ITTO20070341A1 - PROCEDURE AND DEVICE FOR ELECTRIC TRANSDUCTION FOR THE DETECTION OF BIO-RECOGNITION EVENTS IN BIOMOLECULAR INTERACTION PROCESSES FOR GENOMIC / PROTEOMIC ANALYSIS - Google Patents

Description

DESCRIPTION of the industrial invention entitled: "Procedure and electrical transduction device for the detection of bio-recognition events in biomolecular interaction processes for genomic / proteomic analysis"

DESCRIPTION

The present invention relates to the detection of bio-recognition events in biomolecular interaction processes, and more specifically to a method and a device for the detection of bio-recognition events, respectively according to the preamble of claim 1 and claim 31.

In the last decade there has been the development of so-called bio-chips, essentially planar complex structures on whose surface biomolecules are immobilized (for example DNA, proteins, cells) capable of carrying out a selective recognition of one or more molecular species of interest ( identified as target species or, more generally, analytes).

The advent of bio-chips has greatly contributed to knowledge of genomics and proteomics, experimental and clinical diagnostics, pharmacology and pharmacogenomics.

The transducers that allow to selectively detect the presence of the molecular species of interest are mainly of the optical type and are based on the measurement of the fluorescence induced in the bio-recognition site following the binding reaction between probe molecular species and interacting target molecular species. Generally, one or both of the interacting species are conjugated to a fluorescent marker (fluorophore).

The different types of known bio-chips share a significant disadvantage. In fact, they perform an exclusively qualitative recognition (i.e., of the on-off type, to ascertain the presence or absence of the target species), but do not allow the extraction of quantitative information in real time (i.e. indicative of the concentration of the analyte or target species).

Optical detection requires instrumentation for reading signals including CCD imaging devices, photo-multiplier tubes, laser scanning devices, which are sensitive, expensive and difficult to transport devices, generally available in an equipped laboratory. It also requires expensive fluorescence markers, the emission of which is extinguished in a relatively short time. It is therefore not very suitable for the realization of equipment intended for commercial use.

The object of the present invention is to propose a methodology for the detection of bio-recognition events which allows quantitative measurements even in the presence of a small quantity of analyte, and potentially the detection also of single bio-recognition events.

A further object of the invention is to provide a device for detecting bio-recognition events that is inexpensive and easy to manufacture, suitable for point-of-care applications, possibly for single use.

For this purpose, the subject of the invention is a process for the detection of bio-recognition events having the characteristics referred to in claim 1, and a device for the detection of bio-recognition events having the characteristics referred to in claim 31.

Particular embodiments are the subject of the dependent claims.

A further object of the invention is an on-chip arrangement for the simultaneous detection of bio-recognition events as claimed.

In summary, the subject of the invention is a methodology for detecting bio-recognition events in biomolecular interaction processes based on an electrical transduction of these events by a transduction bio-receptor system comprising molecular probes immobilized in one or more nano devices. -junction, preferably made according to a quantum hole technology, suitable for interacting with respective target molecular species.

Different molecular probes, for example ssDNA, antibodies, receptors are able to react with corresponding analytes, such as DNA, proteins, ligands, to investigate biomolecular interaction processes or identify analytes of interest.

Each analyte or biomolecule to be identified is labeled with at least one conductive nanoparticle (for example a metal nanoparticle such as a gold nanoparticle) or is capable of selectively interacting (on the one hand) with molecular probes and (on the other) with molecular signalers coupled to a conductive nanoparticle. For example, the analyte can be a DNA strand consisting of two contiguous recognition elements that are complementary to the molecular probes in one end and to the molecular signaler in the other. The conductive nanoparticles are conjugated to the analyte or to the molecular marker through a strong bond, for example a covalent bond, and selected according to the molecular species under examination.

The nano-junction devices are formed by a pair of electrodes separated by a gap of the order of magnitude of the size of the nanoparticles conjugated to the analyte or molecular marker, i.e. a few nanometers, more specifically between 5 and 30 nanometers, for example 20 nanometers.

The immobilization of the molecular probes occurs on the surface of the electrodes of the nano-junction devices.

