned conditions until their use for the biosensor preparation is required. Example 7: HRP vs. poly-HRP

The sensitivity achieved by performing the labelling of the AbS9.6 with a conventional ProtA-HRP instead of the ProtA-HRP40 was compared (see Figure 6). Results achieved demonstrated an improvement in the biosensor sensitivity of 120 times.

Important advantages offered by this biosensing approach compared to previously developed methodology (Torrente-Rodriguez et al. ACS Sensors. 1 (2016) 896-903) included a much simpler working methodology reducing the steps number from 3 to 1 and the total assay time from 120 to 30 min. Interestingly, the great improvement in sensitivity demonstrated by the use of AbS9.6 for detection purposes and the ProtA-HRP40 for labeling steps demonstrated the possibility to tailor the sensitivity of this approach for the particular application (concentration level of the target miRNA to be detected) by enlarging using additional probes the length of the DNA/miRNA heteroduplexes which led to a higher numbers of AbS9.6 attached by these hybrids and performing the labelling with ProtA conjugated with HRP homopolymer with higher numbers of HRP (Poly-HRP80 ).

Example 8: Further reducing the time-to-result

The possibility of reducing even more the time-to-result was evaluated and, therefore, the step of incubation of the miRNA + ProtA-HRP40-AbS9.6 conjugate mixture solution was shortened up to 15 min. Furthermore, the possibility of minimizing the loss of sensitivity was assessed by employing a larger concentration of ProtA-HRP40 and/or AbS9.6. Interestingly, as can be observed in Table 5, the shortening of the assay time provoked only a 37% loss in sensitivity, working in optimized conditions regarding ProtA-HRP40 and AbS9.6 concentrations, and in only a 2.4% loss in the case of employing a double concentration of ProtA-HRP40, although these latter conditions would involve an increase of the price per assay. These relevant results outlined the potentiality of the developed methodology to be employed as a rapid method for the sensitive determination of miRNAs.

Table 5. Slope and intercept data obtained for each working conditions in the assessment of assay time reduction.

Incubation Slope, nA Intercept,

Recognition and labeling conditions

time, min nM ¹ nA

AbS9.6 (2 μζ/mL) + ProtA-HRP40 (2

30 55,214 ± 921 228 ± 45

μΒ/mL)

AbS9.6 (2 μζ/mL) + ProtA-HRP40 (2 34,843 ±

208 ± 82 μΒ/mL) 2,542

AbS9.6 ^g/mL) + ProtA-HRP40 30,844 ±

82 ± 145 ^g/mL) 4,494

15

AbS9.6 ^g/mL) + ProtA-HRP40

53,899 ± 659 23 ± 21 ^g/mL)

AbS9.6 ^g/mL) + ProtA-HRP40 48,862 ±

93 ± 138 ^g/mL) 4,269

Example 9: Selectivity of the approach

Since the magneto biosensor must be able to differentiate the target miRNA from other miRNAs in complex mixtures, the evaluation of the selectivity of the developed methodology was required. Thus, this study was carried out by comparing the signal measured for the target miRNA-21 with the those obtained for a single central base mismatched (l-m(c)), a single terminal base mismatched (l-m(t)) and two fully non-complementary (NC) sequences (miRNA- 155 and miRNA-223). All these sequences are included in Table 1. The current values provided by the biosensor were measured in the absence or in the presence of 0.025 nM of all these synthetic RNA sequences. As can be observed in Figure 7, the currents with the NC sequences were similar to that measured in the absence of target miRNA, while l-m(c) and l-m(t) sequences gave 48 and 65% of the response provided by the target miRNA, respectively. Example 10. Determination of mature miRNA-21 in RNAt extracted from cancer cells and tumor tissues.

The developed methodology was applied to the determination of the endogenous content of mature miRNA-21 in raw RNAt extracted from breast cancer (MCF-7) vs non-tumorigenic epithelial (MCF-IOA) cells and human tumor (T) vs paired normal adjacent (NT) breast tissues.

