ES2351495A1 - Method for producing biosensors - Google Patents

Abstract

The invention relates to a method for producing biosensors that include a substrate of TiO2, that includes: a first stage of ionic irradiation of certain areas of a substrate of rutile monocrystalline titanium oxide, resulting in amorphous TiO2 in the areas of the irradiated substrate a second stage of depositing a biological or chemical substance or matter on at least one of the areas of amorphous TiO2 produced in the preceding stage in order to manufacture microelectronic or biotechnology devices such a biological microarray or microchip.

Description

Procedure for obtaining biosensors.

Field of the Invention

The present invention is encompassed within the field of manufacturing devices on a substrate, more specifically, to the definition and preparation of surfaces structured in order to be applicable in processes of analysis of substances in chemistry and biotechnology.

In this last aspect, and in a more concrete way the invention relates, in general, to a method of Obtaining and applying a biosensor based on immobilization of molecules or biological materials on TiO2 surfaces, being able to be applied in the detection and characterization of nucleic acid molecules in general and / or other substances or compounds, such as prokaryotic cells (bacteria), cells eukaryotic and viruses of biotechnological, biosanitary interest, clinical, veterinary, environmental, agricultural or food.

Background of the invention

In the field of biotechnology it has meant a important advance the recent development of the technology of microarrays of different biological elements such as those previously presented, among others. These microarrays are also called chips or microchips According to this technology, thousands of probes molecular with the ability to specifically recognize molecules target of different nature can be covalently fixed to a solid support (glass, nitrocellulose, nylon etc.). Through these microarrays can be performed for example expression experiments gene, nucleotide polymorphism studies (SNPs), mini-sequencing and genotyping of microorganisms and genes eukaryotic Different technologies have been applied for the manufacture of these microarrays [Chun et al., 2009; Yarmush and King, 2009; Barbulovic-Nad et al., 2006; Truskett and Watts, 2006]. However, these have very limited limitations. important as are the resolution of the same (which prevents obtaining very high density arrays or nanoarrays) and the number of points of Recognition that can be manufactured simultaneously. Such circumstances are mainly due to the difficulty of immobilize biological materials, usually present in form of liquid dispersion, without these liquid deposits being overlap each other. To this difficulty is added the deposit small volumes of liquid precisely at high speed. In these techniques, robots with needles, microscopes of modified atomic force (AFM) or elastomeric seals that they impregnate with solutions that contain the biological material of interest and contact the receiving substrate. These solutions are deposited in precise areas to facilitate their possibility of reaction with the surface in the contact areas. To facilitate deposition without mixing the liquids on the substrate is possible to resort to substrates endowed with patterns three-dimensional or wells where to deposit the material [WO 2007/011405; US 2008/0245109]. However, with the current manufacturing techniques and materials of these substrates, usually based on silicon or polymeric materials, the depth and dimensions of these wells are limited by The methods of obtaining. These substrates have serious difficulties or limitations for its use and its reuse this is: optical signal, chemical and physical stability. In many cases the surface of these materials is coated with others for the anchoring the biological material on its surface or improving its reflectance

Titanium dioxide is a material. biocompatible, which has been studied as a support for adhesion of oligonucleotides, viruses and cells among others. This titanium dioxide takes the form of thin sheets, covering other materials, or micro and nanoparticles, in isolation or also coating other materials [WO 2007/009994; Bo Li et al. 2009].

The most widespread lithographic techniques that currently allow three-dimensional patterns to be produced with scales below the mine are based on focused beams of electrons (e-beam) or ions (FIB) [CJ Lo et al., 2006; D. Stein et al., 2002; H. Chang et al., 2006]. The focused beams of electrons constitute an intermediate step in the process of generating three-dimensional patterns on other substrates, since they produce partially modified materials, usually polymers, which are subsequently removed by chemical processes, in order to obtain patterns, which in turn will serve like masks over other materials. The ion-focused beam technique cited in the second place represents a suitable method for the generation of three-dimensional patterns directly on the substrates, since it uses as an active element an ion beam that modifies and eliminates solid parts of the material. However, at present this technique is not appropriate for its implementation on an industrial scale due to the time required to create nanostructures over large areas on a macroscopic scale and the low aspect ratio (depth / lateral dimension) of the structures created. In this technique, Ga ions of 10-50 keV of energy are typically employed. These ions have a notable lateral dispersion as they pass through the material, due to predominant interactions with the nuclei of the atoms of the material. As a consequence, these ions remain
implanted to tens of nanometers of the created structures being able to originate unwanted phenomena in these.

