WO2008139421A2

WO2008139421A2 - Electrical transduction method and device for the detection of biorecognition events in biomolecular interaction processes for genome/proteome analysis

Info

Publication number: WO2008139421A2
Application number: PCT/IB2008/051900
Authority: WO
Inventors: Giuseppe Maruccio; Elisabetta Primiceri; Pasquale Marzo; Valentina Arima; Roman Krahne; Teresa Pellegrino; Antonio Della Torre; Franco Calabi; Roberto Cingolani; Rosaria Rinaldi
Original assignee: Consiglio Nazionale Delle Ricerche - Infm Istituto Nazionale Per La Fisica Della Materia; Fondazione Istituto Italiano Di Tecnologia
Priority date: 2007-05-15
Filing date: 2008-05-14
Publication date: 2008-11-20
Also published as: ITTO20070341A1; WO2008139421A3

Abstract

A method is described for detecting biorecognition events in biomolecular interaction processes, including the provision, on at least one biorecognition site, of molecular probes (P) adapted to interact with target molecular species (T) which are coupled to conductive nanoparticles (NP), in which the probes (P) are associated with a nanojunction transducer device (12) comprising a pair of conductive electrodes (22, 24) separated by a nanometric gap of the same order of magnitude as the dimensions of said conductive nanoparticles (NP), adapted to detect a condition of coupling between at least one molecular probe (P) and a specimen of a target species (T) which interact with each other. A biorecognition event includes the coupling of a probe (P) to at least one specimen of the target species (T) and results in the positioning of the conductive nanoparticle (NP) coupled to the target species (T) (by being directly conjugated with the latter or with a signal molecule which can interact with it) between the electrodes (22, 24) of the nanojunction device (12), with the consequent creation of a conductive path between the electrodes. The occurrence of biorecognition events is evaluated according to the intensity of the current which is made to flow between the electrodes (22, 24) of the nanojunction device (12) by the application of a potential difference.

Description

Electrical transduction method and device for the detection of biorecognition events in biomolecuiar interaction processes for genome/proteome analysis

The present invention relates to the detection of biorecognition events in biomolecuiar interaction processes, and more specifically to a method and a device for detecting biorecognition events, according to the preambles of Claim 1 and Claim 31, respectively.

The last decade has seen the development of so-called biochips, which are essentially planar complex structures on whose surfaces biomolecules (such as DNA, proteins, or cells) are immobilized for the selective recognition of one or more molecular species of interest (referred to as target species, or, more generally, analytes).

The advent of biochips has made a major contribution to knowledge in genomics and proteomics, experimental and clinical diagnosis, pharmacology and pharmacogenomics.

The transducers used for the selective detection of the presence of the molecular species of interest are principally of the optical type and are based on the measurement of the fluorescence induced at the biorecognition site as a result of the binding reaction between probe molecular species and target molecular species that interact with each other. Generally, one or both of the interacting species are conjugated with a fluorescent marker (fluorophore).

The various known types of biochip share a considerable drawback. They carry out an exclusively qualitative recognition (of the on-off type, in other words, based on determination of the presence or absence of the target species), but cannot be used for the real-time extraction of quantitative data (in other words, data indicating the concentration of the analyte or target species).

Optical detection requires instruments for reading the signals, including CCD imaging devices, photomultiplier tubes and laser scanning devices, which are sensitive, expensive and difficult to transport, and are generally to be found in a fully equipped laboratory. There is also a need for expensive fluorescent markers whose emission ceases in a relatively short time. This method is therefore rather unsuitable for the production of equipment for commercial use.

The object of the present invention is to propose a method for detecting biorecognition events which enables quantitative measurements to be made even in the presence of minute quantities of analyte, and potentially permits the detection of individual biorecognition events.

A further object of the invention is to provide a device for detecting biorecognition events which is economical and simple to manufacture, can be used for point-of-care applications, and may be disposable.

For this purpose, the invention proposes a method for detecting biorecognition events having the characteristics claimed in Claim 1, and a device for detecting biorecognition events having the characteristics claimed in Claim 31.

Specific embodiments form the subject of the dependent claims.

The invention also proposes a chip arrangement for the simultaneous detection of biorecognition events as claimed.

In summary, the invention proposes a method for detecting biorecognition events in biomolecular interaction processes, based on the electrical transduction of these events by a transduction bioreceptor system comprising molecular probes immobilized in one or more nanojunction devices, preferably formed by quantum well technology, adapted to interact with corresponding target molecular species.