The amperometric responses obtained for the analysis of 250 ng of RNAt extracted from these samples are shown in Figure 8 and indicate overexpression of miRNA-21 in cancer cells and breast tumor tissues compared to normal cells and healthy tissues, which is in agreement with the oncogenic function of miRNA-21 in breast cancer. The possible existence of matrix effect for quantification in these samples was tested by constructing a calibration plot prepared by spiking 250 ng of extracted RNAt samples with growing concentrations of synthetic miRNA-21 up to 10 pM. The slope value of the linear calibration plot was significantly lower, approximately 10 %, than that calculated from the calibration graph constructed with the synthetic target miRNA-21 in the buffered solutions. Therefore, the existence of a matrix effect was concluded, possibly due to a hindered efficiency of the b-DNACp-miRNA-21 heterohybrids recognition by the antibody and ProtA-PolyHRP in the RNAt samples. Accordingly, the endogenous concentration of the mature target miRNA in all these samples was determined by applying the standard additions method. The results obtained are summarized in Table 6 below.

Table 6. Determination of the endogenous content of miRNA-21 (in amol per ng of RNAt) in human cells and breast tissues. miRNA-21 _{^ r}

Sample , T/ T ratio Contents found by other authors

(n = 3)

0.93 [34]

MCF-IOA 0.79 ± 0.14

1.02 [25]

Cells

3.3 [34]

MCF-7 2.33 ± 0.54

3.1 [25]

NT1 0.94 ± 0.12

0.1-1.5 [34]

NT2 0.80 ± 0.20

Breast tissues _^ „ „„„

Tl 2.81 ± 0.43

0.4-3.0 [34]

T2 1.65 ± 0.20

It is important to mention here that, apart from the matrix effect, a significant decrease in the amperometric responses were observed when RNAt amounts larger than 250 ng were used. This was attributed to a hook effect occurring when the amount of the endogenous target miRNA exceeds in a large amount that of the bDNACp immobilized on the MBs. It is important to note also that no significant matrix effect was observed when the hybridization and labelling steps were performed sequentially. Indeed, slope values of (20.3 · 0.7), (20 · 3) and (21 · 2) nA pM-1 were obtained for synthetic microRNA and in the presence of 250 ng of RNAt extracted from MCF-IOA cells and Tl, respectively. These results indicate that the matrix effect was due to the worse efficiency of the labelling step in the presence of RNAt. Nevertheless, despite the existence of the matrix effect, we think that the 1-step protocol can be considered as more advisable to perform the miRNA determination because of its rapidity, straightforwardness and higher sensitivity.

Claims

Claims

1. A method for detecting and/or quantifying target miRNA, RNA or DNA molecules in an isolated sample comprising the steps of:

a) incubating the isolated sample with a mixture comprising:

(i) a magnetic particle coated with at least one polynucleotide which in turn comprises at least one complementary DNA molecule, wherein the complementary DNA molecule is complementary to the target miRNA, RNA or DNA,

(ii) an antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo-/hetero-duplexes conjugated to a poly-HRP through one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody or fragment thereof covalently or non-covalently;

b) adding hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen and poly-HRP and be detected and/or quantified by an electrochemical sensor; c) using a magnet to capture the mixture obtained from step (b) onto an electrochemical sensor; and

d) detecting and/or quantifying an electrochemical signal produced by the complex of step (c), thereby detecting and/or quantifying the target molecule; wherein the antibody fragment is selected from the group consisting of "Fab" fragments, "F(ab')2 fragments, "Fv" fragments, single chain Fv fragments or "scFv", "Diabodies" and "bispecific antibodies" (Bab); and wherein the electrochemical sensor is any rigid or flexible support which can function as a working electrode in an electrochemical transduction.

2. The method according to claim 1, wherein the magnetic particle is coated with one or more polynucleotide sequences each comprising more than one, preferably at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 100, complementary DNA molecules each designed to bind to the specific target sequence, before being incorporated into the mixture.

3. The method according to any one of the preceding claims, wherein the antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo-/hetero-duplexes, is conjugated to a poly-HRP40 or poly-HRP80, through (iv) one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody or fragment thereof covalently or non-covalently.