The use of masks in implantation techniques Ionic is very common in the semiconductor industry. One of the most recent alternatives is called Lithography by Ion Projection (IPL) [F. Watt and col., 2005], in which lithographic masks of non contact (stencil masks) [T. Shibata et al., 2002] and an optician electromagnetic reducer that focuses the ion beam. Without However, this technique presents difficulties due to the fact that it should maintain alignment between the ion beam, the mask and the substrate [A. A. Tseng. 2005).], For which it uses systems External alignment and vibration reduction. This system Lithographic has been used with ions in the energy range of hundreds of keV We have no knowledge, so far, of your use with higher energy ions.

On the other hand it is possible to use ions with a MeV energy range to generate modifications as it passes through the materials, in which it generates zones with characteristics other than those obtained by lower energy ions. These modifications are due to the favoring of interactions electron ions with the electrons of the atoms of the material. These energy range ions of MeV, referred to in the fast heavy ion scientific literature (R. Spohr, "Ion tracks and microtechnology. Basic principies and applications ", Vieweg, Wiesbaden, (1990)) present as they pass through the material a minimum lateral deviation at the beginning of its path through the material, therefore including the section of interest to be used in lithographic processes, which is of the order of a third of the scope of the ion in the material. The maximum depth at which ions are detained is high, so their scope constitutes Another advantage of using heavy ions with MeV energy over the structures created with lower energy ions. Ions implanted in this way remain at distances of the order of micrometer of the surfaces of the generated structures. These structures, in practice, are therefore exempt from Inclusion of the ions used. Heavy ion irradiation rapid has been used successfully in recent decades to induce isolated modifications in sensitive materials. When an ion passes through the material, induces a trace of modified material or latent traces. These modifications to be eliminated by chemical attacks, generate pores on materials such as polymers, alloys and crystals. Irradiation with heavy ions Rapids has also been used for generating nanostructures in various materials sensitive to this radiation without removing the affected material. The Difficulty in selecting the area to radiate at scales below the miera can be resolved without resorting to the focus of the beam of ions using solid lithographic masks that restrict areas exposed to irradiation.

The effects of irradiation of dioxide monocrystalline titanium in rutile phase and other materials with ions heavy in the MeV range for the generation of latent traces, both isolated and overlapping, as well as its subsequent attack to generate micro and nanostructures with depths of several micrometers and with aspect ratios (lateral depth-dimension) greater than 25, have been studied in depth by various authors and collected by R. Sanz, "Nanostructures based on TiO2 and ZnO obtained by ionic irradiation ", Doctoral Thesis, University Autonomous of Madrid, March 2009 (http://hdl.handle.net/10261/22699).

These works describe and indicate the threshold energy values of the ions used, creep for achieve volumetric amortization of the material and parameters for its dissolution, both in isolated trace regimes and in overlapping of these. The volumes of titanium dioxide affected by irradiation they have amorphous character and not all this material affected can be eliminated by a selective acid attack. This amortized material has different qualities than the one obtained by other methods of synthesis both chemical and physical.

On the other hand, the substrate described in US 2006157873 allows the deposit and fixation of multiple substances after the chemical functionalization of its Si-based surface, but its lack of transparency hinders its use in examinations of microscopy. Added to this capacity is the relative to the no fluorescence emission of titanium oxide in the range of wavelengths normally used by markers usual biological.

Also substrates with wells obtained by optical lithography (WO2010011939) do not have the ability to fix biological substances and emit fluorescence.

Description of the invention

The present invention seeks to solve the problems arising from the deposition of materials on substrates through a lithography process with ions on substrates of titanium dioxide that generates three-dimensional structures with different physicochemical characteristics. The zones exposed to irradiation have the capacity of be functional for the immobilization or support of materials chemical or biological, avoiding their overlapping and facilitating your examination by using optical microscopy techniques, fluorescence and / or AFM.

The use of ionic irradiation for generation of structures in amorphous titanium oxide and its use as element in photonic crystals is known, however, nothing does show the state of the art its application for deposition of biological or chemical material.

The TiO2 substrate irradiated from the invention gives rise to the amorphous structure of TiO_ {2} with a hydrophilic behavior, compared to the hydrophobic behavior of non-irradiated material, as well as with a large capacity for support of biological material, and immobilization of material Chemical and biological These characteristics and capabilities do not result evident given the variations presented by the amorphous structure compared to those previously obtained through other processes Chemicals as physicists. In addition to this, the optical properties of the material, wavelength transparency of the visible spectrum and the fact of not emitting fluorescence at the wavelengths in the that emit fluorescent markers commonly used for biological material tightening, make titanium oxide more suitable than other materials used as a support such as glass, Si, SiO2 and polymers. As an example to illustrate these advantages, we can indicate the possibility of illuminating the surface opposite to the surface where the material deposit was made (lighting in transmission). This greatly simplifies the lighting and detection process to be the light sources and the detectors located in opposite places, for example but not limiting in tests with confocal microscopy.