Different molecular probes, such as ssDNA, antibodies, and receptors, are adapted to react with corresponding analytes, such as DNA, proteins and ligands, for the investigation of biomolecular interaction processes or the identification of analytes of interest.

Each analyte or biomolecule to be identified is marked with at least one conductive nanoparticle (for example, a metallic nanoparticle such as a gold nanoparticle), or can interact selectively (on the one hand) with the molecular probes and (on the other hand) with signal molecules coupled to a conductive nanoparticle. For example, the analyte can be a DNA strand composed of two contiguous recognition elements which are complementary to the molecular probes at one end and to the signal molecule at the other. The conductive nanoparticles are conjugated with the analyte or with the signal molecule by a strong bond, for example a covalent bond, and are selected according to the molecular species to be examined.

The nanojunction devices are formed by a pair of electrodes separated by a gap of the same order of magnitude as the dimensions of the nanoparticles conjugated with the analyte or with the signal molecule, i.e. of a few nanometres, and more specifically in the range from 5 to 30 nanometres, for example 20 nanometres.

The molecular probes are immobilized on the surfaces of the electrodes of the nanojunction devices.

As a result of the specific interaction of the analyte (DNA, protein or ligand) to be determined with one or more molecular probes (the biorecognition event) associated with the electrodes of the nanojunction devices, or as a result of the action of the analyte as a bridge between the molecular probes and the signal molecules, one or more particles conjugated with the analyte or with the corresponding signal molecule are positioned between the electrodes of the nanojunctions, thus interconnecting them and creating a flow (or increase) of current between the electrodes, which is easily measurable by the application of a predetermined potential difference, owing to the conductive properties of the nanoparticles conjugated with the analyte or with the signal molecule, thus providing evidence of the biorecognition event.

By monitoring the current variation as a function of the voltage applied in a controlled way to the terminals of the electrodes, the biorecognition processes that take place in the nanojunction device can be determined quantitatively in the absence of a background signal which would potentially interfere with the detection. An array of nanojunction devices, each with a width of a few nanometres, can be formed at low cost by means of simple photolithographic and quantum well chemical etching methods, and can be used for the parallel detection of single biorecognition events between a plurality of analytes/targets and corresponding molecular probes by detecting a current increase in the corresponding dedicated devices.

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

Advantageously, the detection method proposed by the invention allows the practical manufacture of simple and economical devices.

Using the nanojunction devices proposed by the invention, it is possible to carry out accurate detection of biorecognition events by means of a flow of current which takes place as a result of even a single biorecognition event, and thus it is possible to eliminate any background signal generated by free species in solution, by contrast with the systems available at present.

Because of the high selectivity and extreme sensitivity of the detection, it is possible to make quantitative measurements, and the detection of even a single recognition event makes it possible, for example, to identify target molecules (such as specific DNA sequences) present in very small quantities; consequently, there is no need for an amplification stage such as a PCR process in cases in which the number of specific DNA sequences in a biological specimen is very small, although this amplification process is not necessarily ruled out for other applications.

The simplicity and low cost of manufacturing the nanojunction devices are such that mass production is possible. Furthermore, such devices can easily be integrated with microelectronic circuits for signal processing (by amplification, filtering, modulation, or analogue-digital conversion for noise reduction). Finally, the electrical signals make it possible to integrate storage and display systems for the data which is conveyed. Advantageously, it is possible to integrate the recognition process on a large scale in arrays of devices for simultaneous analysis in parallel on different analytes, thus forming "lab-on- chip" devices in multiplexing mode. The possibility of increasing the number of different probes per unit of area enables a faster and more efficient response to be achieved.

For example, the proposed approach is an efficient way of producing DNA chips which are economical, easy to use, and offer high performance and rapid response for rapid large- scale analysis of nucleic acid specimens in the fields of diagnosis of genetic diseases, detection of infectious agents, differential gene expression and environmental analysis, thus providing opportunities for "point-of-care" analysis without the need to transfer specimens for analysis to a specialized laboratory or to use additional instruments and/or reagents.