Following the specific interaction of the analyte (DNA, protein, ligand) to be determined with one or more molecular probes (bio-recognition event) associated with the electrodes of the nano-junction devices, or following the action of the analyte as a bridge between the molecular probes and the molecular signalers, one or more particles conjugated to the analyte or to the respective molecular signaler are arranged between the electrodes of the nano-junctions interconnecting them, causing a passage (or an increase) of current between them easily measurable following the application of a predetermined potential difference, thanks to the conductive properties of the nanoparticles conjugated to the analyte or molecular marker, which highlights the bio-recognition event.

By monitoring the variation of the current as a function of the voltage applied in a controlled manner to the ends of the electrodes, it is possible to quantitatively determine the bio-recognition processes that take place in the nano-junction device in the absence of a background signal which would constitute a potential detection disturbance. An array of nano-junction devices, each a few nanometers wide, is achievable at low cost through simple photolithographic and quantum-hole chemical etching techniques and allows the parallel detection of single bio-recognition events among a plurality of analytes / target and respective molecular probes by verifying an increase in current in the respective dedicated devices.

It is therefore possible to realize an on-chip detection arrangement, comprising an array of nano-junction devices functionalized with different molecular probes according to the biomolecular interaction processes to be investigated or the analytes to be identified.

Advantageously, the detection methodology object of the invention makes possible the practical realization of simple and inexpensive devices.

Through the nano-junction devices object of the invention it is possible to carry out accurate revelations of bio-recognition events through a current flow that also occurs following a single bio-recognition event, so that any background signal generated by species can be eliminated. free in solution, as opposed to currently available systems.

The high selectivity and extreme sensitivity of the detection make quantitative measurements possible and the detection of even a single recognition event makes it possible, for example, to identify target molecules (for example, specific DNA sequences) present only in number very small, so no amplification step is required, such as a PCR process in the case in which the number of specific DNA sequences in a biological sample is very low, even if this amplification process is not to be excluded a priori for other applications.

The simplicity and low manufacturing cost of nano-junction devices allows for mass production. Furthermore, such devices can be easily integrated with microelectronic circuits for the processing (amplification, filtering, modulation, analog / digital conversion for noise reduction) of the signals. Finally, electrical signals allow the integration of storage and display strategies for the information conveyed.

Advantageously, it is possible to integrate the recognition process on a large scale in arrays of devices for simultaneous analyzes in parallel on different analytes, so as to realize lab-on-chips in multiplexing mode. The possibility of increasing the number of different probes per unit area allows for a faster and more efficient response.

For example, the proposed approach effectively lends itself to the realization of inexpensive, easy-to-use, high-performance, short-response DNA-chips for rapid large-scale analysis of nucleic acid samples in the field of genetic disease diagnosis. detection of infectious agents, differential gene expression, environmental analyzes, opening the way to "point-of-care" analyzes, without the need to transfer the samples to be analyzed in a specialized laboratory, nor to use instruments and / or reagents additional.

Further characteristics and advantages of the invention will be set out in more detail in the following detailed description of an embodiment thereof, given by way of non-limiting example, with reference to the attached drawings, in which:

Figure 1 schematically shows a plan view of an array arrangement of nano-junction devices according to the invention;

Figure 2 is a three-dimensional schematic representation of an array arrangement of nano-junction devices according to the invention;

Figures 3a-3c show in section, schematically, the manufacturing steps of the structures forming the nano-junction devices object of the invention;

Figure 4 shows graphs of the eddy current trend in a nano-junction device as a function of the oxidation temperature and time, as well as the characteristic current-voltage at room temperature and in the dark for nano-junction devices with different degrees of oxidation;

Figures 5a and 5b show a nano-junction device object of the invention, respectively with a molecular probe immobilized before the molecular interaction with a target species and after this interaction, in the case of the example being the hybridization of a DNA strand; and figure 6 shows a series of current-voltage characteristics relating to three different nano-junction devices, respectively before an interaction phenomenon and after an interaction.

The procedure for detecting bio-recognition events in biomolecular interaction processes covered by the patent is based on the following operations:

- the conjugation of conductive nanoparticles with the target molecular species to be identified, more generally defined in the following analyte, or with associated molecular signalers {or better, the predisposition of conductive nanoparticles capable of coupling with said target species in a sample suspected of including said species);

- the preparation of transducer devices with metallic nano-junction, for the electrical transduction of bio-recognition events, and the immobilization, possibly spatially controlled, of molecular probes on the nano-junction, more precisely on the surface of the electrodes of the nano-junction junction; And

the detection of bio-recognition events between molecular probes (such as, for example, DNA, antibodies, receptors) and analytes or molecular targets (such as, for example, DNA, protein, ligand) for measuring the passage of current between the electrodes of the device nanojunction.