The present invention relates to a method of manufacture of a biosensor that can be applied to multiple tests, detections and reactions of biological samples and / or Chemicals with high efficiency, high fidelity, low cost and compatibility between usual detection techniques.

The biosensor obtained by the procedure of the invention is a substrate of total exposed titanium oxide or partially from its surface to at least one irradiation process of heavy ions in the energy range of MeV, hereinafter ionic irradiation, which sustains or to which one or several deposits of chemical or biological material that can be performed both on areas of the titanium oxide substrate virgins as in those that have been modified both superficial as volumetrically by ionic irradiation and exposed or not to the effect of a chemical attack that eliminates part of these volumes

The procedure comprises the following stages:

1) Ionic irradiation

A first stage of ionic irradiation on certain areas of a monocrystalline titanium oxide substrate in the rutile phase, giving rise to amorphous TiO_ {2} in the zones of the irradiated substrate.

The type of irradiation can be of any heavy ion accelerated, meaning heavy ion any mass higher than that of H, which deposits through interactions inelastic, with the electronic cloud of atoms, at least in surface, an energy greater than 5.1 KeV per nm traveled.

Irradiation, as it passes through a substrate of TiO_ {2} monocrystalline in rutile phase, will generate a volume of Damaged material called trace. The shape of this trace can be continuous or discontinuous depending on the energy of the ion, in one case general can be associated with a cylindrical shape, whose radius and depth will depend on the total energy deposited by the ion. Yes ionic irradiation exceeds a threshold creep, this is number of ions that cross a surface, the superposition of these traces, in principle singular, forming a related volume liable to be dissolved by a chemical attack composed of an aqueous solution of HF.

The value of the required threshold creep depends on of the energy of the ion used. For example, for irradiation with Br ions of energies from 9 MeV to 50 MeV, the threshold creep must be equal to or greater than 8 \ 10 13 cm -2. However experiments performed with major ions energy, for example Cu ions of 84.5 MeV, show creep thresholds of 5 · 13 <13> cm <2>.

For which the ionic radiation is in some specific areas of the TiO2 substrate, a stage is performed prior fixation on the titanium oxide substrate, of at least one element that acts as a mask whose difference from density and thickness of motifs allow selective braking of ions

Any material capable of slowing a flow of ions can be used to compose the mask to use, depending on the desired braking capacity. The ones are preferable solid materials that resist ionic flow without suffer high deformations, for example metals such as Au, Cu, Cu / Ni, oxides such as Al 2 O 3, SiO 2. It is possible to wear masks spatially heterogeneous of different materials to take advantage of the different braking capacities of each material. Within Materials are preferred metals or masks whose first layer exposed to the flow of ions of a metallic nature, to diffuse more easily generated heat. Fixing the mask to titanium oxide substrate can be by physical elements or Chemicals

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2) Deposit of chemical or biological substance or material

A second stage of deposit of a substance or a material selected from chemical or biological, such as a chemical solution of organic molecules or a suspension of cells, oligonucleotides, fatty acids or proteins including enzymes and antibodies, over the entire surface or at least in one of the three-dimensional structures generated from the amorphous TiO_ {2} obtained.

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3) Optional UV fixation

The chemical or biological material deposited in the second stage can be fixed in a third stage with ultraviolet light for adhesion of, for example, oligonucleotides

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4) Intermediate acid attack between the first and second stage

The areas subjected to ionic irradiation they can be exposed later to the effect of at least one chemical attack that eliminates part of the volumes corresponding to those areas to generate a three-dimensional pattern in depth, thus, optionally, an intermediate stage between the first and second, in which, after the irradiation process ionic, the substrate is subjected to a selective acid attack with object to eliminate part of the exposed material, being able to apply how many ionic irradiation processes and acid attacks are necessary with the removal or replacement of different masks if required, until obtaining the three-dimensional structure with the desired topographic profiles on the substrate of TiO_ {2}. The time required for the dissolution of TiO2 affected will depend on the concentration of the acid used. By For example, a 20% volume HF solution will attack the entire of the volume susceptible to dissolution in 25 minutes at temperature ambient.