4. The method according to any one of the preceding claims, wherein the target molecules are miRNAs.

5. The method according to any one of the preceding claims, wherein the adaptor molecule is protein A which is conjugated to poly-HRP40 or poly-HRP80. 6. The method according to any one of the preceding claims, wherein the antibody or fragment thereof is S9.

6.

7. The method according to any one of the preceding claims, wherein the electrochemical mediator molecule is hydroquinone.

8. The method according to any one of the preceding claims, wherein the isolated sample is a tissue sample or biological fluid such as blood, preferably whole blood, serum, plasma, cerebrospinal fluid, saliva or urine.

9. The method according to any one of the preceding claims, wherein the electrochemical sensor of step (c) comprises:

(i) a solid electrode made of a material selected from the list consisting of: gold, carbon, platinum, CD-trode, screen-printed electrodes, silver, mercury, graphite, glassy carbon, carbon nanotubes, gold nanowires, gold nanoparticles, metallic oxide nanoparticles, carbon paste, boron-doped diamond and composites, and

(ii) a magnet, preferably a neodymium magnet.

10. A device or kit comprising:

(a) a magnetic particle coated with at least one polynucleotide which in turn comprises at least one complementary DNA molecule, wherein the complementary DNA molecule is complementary to a target miRNA, RNA or DNA,

(b) an antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo- /hetero-duplexes, wherein, the antibody fragment is selected from the group consisting of "Fab" fragments, "F(ab')2 fragments, "Fv" fragments, single chain Fv fragments or "scFv", "Diabodies" and "bispecific antibodies" (Bab), conjugated to a poly-HRP, through one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody covalently or non-covalently,

(c) hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor,

(d) an electrochemical sensor, wherein the electrochemical sensor is any rigid or flexible support which can function as a working electrode in an electrochemical transduction, and

(e) a magnet, preferably a neodymium magnet.

11. The device or kit according to claim 10, wherein the at least one polynucleotide comprising at least one DNA molecule is modified with biotin or a biotin analogue and the magnetic particle is coated with a biotin or biotin analogue-binding protein.

12. The device or kit according to any one of claims 10-11, wherein the adaptor molecule is protein A which is conjugated to poly-HRP.

13. The device or kit according to any one of claims 10-12, wherein the antibody or fragment thereof is S9.6.

14. The device or kit according to any one of claims 10-13, wherein the electrochemical mediator molecule is hydroquinone.

15. The device or kit according to any one of claims 10-14, wherein the electrochemical sensor comprises a solid electrode made of a material selected from the list consisting of: gold, carbon, platinum,

CD-trode, screen-printed electrodes, silver, mercury, graphite, glassy carbon, carbon nanotubes, gold nanowires, gold nanoparticles, metallic oxide nanoparticles, carbon paste, boron-doped diamond and composites.

16. A combination product comprising:

(a) an antibody or fragment thereof which specifically binds RNA/DNA or DNA/DNA homo-/hetero- duplexes, wherein, the antibody fragment is selected from the group consisting of "Fab" fragments, "F(ab')2 fragments, "Fv" fragments, single chain Fv fragments or "scFv", "Diabodies" and "bispecific antibodies" (Bab),

(b) a poly-HRP, and

(c) one or more adaptor molecule(s) which bind(s) the poly-HRP and antibody covalently or non- covalently.

17. The combination product according to claim 16, wherein the combination product further comprises a magnetic particle which is coated with at least one polynucleotide comprising at least one modified DNA molecule which is complementary to a target miRNA, RNA or DNA molecule.

18. The combination product according to claim 16 or 17, wherein the combination product further comprises hydrogen peroxide and an electrochemical mediator molecule which can be oxidized in the presence of hydrogen peroxide and poly-HRP and detected and/or quantified by an electrochemical sensor.

19. The combination product according to any one of claims 16-18, wherein the adaptor molecule is protein A which is conjugated to poly-HRP.

20. The combination product according to any one of claims 16-19, wherein the antibody or fragment thereof is S9.6.

21. The combination product according to any one of claims 18-20, wherein the electrochemical mediator molecule is hydroquinone.

22. Use of the device or kit according to any one of claims 10-15 or the combination product according to any one of claims 16-21 for implementing the method according to any one of claims 1-9.