Figure 1 schematically shows a plan view of an arrangement 10 comprising an array of nano-junction transducer devices 12 according to the invention.

The arrangement has a "mesa" formation 20, comprising superimposed layers forming a traditional quantum hole (QW) structure, on which a first contact or common electrode 22 of conductive material is formed, and in the vicinity of which a plurality of second contacts or electrodes 24 of conductive material.

The first common electrode 22 essentially has the shape of an elongated brush, extended along the largest dimension of the formation 20, while the electrodes 24 essentially have the shape of a square or rectangular pad, different geometries being naturally possible without thereby departing from the scope of the present invention. invention.

Each electrode 24 is connected to the common electrode 22 through a conductive bridge 26 which extends from the respective pad on the inclined side of the "mesa" formation 20, and defines a nano-junction presenting an interruption in the conductive path which constitutes an electrical separation between the two electrodes in an inactive condition of the device, as it will be better understood from figure 2.

Figure 2 is a partial three-dimensional schematic representation of the arrangement 10 of Figure 1, in which two nano-junction devices 12 are represented.

SUB indicates the substrate or buffer layer on which the "mesa" formation 20 has grown and the electrodes 24 are deposited, 30 indicates a first lower barrier layer, 32 indicates a thin layer forming the quantum hole and 34 indicates a second upper barrier layer, oxidized at the top (oxidation depth indicated by hatching).

The conductive bridge 26 connecting the electrodes 22 and 24 placed on the side of the "mesa" formation 20 is interrupted at the quantum hole layer, and has dimensions comparable to those of the quantum hole, typically of the order of a few nanometers or a few tens of nanometers, further reduced by the thickness of the metal layer itself.

The process for manufacturing a nano-junction device or an array of devices as described is based on photolithography and wet chemical etching techniques of an AlGaAs / GaAs quantum well (QW), and explained below with reference to figures 3a-3c.

The process includes the traditional steps of forming a quantum hole of AlGaAs / GaAs grown on GaAs substrates by metal-organic chemical vapor deposition (MOCVD). As shown in the figures, the structure of the quantum hole includes the following layers: a GaAs buffer layer, for example 200 nanometers thick, an AlGaAs barrier, for example 300 nanometers thick, a GaAs layer (quantum hole) , for example with a thickness of 20 nanometers, an AlGaAs barrier, for example with a thickness of 100 nanometers and a covering layer of GaAs, for example with a thickness of 10 nanometers.

All layers are preferably grown at 750 ° C and are not doped.

The thickness of the GaAs quantum hole can be varied as needed to control the separation between the electrodes of the nanojunction.

Quantum holes made of other materials or grown with different procedures, for example molecular beam epitaxy, can also be used as long as it is possible to carry out a selective chemical attack of the quantum hole in order to obtain a nanometric gap on which to subsequently make the nano-junction by evaporation of contacts.

After a cleaning phase with acetone and isopropyl, mesa structures (about 250 nanometers high) are defined by optical lithography and wet chemical etching, for example in a Η20 / Η30Ξ / Η2Ρ04 solution with a 50: 1: 1 ratio for 120 seconds at 24 ° C, using a predefined mask.

The result is mesa structures with an oblique profile as shown in three-dimensional view in Figure 2.

Consequently, the quantum hole of GaAs is exposed and, after removal of the photoresist, it is subsequently selectively attacked by means of a 5: 1 solution of citric acid and water to remove a few tens of nanometers and create an effective separation between the barrier layers. equal to the thickness of the quantum hole (figure 3b) thanks to the high selective between GaAs and AlGaAs of the chemical etching process (nominally, 100: 1).

To reduce the eddy currents in the final nanojunctions, a selective oxidation of AlGaAs is preferably performed according to the following reaction:

in an oven heated by the Joule effect, for example by bubbling a flow of nitrogen in deionized water (at 3.3 1 / min <'1> and 80 ° C respectively) in order to transport water vapor into the oven and make it react with the 'AlAs present in the AlGaAs.

The sample can be placed in the oven a few minutes after starting the nitrogen flow, and a thermocouple allows you to control the temperatures in the chamber at the location of the sample.