Further characteristics and advantages of the invention will be disclosed more fully in the following detailed description of one embodiment of the invention, provided by way of non-limiting example, with reference to the attached drawings, in which:

Figure 1 shows a schematic plan view of a nanojunction device array arrangement according to the invention;

Figure 2 is a schematic three-dimensional view of a nanojunction device array arrangement according to the invention;

Figures 3a-3c are schematic cross-sectional views of the stages for manufacturing the structures forming the nanojunction devices according to the invention;

Figure 4 shows graphs of the variation of the parasitic current in a nanojunction device as a function of the temperature and oxidation time, together with the current- voltage characteristic at ambient temperature and in darkness for nanojunction devices with different degrees of oxidation;

Figures 5 a and 5b show a nanojunction device according to the invention, with, respectively, a molecular probe immobilized before the molecular interaction with a target species and after this interaction, in the hybridization of a DNA strand in this exemplary case, and

Figure 6 shows a set of current-voltage characteristics relating to three different nanojunction devices, before an interaction phenomenon and after the interaction respectively.

The method for detecting biorecognition events in biomolecular interaction processes proposed by this patent is based on the following operations:

- the conjugation of conductive nanoparticles with the target molecular species to be identified, referred to more generally below as "analytes", or with associated signal molecules (or more precisely, the provision of conductive nanoparticles adapted to couple to said target species in a specimen suspected of including said species);

- the provision of metal nanojunction transducer devices for the electrical transduction of the biorecognition events, and the immobilization, which may be spatially controlled, of molecular probes on the nanojunction, or more precisely on the surfaces of the electrodes of the nanojunction device; and

- the detection of the biorecognition events between molecular probes (such as DNA, antibodies or receptors) and analytes or target molecules (such as DNA, a protein or a ligand) by measuring the flow of current between the electrodes of the nanojunction device.

Figure 1 shows a schematic plan view of an arrangement 10 comprising an array of nanojunction transducer devices 12 according to the invention.

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

The first common electrode 22 essentially takes the form of an elongated pad extending along the larger dimension of the formation 20, while the electrodes 24 essentially take the form of square or rectangular pads, although obviously different geometries can be used without departing from the scope of the present invention.

Each electrode 24 is connected to the common electrode 22 by a conductive bridge 26 which extends from the corresponding pad to the inclined side of the "mesa" formation 20, and forms a nanojunction having an interruption in the conductive path which forms an electrical separation between the two electrodes in an inactive condition of the device, as shown more clearly in Figure 2.

Figure 2 is a partial schematic three-dimensional representation of the arrangement 10 of Figure 1, showing two nanojunction devices 12.

The reference SUB indicates the substrate or buffer layer on which the "mesa" formation 20 is grown and on which the electrodes 24 are deposited, while 30 indicates a first lower barrier layer, 32 indicates a thin layer forming the quantum well, and 34 indicates a second upper barrier layer, the top of which is oxidized (the depth of oxidation being indicated by a broken line).

The conductive bridge 26, which interconnects the electrodes 22 and 24 and is deposited on the side of the "mesa" formation 20, is interrupted at the position of the quantum well layer, and has dimensions comparable with those of the quantum well, being typically of the order of a few nanometres or a few tens of nanometres, reduced further by the thickness of the metal layer itself.

The method for the fabrication of a nanojunction device or of an array of devices as described is based on methods of photolithography and wet chemical etching of an AlGaAs/GaAs quantum well (QW), and is described below with reference to Figures 3 a- 3c.

The method includes the conventional stages of forming an AlGaAs/GaAs quantum well grown on GaAs substrates by metal organic chemical vapour deposition (MOCVD). As shown in the drawings, the quantum well structure comprises the following layers: a buffer layer of GaAs, having a thickness of 200 nanometres for example, a barrier of AlGaAs, having a thickness of 300 nanometres for example, a layer of GaAs (quantum well), having a thickness of 20 nanometres for example, a barrier of AlGaAs, having a thickness of 100 nanometres for example, and a cover layer of GaAs, having a thickness of 10 nanometres for example. All the layers are preferably grown at 750°C and are not doped.

The thickness of the GaAs quantum well can be varied as required in order to control the separation between the electrodes of the nanojunction.

Quantum wells made from other materials or grown by different procedures such as molecular beam epitaxy can also be used, provided that selective chemical etching of the quantum well can be carried out in order to obtain a nanometric gap on which the nanojunction can be formed subsequently by evaporation of the contacts.

After a stage of cleaning with acetone and isopropyl alcohol, mesa structures are formed (with a height of about 250 nanometres) by optical lithography and wet chemical etching, for example in an H₂O/H₂θ₂/H₂PO₄ solution with a ratio of 50:1 :1 for 120 seconds at 24°C, using a previously designed mask.