As the covering layer of GaAs is removed, the AlGaAs exposed to water vapor in the furnace is oxidized directly.

Selective oxidation temperatures and times are optimized at 450 ° C and 240 minutes.

The aluminum concentration in the two layers of AlGaAs must be higher than 70% to obtain a good oxidation.

Then, using the configuration of the quantum hole structure as a "mask" or "guide", the electrode arrays for optical lithography and evaporation of a metallic layer of 15 nanometers of Cr / Au are defined. The deposition of the metallization preferably takes place in a direction perpendicular to the surface of the sample (Figure 3c), so as to produce the electrodes substantially by vertical projection on the oblique profile of the quantum hole structure, which thus defines the geometry of the nanjunction.

The result is a nano-junction device in which the distance between the electrodes is determined with sub-nanometer precision by the thickness of the GaAs quantum hole, the evaporated metal layer and the surface roughness of the chemically attached AlGaAs / GaAs interface ( experimentally less than 1 nanometer for low attack times).

The manufacturing process advantageously allows to produce pairs of electrodes with separations of a few nanometers, more precisely between 5 and 30 nanometers, by controlling the distance of the electrodes with subnanometric precision without the use of expensive electron beam devices, and by reducing eddy currents. (which would otherwise hinder the electrical detection) by at least six orders of magnitude with respect to the prior art through the selective oxidation of the AlGaAs layer (as shown in Figure 4).

Conveniently, the illustrated technique allows the simultaneous and economical fabrication, using low cost processes such as chemical etching and lift-off optical lithography, of extensive arrays of nano-junction devices, for example in an overall arrangement 10 as illustrated in Figure 1, and possibly of arrays of arrangements 10 for the formation of a multiplicity of side-by-side "mesa" structures, ultimately reaching a very large number of devices, since all the processes described can be performed simultaneously on a silicon wafer, satisfying an essential condition for a production mass of circuits at the nanoscale.

Figures 5a and 5b show a single nano-junction device object of the invention, respectively in an inactive condition with an immobilized molecular probe before the biomolecular interaction with a target species, and in an operating condition subsequent to the biomolecular interaction, in the case of the example being the hybridization of a DNA strand;

The nano-junction transducer device 12 comprises a pair of electrodes 22, 24 separated by a nanometric gap, of the order of magnitude of a few nanometers, i.e. between 5 and 30 nanometers, and at least one biological recognition element or probe molecular P, adapted to interact with an analyte / target T to be identified, immobilized on at least one of the electrodes.

Different molecular probes (e.g. ssDNA, antibodies, receptors) depending on the biomolecular interaction processes to be investigated, or the analytes to be identified (e.g. DNA, proteins, ligands), can be immobilized on different nano-junction devices of an arrangement terraced.

The target analytes or molecular species to be identified, represented in Figure 5a separately from the nano-junction transducer device, are correspondingly coupled with conductive NP nanoparticles, for example metal nanoparticles, or - in an alternative embodiment not shown - capable of acting bridge to connect one or more molecular signalers to which conductive nanoparticles are coupled to the molecular probes. Otherwise said, the target molecular species are labeled with conductive nanoparticles by direct conjugation of the nanoparticles with the target species or by coupling the nanoparticles with molecular markers capable of interacting with the target species.

The nanoparticles bound to the biomolecules can be of different types, as long as they are metallic or more generally conductive (for example, gold, silver, platinum, cobalt nanoparticles).

In particular, gold nanoparticles are the most stable among all metallic nanoparticles and the gold bonding chemistry is very well known and advanced, in particular it is known that thiol end groups allow an extremely stable covalent bond of gold molecules. and biomolecules. They can be synthesized in the organic phase or in the aqueous phase, but for biological applications that involve the interaction of nanoparticles with biomolecules such as DNA or proteins it is convenient to pass, after synthesis, in aqueous solution, since biological processes usually take place in water.

Aqueous nanoparticles with good size distribution are currently commercially available from companies such as Ted Pella or Sigma-Aldrich.

Hybrid materials composed of biomolecules such as DNA or proteins and inorganic substances can be obtained using different strategies such as: i) assembly by electrostatic interaction; iì) assembly by means of a sulfur-gold bond between gold nanoparticles and a thiol group, or a natural or synthetic disulfide bridge of the biomolecule; iii) assembly driven by the high affinity interaction between biotin / avidin or antigen / antibody.