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

The GaAs quantum well is thus exposed, and, after removal of the photoresist, it is etched selectively with a 5:1 citric acid and water solution to remove a few tens of nanometres from it and create an effective separation between the barrier layers, this separation being equal to the thickness of the quantum well (Figure 3b), because of the high selectivity of the chemical etching process between GaAs and AlGaAs (nominally 100:1).

In order to reduce the parasitic currents in the finished nanojunctions, it is preferable to carry out a selective oxidation of the AlGaAs according to the following reaction:

2AlAs+6H₂O --> Al₂O₃+As₂O₃+6H₂

in a furnace heated by the Joule effect, for example by bubbling a nitrogen flow through deionized water (at 3.3 1/min^"1 and 80°C respectively) so as to carry water vapour into the furnace and make it react with the AlAs present in the AlGaAs. The specimen can be placed in the furnace a few minutes after the nitrogen flow has started, and a thermocouple is used to monitor the temperature in the chamber at the position of the specimen.

Since the cover layer of GaAs has been removed, the AlGaAs exposed to the water vapour in the furnace is directly oxidized.

The selective oxidation temperature and time are set at the optimal levels of 450°C and 240 minutes.

The concentration of aluminium in the two AlGaAs layers must be greater than 70% in order to achieve thorough oxidation.

The configuration of the quantum well structure is then used as a "mask" or "guide" for the formation of the electrode arrays by optical lithography and evaporation of a metal layer of 15 nanometres of Cr/ Au. The metal coating is preferably deposited in a direction perpendicular to the surface of the specimen (Figure 3 c), in order to form the electrodes substantially by vertical projection on to the oblique profile of the quantum well structure, which thus determines the geometry of the nanojunction.

The result is a nanojunction device in which the distance between the electrodes is determined with sub-nanometric precision by the thickness of the GaAs quantum well and of the evaporated metallic layer, and by the surface roughness of the chemically etched AlGaAs/GaAs interface (which has been found experimentally to be less than 1 nanometre for short etching times).

The fabrication method can be used advantageously to form pairs of electrodes with separations of a few nanometres, more precisely in the range from 5 to 30 nanometres, while controlling the electrode spacing with sub-nanometric precision without using expensive electron beam equipment, and reducing the parasitic currents (which would otherwise impede the electrical detection) by at least six orders of magnitude by comparison with the known art, by the selective oxidation of the AlGaAs layer (as shown in Figure 4).

Conveniently, the illustrated method can be used for the simultaneous and economical fabrication, using low-cost processes such as optical lithography, chemical etching and liftoff, of extensive arrays of nanojunction devices, for example in an overall arrangement 10 as shown in Figure 1, and, if necessary, of arrays of arrangements 10 to form a multiplicity of adjacent "mesa" structures, resulting in a fairly high number of devices, since all the processes described can be carried out simultaneously on a silicon wafer, thus meeting an essential condition for the mass production of circuits on a nanometric scale.

Figures 5a and 5b show a single nanojunction device according to the invention, respectively in an inactive condition with a molecular probe immobilized before the molecular interaction with a target species, and in an operating condition after the molecular interaction, being the hybridization of a DNA strand in this exemplary case.

The nanojunction transducer device 12 comprises a pair of electrodes 22 and 24, separated by a gap of nanometric dimensions, of the order of a few nanometres, in other words in the range from 5 to 30 nanometres, and at least one biological recognition element or molecular probe P immobilized on at least one of the electrodes, this probe being adapted to interact with an analyte/target T to be identified.

Different molecular probes (for example ssDNA, antibodies and receptors) can be immobilized on different nanojunction devices of an array arrangement, according to the biomolecular interaction processes to be investigated, or the analytes to be identified (for example DNA, proteins and ligands).

The analytes or target molecular species to be identified, shown in Figure 5a separately from the nanojunction transducer device, are correspondingly coupled to conductive nanoparticles NP, for example metallic nanoparticles, or, in an alternative embodiment which is not shown, can act as bridges to connect the molecular probes to one or more signal molecules to which conductive nanoparticles are coupled. In other words, the target molecular species are marked, or can be marked, with conductive nanoparticles by the direct conjugation of the nanoparticles with the target species or by the coupling of the nanoparticles to signal molecules which can interact with the target species.

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

In particular, gold nanoparticles are the most stable of all metallic nanoparticles, and the chemistry of the gold bond is very well known and advanced; in particular, it is known that thiol terminal groups can provide a highly stable covalent bond between gold molecules and biomolecules. They can be synthesized in the organic phase or in the aqueous phase, but for biological applications requiring the interaction of nanoparticles with biomolecules such as DNA or proteins it is convenient to bring them into aqueous solution after synthesis, since biological processes usually take place in water.