The physical adsorption of biomolecules to the surface of nanoparticles can involve, especially in the case of proteins, problems of instability or inactivation, so it is convenient to bind the molecules in a covalent way. For example, the thiol groups of cysteines present in a protein can be exploited to bind it to gold nanoparticles. In the absence of accessible cysteines, thiol groups can be introduced either by chemical methods or by genetic engineering. Also for DNA it is possible to synthesize oligonucleotides linked to alkantiols which are able to bind covalently with gold nanoparticles.

Another strategy to create hybrid nanoparticle / biomolecule systems is to use high affinity systems in which one of the two components is bound to the nanoparticle, while the other is conjugated with the biomolecule. For this purpose, different conjugable systems can be exploited such as biotin and streptavidin, antibody and specific antigen, or protein A which recognizes the constant portion of antibodies.

The electrical translation of bio-recognition events is based on monitoring the current flowing through the nano-junction by applying a controlled potential difference between the electrodes of the device.

Following the specific interaction of the analyte (DNA, protein, ligand) to be determined with one or more probes (bio-recognition event), one or more nanoparticles conjugated to the analyte are immobilized between the electrodes of the nanjunctions, interconnecting them directly, since of comparable dimensions, and such events are identifiable by observing a passage or increase of current in the nano-junction, easily measurable.

In the embodiment shown here in Figures 5a and 5b, a hybridization sensor for DNA sequencing is provided. A single-stranded DNA fragment is immobilized on the surface of the nano-junction device and the formation of the double helix between the immobilized probe and the complementary target in solution, conjugated to a metal nanoparticle, is detected. In this case the molecular probes and the target are both single-stranded DNA fragments (ssDNA) and must be stably bound to the nanoparticles and to the nano-junction device respectively.

In the exemplary embodiment, nano-junctions consisting of gold electrodes were fabricated and gold nanoparticles were used as conductive nanoparticles bonded to the analyte.

Oligonucleotides modified at one end with thiol molecules (for example Cs-SH) able to bind stably both to the surface of the electrodes of the nano-junction device and to the metal nanoparticles through the formation of gold-sulfur bonds were used as probes and targets.

This solution reduces the release of probes during the analysis and allows to maintain the bio-recognition functions of the molecules unaltered, in this case the ability of the immobilized / conjugated ssDNA to hybridize with a complementary strand.

Commercial gold nanoparticles of 20 nanometers in diameter were used as markers of the analyte, which can be purchased in aqueous solutions stabilized with citrate, for example from Ted Pella or Sigma-Aldrich. As the nanoparticles were not stable in micromolar concentrations or in saline solutions, a first step was carried out consisting in stabilizing them with surfactant molecules such as phosphines (for example bis (psulfonatophenyl) phenylphosphines), which by replacing the initial surfactant (citrate) on the surface of the nanocrystals, thanks to a greater bonding affinity, create a coating of negative charges that prevents aggregation thanks to the electrostatic repulsion forces induced between the particles.

The gold nanoparticles were then conjugated with thiol functional oligonucleotides (C6-SH). The amount of DNA bound to the gold nanoparticles has been optimized so as not to hinder the hybridization due to a lack of DNA at the surface of the gold nanoparticles or excessive packing between the oligonucleotides.

For this purpose, conjugation reactions were carried out by incubating the DNA and the gold nanoparticles in different ratios (with 4, 40, 200, 400 DNA equivalents) for a time of about 18 hours and the amount of immobilized DNA per nanoparticles is was evaluated by agarose gel. It has been noted that the reaction reaches saturation at 200 equivalents and it has been chosen to use an intermediate ratio of gold nanoparticles to DNA equal to 1:40. Gels performed on 1:40 ratio samples incubated 42 hours and 18 hours gave similar results demonstrating that there are no other unreacted oligonucleotide molecules in solution that could exchange gold nanoparticles on the surface and / or interfere in the subsequent hybridization step. . Otherwise, a difference in migration between the two gels would have been observed.

In order to optimize the functionalization of gold surfaces with oligonucleotides with thiol functionalities, which constitute the molecular probes, oligonucleotides complementary to those conjugated to gold nanoparticles were immobilized on substrates of gold on mica. In particular, there are three fundamental prerequisites that the immobilized probes must satisfy:

1) be stably bonded to the surface of the electrodes;

2) maintain their own bio-recognition functions unaltered, which favors an <1> ordered orientation of the molecules in such a way as to avoid a reduction in the efficiency of bio-recognition due to sterile problems;

3) have a configuration and an orientation that do not prevent bio-recognition events (in the case in question, the hybridization reaction).

It was therefore decided to make self-assembling monolayers using again oligonucleotides with thiol functionalities capable of forming a strong covalent bond with a gold surface and self-assembling in monolayers on it in an ordered way with a defined orientation.

Consequently, the preferred procedure for the manufacture of probes in the example described provides for the realization of mixed self-assembling monolayers of thiolated oligonucleotides and other thiol molecules (mercaptoethanol or mercaptohexanol) which act as spacers between the DNA molecules, so as to control the density of packing of the probes and improve their accessibility, overcoming problems of spherical dimensions that could lead to a reduced efficiency of bio-recognition due to poor degrees of hybridization.

The devices are incubated first for two hours in an aqueous solution of DNA diluted to 1 μΜ in a 1M solution of KH2PO4 and then for 1 hour with a 1 mM solution of mercaptoethanol. In order to evaluate and demonstrate the functioning of the electrical detection microdevice, the junctions were subsequently incubated for hybridization in a 0.5 nM solution of gold nanoparticles conjugated in a ratio of 1:10 with thiol-modified oligonucleotides complementary to those immobilized on the electrodes for 15 hours. After 1 <1> hybridization the sample was rinsed in phosphate buffer and ammonium acetate solution to remove nonspecifically bound DNA. Current-voltage measurements were then followed to demonstrate the detection capability and verify the passage of current between the electrodes.

The results obtained are shown in figure 6, where curve A shows the current flowing in the nano-junctions before hybridization (background signal) almost zero, while curves B, C, D show the current flowing in three different nano-junctions after hybridization.

As can be observed, the current flowing in the nano-junctions functionalized with the molecular probes in an inactive condition is very low, of the order of pA, and comparable to the eddy currents present in this type of device. Following bio-recognition events between molecular probes and analyte, the gold nanoparticles are immobilized between the electrodes acting as a conductive bridge and consequently we observe the passage of a much higher current, of the order of nA, therefore an increase of a order of magnitude.

In the three test examples carried out, the shape of the curves after 1 <1> hybridization is of the sigmoid type and no non-conductive gap is observed, as would be expected for metal nanoparticles in the case of an ohmic contact. It is interesting to note how the observed current values appear quantized by 3 nA, respectively (this is shown in the insert of the figure). This means that one, two and three gold nanoparticles respectively are immobilized in the three electrodes. If the analyte has two different bases with respect to the complementary sequence of the molecular probes, no significant current flow is observed (curve E).

Conveniently, this means that the device object of the invention makes it possible to carry out quantitative measurements and also detect a very limited number of bio-recognition events, at most a single event.

This very high sensitivity makes it possible to identify specific DNA sequences in a biological sample even if they are present only in a very small number of copies, so that no analyte amplification step such as PCR is needed. PCR reactions or comparable amplification systems are currently required with the DNA-chips now on the market, but require additional instrumentation and reagents that are not ideal for point-of-care or field use.

Although not essential for the purposes of the invention, the device can optionally integrate a PCR chamber.

To induce thermal cycles it is possible to conjugate DNA with magnetic nanoparticles, for example of cobalt, and to use radiofrequencies to increase the temperature by orienting the magnetic moments of the nanoparticles. In this way it is possible to make the PCR reaction more selective since only the DNA conjugated to the nanoparticles would be subject to the correct thermal cycle and would be amplified. The same conductive cobalt particles could subsequently be used for the electrical detection of the hybridization as explained above.

Naturally, the principle of the invention remaining the same, the embodiments and construction details may be widely varied with respect to what has been described and illustrated purely by way of non-limiting example, without thereby departing from the scope of protection of the present document. invention defined by the appended claims.