Nanoparticles in aqueous solution with a good distribution of dimensions are currently available on the market from companies such as Ted Pella and Sigma- Aldrich.

Hybrid materials composed of biomolecules such as DNA or proteins and inorganic substances can be produced by various procedures, such as: i) assembly by electrostatic interaction; ii) assembly using the sulphur-gold bond between gold nanoparticles and a thiol group, or a natural or synthetic disulphide bridge of the biomolecule; iii) assembly guided by the high-affinity interaction between biotin and avidin, or between an antigen and an antibody.

The physical adsorption of biomolecules on the surface of nanoparticles can cause problems of instability or inactivation, especially in the case of proteins, and therefore it is convenient to bind the molecules covalently. For example, it is possible to use the thiol groups of cysteines present in a protein to bind it to gold nanoparticles. If there are no accessible cysteines, thiol groups can be introduced either by chemical methods or by genetic engineering. For DNA, it is also possible to synthesize oligonucleotides bound to alkanethiols which can form covalent bonds with the gold nanoparticles. Another procedure for creating hybrid nanoparticle/biomolecule systems is the use of 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 it is possible to use various conjugatable systems such as biotin and streptavidin, antibody and specific antigen, or protein A which recognizes the constant portions of antibodies.

The electrical transduction of biorecognition events is based on the monitoring of the current flowing through the nanojunction by the application of a controlled potential difference between the electrodes of the device.

As a result of the specific interaction of the analyte (DNA, protein, or ligand) to be determined with one or more probes (the biorecognition event), one or more nanoparticles conjugated with the analyte are immobilized between the electrodes of the nanojunctions, thus interconnecting them directly, since they have comparable dimensions, and these events can be identified by observing a flow or increase of current in the nanojunction, which is easily measured.

In the embodiment shown in Figures 5 a and 5b, a hybridization sensor for DNA sequencing is formed. A single-strand fragment of DNA is immobilized on the surface of the nanojunction device and the formation of the double helix between the immobilized probe and the complementary target in solution, conjugated with a metallic nanoparticle, is observed. In this case, the molecular probes and the target are both single-strand fragments of DNA (ssDNA) and must be bound in a stable way to the nanoparticles and to the nanojunction device, respectively.

In the exemplary embodiment, nanojunctions formed by gold electrodes were fabricated, and gold nanoparticles were used as the conductive nanoparticles bound to the analyte.

The probes and targets used were oligonucleotides modified at one end with thiol molecules (such as C₆-SH) which can bind stably to the surfaces of the electrodes of the nanojunction device and also to the metal nanoparticles by the formation of gold-sulphur bonds. This solution reduces the release of probes during the analysis and allows to maintain unaltered the biorecognition functionality of the molecules, in this case the ability of the immobilized/conjugated ssDNA to hybridize with a complementary strand.

Commercially available gold nanoparticles with a diameter of 20 nanometres, which can be obtained in aqueous solution stabilized with citrate, from Ted Pella or Sigma-Aldrich for example, were used as the analyte markers. Since the nanoparticles are unstable in micromolar concentrations or in saline solution, a first stage was carried out in which they were stabilized with surfactant molecules such as phosphines (for example bis(p- sulphonatophenyl)phenylphosphine), which are substituted for the initial surfactant (citrate) on the surfaces of the nanocrystals due to their higher binding affinity, and thus create a negative charge coating which prevents the aggregation of the particles because of the electrostatic repulsive forces induced between them.

The gold nanoparticles were then conjugated with oligonucleotides having thiol functionality (C₆-SH). The quantity of DNA bound to the gold nanoparticles was optimized in order to ensure that hybridization was not impeded by a scarcity of DNA on the surface of the gold nanoparticles, or by excessive packing of the oligonucleotides.

For this purpose, conjugation reactions were conducted by incubating the DNA and the gold nanoparticles in different ratios (with 4, 40, 200 and 400 DNA equivalents) for about 18 hours, and the quantity of DNA immobilized per nanoparticle was determined using agarose gel. It was found that the reaction reached saturation at 200 equivalents, and an intermediate ratio of 1 :40 between gold nanoparticles and DNA was chosen for use. Gel tests conducted on specimens with a ratio of 1 :40, incubated for 42 hours and 18 hours, yielded similar results, demonstrating that the solution contained no other unreacted oligonucleotide molecules which might exchange at the gold nanoparticles surface and/or interfere with the subsequent hybridization stage. If this were not the case, a difference in migration between the two gels would have been observed.