Claims (45)

CLAIMS 1. Process for the detection of bio-recognition events in biomolecular interaction processes, including the preparation, in at least one bio-recognition site, of molecular probes (P) capable of interacting with target molecular species (T), and of associated transduction means suitable for detecting a coupling condition between at least one molecular probe (P) and a specimen of interacting target species (T), characterized in that it includes the following operations: - predisposition, in a sample suspected of including target molecular species (T), of conductive nanoparticles (NP) capable of coupling with said target species (T); - arrangement of at least one molecular probe (P) in association with a nano-junction transducer device (12) comprising a pair of conductive electrodes (22, 24) separated by a nanometric gap of the order of magnitude of the dimensions of said conductive nanoparticle (NP); exposure of said bio-recognition site to a solution containing said sample, the bio-recognition event of a target species (T) by a probe (P) involving the coupling between said probe (P) and at least one specimen of said target species (T) so as to determine the arrangement of said conductive nanoparticle (NP) coupled to the target molecular species (T) between the electrodes (22, 24) of the nano-junction device (12) and the consequent creation of a path conductive between them; - application of a predetermined potential difference to the electrodes (22, 24) of the nano-junction device (12); - detection of the current flow between the electrodes (22, 24) of the nano-junction device (12); And evaluation of the occurrence of bio-recognition events as a function of the detected current intensity. 2. Process according to claim 1, wherein said conductive nanoparticles (NP) are capable of conjugating directly with the target molecular species (T). 3. Process according to claim 1, wherein said conductive nanoparticles (NP) are suitable for conjugating with a molecular marker capable of selectively interacting with the target molecular species (T). 4. Process according to claim 2 or 3, wherein said conductive nanoparticles (NP) are metallic nanoparticles. 5. Process according to claim 4, wherein said metal nanoparticles are gold nanoparticles. 6. Process according to claim 5, wherein said nanoparticles (NP) are suitable for conjugating with the target molecular species (T) or respective molecular signalers through a covalent bond with thiol end groups or disulfide bridges of the target species. 7. Process according to claim 4 or 5, wherein said nanoparticles (NP) are suitable for conjugating with the target molecular species (T) or respective molecular markers by physical adsorption. 8. Process according to claim 4 or 5, wherein said nanoparticles (NP) are capable of conjugating with the target molecular species (T) or respective molecular signalers by electrostatic interaction. 9. Process according to claim 4 or 5, wherein said nanoparticles (NP) are suitable for conjugating with the target molecular species (T) or respective molecular signalers by high affinity interaction between a first component bound to the nanoparticle (NP) and a second component conjugated with the biomolecule (T). 10. Process according to claim 1, 2 or 3, wherein said conductive nanoparticles (NP) are magnetic nanoparticles. 11. Process according to claim 10, wherein said magnetic nanoparticles are cobalt nanoparticles. 12. Process according to claim 10 or 11, comprising the orientation of the magnetic moments of said nanoparticles (NP) by means of a variable external magnetic field for the induction of a thermal cycle and a PCR reaction for amplification of a target molecular species (T ) of the ssDNA type. 13. Process according to any one of the preceding claims, comprising the stabilization of the nanoparticles (NP) conjugable with target molecular species (T) of the ssDNA type with surfactant molecules. 14. Process according to any one of claims 1 to 12, comprising the stabilization of the nanoparticles (NP) that can be conjugated with target molecular species (T) of the ssDNA type with oligonucleotides with thiolic functionality. Method according to claim 1, wherein the arrangement of the molecular probe (P) in association with the nano-junction device (12) comprises the spatially controlled immobilization of the probe (P) on the surface of at least one electrode of the device (22 ; 24), according to an oriented arrangement. 16. Process according to claim 15, wherein said molecular probe (P) is ssDNA. Method according to claim 16, wherein the arrangement of the molecular probes (P) in association with a nano-junction device (12) comprises the immobilization of at least one probe (P) on the surface of at least one electrode (22; 24) through a covalent bond of self-assembling monolayers of oligonucleotides with thiol functionalities. 18. Process according to claim 17, comprising the interposition of thiol molecules acting as spacers between the ssDNA molecules. 19. Process according to claim 15, wherein said molecular probe (P) is an antibody, adapted to interact with a sample suspected of including target molecular species (T) antigens. 