In order to optimize the functionalization of gold surfaces with oligonucleotides having thiol functionality, which constitute the molecular probes, oligonucleotides complementary to those conjugated with the gold nanoparticles were immobilized on substrates of gold on mica. There are three basic requirements which the immobilized probes must satisfy:

1) they must be bound stably to the surfaces of the electrodes;

2) they must keep their biorecognition functionality unaltered, and therefore it is preferable to have an ordered orientation of the molecules so as to avoid a reduction of the efficiency of the biorecognition due to steric problems;

3) they must have a configuration and orientation which do not impede the biorecognition events (the hybridization reaction in the present case).

It was therefore decided to form self-assembling monolayers by using, once again, oligonucleotides with thiol functionality, which can form a strong covalent bond with a gold surface and which can self-assemble into monolayers on the gold surface in an ordered way with a specified orientation.

Consequently, the preferred procedure for the fabrication of probes in the described example includes the formation of mixed self-assembling monolayers of thiolated oligonucleotides and other thiol molecules (mercaptoethanol or mercaptohexanol) which act as spacers between the DNA molecules in order to control the packing density of the probes and improve their accessibility, thus overcoming problems of steric size which could lead to a loss of biorecognition efficiency due to low levels of hybridization.

The devices are initially incubated for two hours in an aqueous solution of DNA diluted to 1 μM in a IM solution of KH₂PO₄, and then for one hour with a ImM solution of mercaptoethanol. hi order to evaluate and demonstrate the operation of the electrical detection microdevice, the junctions were subsequently incubated for hybridization for 15 hours 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. After the hybridization, the specimen was rehydrated in a phosphate buffer and an ammonium acetate solution to remove the non-specifically bound DNA.

Current and voltage measurements were then made to demonstrate the detection capacity and to check the flow of current between the electrodes. The results are shown in Figure 6, where the curve A shows the current flowing in the nanojunctions before hybridization (the background signal) as virtually zero, while curves B, C and D show the current flowing in three different nanojunctions after hybridization.

As can be seen, the current flowing in the nanojunctions functionalized with the molecular probes in the inactive state is very low, of the order of a few pA, comparable with the parasitic currents present in this type of device. As a result of biorecognition events between molecular probes and analyte, the gold nanoparticles are immobilized between the electrodes, acting as a conductive bridge, and there is consequently a flow of current which is markedly higher, of the order of the nA, in other words an increase by an order of magnitude.

In the three examples of tests which were conducted, the shape of the curves after hybridization is sigmoid, and there is no non-conductive gap as would be expected for metallic nanoparticles in the case of ohmic contact. Interestingly, the observed current values appear to be respectively quantized with steps of 3 nA (this is shown in the insert in the figure). This signifies that one, two and three gold nanoparticles respectively are immobilized in the three electrodes. If the analyte has two bases differing from the molecular probes with respect to the complementary sequence, there is no significant flow of current (curve E).

Conveniently, this means that the device according to the invention can be used to make quantitative measurements and to detect a very limited number of biorecognition events, and even a single event.

This very high sensitivity makes it possible to identify specific DNA sequences in a biological specimen even if they are present in a very small number of copies, and therefore it is no longer necessary to use an analyte amplification stage such as PCR. PCR reactions or comparable amplification systems are required at present with the DNA chips available on the market, but they require additional instrumentation and reagents which are not ideal for use at the point of care or in the field. The device can if necessary incorporate a PCR chamber, although this is not essential for the purposes of the invention.

To induce thermal cycles, it is possible to conjugate the DNA with magnetic nanoparticles, such as cobalt, and to use radio frequencies to increase the temperature by orientating the magnetic moments of the nanoparticles. In this way the selectivity of the PCR reaction can be increased, since only the DNA conjugated with the nanoparticles will be subjected to the correct thermal cycle and amplified. The same conductive cobalt particles could be reused for the electrical detection of hybridization, as explained above.

Naturally, the principle of the invention remaining the same, the forms of embodiment and details of construction may be varied widely with respect to those described and illustrated, which have been given purely by way of non-limiting example, without thereby departing from the scope of protection of the present invention as defined by the attached claims.