20. Process according to claim 15, wherein said molecular probe (P) is a receptor, adapted to interact with a sample suspected of including target molecular species (T) ligands. A method according to any one of the preceding claims, comprising the formation of a nano-junction transducer device (12) based on a quantum hole. 22. Process according to claim 21, comprising the steps of: - deposition of superimposed layers (30, 32, 34) of a heterostructure including a quantum hole layer (32) comprised between a pair of barrier layers (30, 34); - definition of "mesa" formations (20) according to a predetermined configuration by lithography and subsequent chemical etching, said chemical etching process determining formations with an oblique profile with an exposed quantum hole (32); - selective chemical etching by wet method of the quantum hole layer (32); And - formation of the electrode contacts (22, 24). 23. Process according to claim 22, wherein said layer deposition occurs through chemical metal-organic vapor deposition. 24. Process according to claim 22, wherein said layer deposition occurs through molecular beam epitaxy. 25. Process according to claim 22, wherein said selective etching is obtained by means of a 5: 1 solution of citric acid and water. The method according to claim 22, wherein the formation of the electrode contacts (22, 24) comprises the definition of the contact configuration by lithography and the evaporation deposition of a conductive material layer in a direction perpendicular to the arrangement of the "mesa" formation. "(20). A process according to claim 22, further including selective oxidation of the upper barrier free layer (34). 28. A method according to claim 27, wherein said quantum well is a Ga-As / AlGaAs quantum well. 29. Process according to claim 28, wherein said oxidation occurs according to the reaction in a room heated to 450 ° C for 240 minutes. 30. Process according to claim 29, wherein the concentration of Aluminum in the top free barrier layer of AlGaAs is greater than 70%. 31. Device for the detection of bio-recognition events in biomolecular interaction processes, including at least one bio-recognition site that houses molecular probes (P) capable of interacting with target molecular species (T), and transduction means associated with said site of bio-recognition, designed to reveal a condition of coupling between at least one molecular probe (P) and a specimen of interacting target species (T), characterized in that said transduction means comprise at least one nano-junction device (12) functionalized with at least one predetermined molecular probe (P), comprising a pair of conductive electrodes (22, 24} separated by a nanometric gap on at least one of the which said molecular probe (P) is arranged, in which the gap between the electrodes (22, 24) is of the order of magnitude of the size of a predetermined conductive nanoparticle (NP) suitable for coupling to the target molecular species (T) to which said bio-recognition site is operationally intended to be exhibited; the bio-recognition event of a target species (T) by a probe (P) involving the coupling between said probe (P) and at least one specimen of said target species (T) so as to determine the interposition of said conductive nanoparticle (NP) between the electrodes (22, 24) of the nano-junction device (12) and the consequent creation of a conductive path between them; And in that the device also includes means for applying a predetermined potential difference to the electrodes (22, 24) of the nano-junction device (12) and means for detecting the current flow between the electrodes (22, 24) of the nano-junction (12). 32. Device according to claim 31, wherein said molecular probe (P) is immobilized in a spatially controlled manner on the surface of at least one electrode (22; 24} of the nano-junction device (12), according to an oriented arrangement. A device according to claim 31 or 32, wherein said molecular probe (P) is ssDNA, adapted to interact with a sample suspected of including complementary ssDNA. 34. Device according to claim 33, wherein said probe {P) is immobilized on the surface of at least one electrode {22; 24) through a covalent bond of self-assembled monolayers of oligonucleotides with thiol functionalities. 35. Device according to claim 34, wherein thiol molecules are interposed as spacers between the ssDNA molecules. A device according to claim 31 or 32, wherein said molecular probe (P) is an antibody, adapted to interact with a sample suspected of including target molecular species (T) antigens. A device according to claim 31 or 32, wherein said molecular probe (P) is a receptor, adapted to interact with a sample suspected of including target molecular species (T) ligands. A device according to any one of claims 31 to 37, wherein said nano-junction device (12) is a quantum hole-based device. A device according to claim 38, wherein said nano-junction device (12) comprises a heterostructure including a quantum hole layer (32) comprised between a pair of barrier layers (30, 34). 40. Device according to claim 39, wherein said heterostructure has a "mesa" formation (20) according to a predetermined configuration, with an exposed quantum hole oblique profile, on which the electrode contacts (22, 24) are formed. A device according to claim 40, wherein the barrier free upper layer (34) is oxidized. 42. Device according to any one of claims 38 to 41, wherein said quantum hole is a GaAs / AlGaAs quantum hole. 43. Device according to claim 42, wherein the concentration of Aluminum in the barrier free upper layer of AlGaAs is greater than 70%. A device according to claim 43, wherein said electrodes (22, 24) include metallic gold contacts. 45. Arrangement on chip (10) for the simultaneous detection of bio-recognition events in biomolecular interaction processes, comprising a matrix of nano-junction devices (12) according to claims 31 to 44, each functionalized with a molecular probe species (P) different.