Claims

1. Method for detecting biorecognition events in biomolecular interaction processes, including the provision, on at least one biorecognition site, of molecular probes (P) adapted to interact with target molecular species (T), and of associated transducer means adapted to detect a condition of coupling between at least one molecular probe (P) and a specimen of target species (T) which interact with each other, characterized in that it includes the following operations:

- the provision, in a specimen suspected of including target molecular species (T), of conductive nanoparticles (NP) adapted to couple to said target species (T);

- the positioning of at least one molecular probe (P) in association with a nanojunction transducer device (12) comprising a pair of conductive electrodes (22, 24) separated by a nanometric gap of the same order of magnitude as the dimensions of said conductive nanoparticle (NP);

- the exposure of said biorecognition site to a solution containing said specimen, the event of biorecognition of a target species (T) by a probe (P) including the coupling between said probe (P) and at least one specimen of said target species (T), resulting in the positioning of the said conductive nanoparticle (NP), coupled to the target molecular species (T), between the electrodes (22, 24) of the nanojunction device (12), and the consequent creation of a conductive path between the electrodes;

- the application of a predetermined potential difference to the electrodes (22, 24) of the nanojunction device (12);

- the detection of the flow of current between the electrodes (22, 24) of the nanojunction device (12);

- the evaluation of the occurrence of biorecognition events according to the intensity of the current detected.

2. Method according to Claim 1, in which said conductive nanoparticles (NP) are adapted to conjugate directly with the target molecular species (T).

3. Method according to Claim 1, in which said conductive nanoparticles (NP) are adapted to conjugate with a signal molecule adapted to interact selectively with the target molecular species (T).

4. Method according to Claim 2 or 3, in which said conductive nanoparticles (NP) are metallic nanoparticles.

5. Method according to Claim 4, in which said metallic nanoparticles are gold nanoparticles.

6. Method according to Claim 5, in which said nanoparticles (NP) are adapted to conjugate with the target molecular species (T) or with corresponding signal molecules by means of a covalent bond with thiol terminal groups or disulphide bridges of the target species.

7. Method according to Claim 4 or 5, in which said nanoparticles (NP) are adapted to conjugate with the target molecular species (T) or corresponding signal molecule by physical adsorption.

8. Method according to Claim 4 or 5, in which said nanoparticles (NP) are adapted to conjugate with the target molecular species (T) or corresponding signal molecule by electrostatic interaction.

9. Method according to Claim 4 or 5, in which said nanoparticles (NP) are adapted to conjugate with the target molecular species (T) or corresponding signal molecules by high- affinity interaction between a first component bound to the nanoparticle (NP) and a second component conjugated with the biomolecule (T).

10. Method according to Claim 1, 2 or 3, in which said conductive nanoparticles (NP) are magnetic nanoparticles.

11. Method according to Claim 10, in which said magnetic nanoparticles are cobalt nanoparticles.

12. Method 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. Method according to any one of the preceding claims, comprising the use of surfactant molecules to stabilize the nanoparticles (NP) which can be conjugated with target molecular species (T) of the ssDNA type.

14. Method according to any one of Claims 1 to 12, comprising the use of oligonucleotides with thiol functionality to stabilize the nanoparticles (NP) which can be conjugated with target molecular species (T) of the ssDNA type.

15. Method according to Claim 1, in which the provision of the molecular probe (P) in association with the nanojunction device (12) comprises the spatially controlled immobilization of the probe (P) on the surface of at least one electrode of the device (22; 24) in an orientated arrangement.

16. Method according to Claim 15, in which said molecular probe (P) is ssDNA.

17. Method according to Claim 16, in which the provision of the molecular probes (P) in association with a nanojunction device (12) comprises the immobilization of at least one probe (P) on the surface of at least one electrode (22; 24) by means of a covalent bond of self-assembling monolayers of oligonucleotides with thiol functionality.

18. Method according to Claim 17, comprising the interposition of thiol molecules acting as spacers between the ssDNA molecules.

19. Method according to Claim 15, in which said molecular probe (P) is an antibody adapted to interact with a specimen suspected of including target molecular species (T) which are antigens.

20. Method according to Claim 15, in which said molecular probe (P) is a receptor adapted to interact with a specimen suspected of including target molecular species (T) which are ligands.

21. Method according to any one of the preceding claims, comprising the formation of a nanojunction transducer device (12) based on a quantum well.

22. Process according to Claim 21 , comprising the stages of:

- depositing superimposed layers (30, 32, 34) of a heterostructure including a quantum well layer (32) enclosed between a pair of barrier layers (30, 34);

- defining "mesa" formations (20) in a predetermined configuration by lithography and subsequent chemical etching, said chemical etching process producing formations with an oblique profile with the quantum well (32) exposed;

- using selective wet chemical etching on the quantum well layer (32); and

- forming the electrode contacts (22, 24).

23. Method according to Claim 22, in which said deposition of layers takes place by metal organic chemical vapour deposition.

24. Method according to Claim 22, in which said deposition of layers takes place by molecular beam epitaxy.

25. Method according to Claim 22, in which said selective chemical etching is carried out using a 5:1 citric acid and water solution.

26. Method according to Claim 22, in which the forming of the electrode contacts (22, 24) comprises the definition of the contact configuration by lithography and the deposition by evaporation of a layer of conductive material in a direction perpendicular to the arrangement of the "mesa" formation (20).

27. Method according to Claim 22, additionally including the selective oxidation of the upper free barrier layer (34).

28. Method according to Claim 27, in which said quantum well is a GaAs/AlGaAs quantum well.

29. Method according to Claim 28, in which said oxidation takes place according to the reaction

2AlAs+6H₂O -> Al₂O₃+As₂O₃+6H₂

in an environment heated to 450°C for 240 minutes.

30. Method according to Claim 29, in which the concentration of aluminium in the upper free barrier layer of AlGaAs is higher than 70%.

31. Device for detecting biorecognition events in biomolecular interaction processes, including at least one biorecognition site which accommodates molecular probes (P) adapted to interact with target molecular species (T), and transducer means associated with said biorecognition site, adapted to detect a condition of coupling between at least one molecular probe (P) and a specimen of target species (T) which interact with each other, characterized in that said transducer means comprise at least one nanojunction 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 which said molecular probe (P) is positioned, the gap between the electrodes (22, 24) being of the same order of magnitude as the dimensions of a predetermined conductive nanoparticle (NP) adapted to couple to the target molecular species (T) to which said biorecognition site is intended to be exposed in operation; the event of biorecognition of a target species (T) by a probe (P) including the coupling between said probe (P) and at least one specimen of said target species (T), so as to cause the interposition of said conductive nanoparticle (NP) between the electrodes (22, 24) of the nanojunction device (12), and the consequent creation of a conductive path between the electrodes; and in that the device also includes means for applying a predetermined potential difference to the electrodes (22, 24) of the nanojunction device (12) and means for detecting the flow of current between the electrodes (22, 24) of the nanojunction device (12).

32. Device according to Claim 31, in which said molecular probe (P) is immobilized in a spatially controlled way on the surface of at least one electrode (22; 24) of the nanojunction device (12) in an orientated arrangement.

33. Device according to Claim 31 or 32, in which said molecular probe (P) is ssDNA, adapted to interact with a specimen suspected of including complementary ssDNA.

34. Device according to Claim 33, in which said probe (P) is immobilized on the surface of at least one electrode (22; 24) by means of a covalent bond of self-assembling monolayers of oligonucleotides with thiol functionality.

35. Device according to Claim 34, in which thiol molecules are interposed to act as spacers between the ssDNA molecules.

36. Device according to Claim 31 or 32, in which said molecular probe (P) is an antibody adapted to interact with a specimen suspected of including target molecular species (T) which are antigens.

37. Device according to Claim 31 or 32, in which said molecular probe (P) is a receptor adapted to interact with a specimen suspected of including target molecular species (T) which are ligands.

38. Device according to any one of Claims 31 to 37, in which said nanojunction device (12) is a device based on a quantum well.

39. Device according to Claim 38, in which said nanojunction device (12) comprises a heterostructure including a quantum well layer (32) enclosed between a pair of barrier layers (30, 34).

40. Device according to Claim 39, in which said heterostructure has a "mesa" formation (20) according to a predetermined configuration, with an oblique profile with the quantum well exposed, on which the electrode contacts (22, 24) are formed.

41. Device according to Claim 40, in which the upper free barrier layer (34) is oxidized.

42. Device according to any one of Claims 38 to 41, in which said quantum well is a GaAs/ AlGaAs quantum well.

43. Device according to Claim 42, in which the concentration of aluminium in the upper free barrier layer of AlGaAs is higher than 70%.

44. Device according to Claim 43, in which said electrodes (22, 24) include metallic contacts made from gold.

45. Chip arrangement (10) for the simultaneous detection of biorecognition events in biomolecular interaction processes, comprising an array of nanojunction devices (12) according to Claims 31 to 44, each functionalized with a different molecular probe (P) species.