The depths obtained after the attack acid, for creep equal to or greater than 8 · 10 13 cm -2 of Br ions with different energies on Rutile phase monocrystalline TiO2 substrates regardless of their surface orientation are collected in the following table:

one

The technique allows to concatenate several processes of ionic irradiation and subsequent chemical attack to increase the depth of the treated areas.

In or on the microstructures or nanostructures obtained after ionic irradiation, such as pillars, or those obtained after at least some of the ionic irradiation and attack processes later chemical such as channels or wells, can be carried to perform or facilitate certain physical, chemical procedures, biological, biophysical, biochemical, etc. These processes can be carried out directly on the structures themselves or on functionalized structures by deposition of others materials. As examples we can indicate the adhesion of oligonucleotides to at least one of these structures and the support of cells on them.

The biosensor obtained from the deposit of matter biological on the monocrystalline titanium dioxide substrate in rutile phase, can be used for the study of interactions specific by deposit, over the entire surface or at least in one of the three-dimensional structures where it has been made the deposition of the biological material, of a complementary oligonucleotide adhered to the substrate in the second stage, by examination with AFM or fluorescence signal if complementary oligonucleotides are equipped with markers fluorescent

The shapes of each structure or group of structures can be adapted to particular needs, to example of isolated and non-limiting structure, square, rectangle, circle or ellipse.

A particular aspect of the invention is that the biosensor obtained by the aforementioned procedure allows it to be illuminated from the bottom base of the substrate. This aspect is a crucial advantage for compatibility with detection techniques common by optical microscopy, such as examining cell proliferation or confocal microscopy analysis, and that Other substrates do not possess.

Another particular aspect of the biosensor obtained by the process of the invention, it is given by its resistance at high temperatures, less than 450 ° C and chemical stability, which gives it the ability to reuse by cleaning by organic solvents, such as acetone, with solutions aggressive, such as H 2 SO 4: H 2 O 2 (1: 1), also allowing autoclaving processes. These properties of resistance to sterilization and cleaning processes give it reuse capabilities, which reduces the cost of operation.

Brief description of the drawings

Then it goes on to describe very brief a series of drawings that help to better understand the invention and that expressly relate to an embodiment of said invention presented as a non-limiting example of is.

Figure 1 shows a scheme of the procedure Obtaining the biosensor with an intermediate stage of attack acid.

Figure 2 shows a scheme of the procedure of the invention without intermediate stage of acid attack.

In the aforementioned figures, identify a series of references that correspond to the elements indicated below, without implying character any limitation:

one.-

biosensor

2.-

TiO_ {2} lithographed ion substrate heavy

3.-

areas exposed to ionic irradiation

4.-

biological deposit

5.-

TiO_ {2} amorphous

6.-

water well

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Detailed description of one embodiment

As can be seen in Figure 2, the manufacturing process of the biosensor of the invention understands:

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a first stage (A) in which an ionic irradiation is applied on certain zones (3) of a monocrystalline TiO2 substrate in the rutile phase giving rise to amorphous TiO_ {2},

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a second stage (B) of deposition of biological material (4) on the Amorphous TiO_2 (5) obtained in the first stage, selected between oligonucleotides, enzymes, proteins or molecules organic.

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Optionally you can perform a stage intermediate (C) enters the first and second stage, as shown in figure 1, in which an acid attack is performed on the ionically irradiated TiO2 substrate (2), resulting in a plurality of wells (7) in which the acid attack itself does not removes all amorphous TiO2 (5) obtained from irradiation of the first stage, as can be seen in figure 1. Of this way, the biological material that is deposited in the second stage, it is still deposited on the amorphous TiO2 (5) obtained from the irradiation.

As an exemplary embodiment, it has been applied the method described in the previous lines in order to check that the invention possesses the oligonucleotide adhesion capacity in irradiated areas against non-adhesion on non-zones irradiated

For this purpose, a 50 base length deoxyoligonucleotide corresponding to the mRNA derived from the rat β-actin mRNA (SEQ. ID. NO: 1) was labeled at the 5 'end with a fluorescent molecule, specifically the Cyclo fluorochrome. After resuspending the labeled oligonucleotide in sterile nuclease-free water, aliquots of one microliter at ten micromolar concentration were deposited on structured substrates in the form of
wells.

For the generation of wells on the titanium dioxide substrate substrates were employed monocrystalline in rutile phase with <100> orientation and of dimensions 10 x 5 x 0.5 mm 3, with the two largest surfaces polished to optical grade (MTI Corp.). The areas exposed to the irradiation were obtained covering part of one of them Through a mask The mask consisted of a TEM grid (Spi supplies Cu-400) and was immobilized to the Crystalbond 509 heat-reversible glue surface (Electron Microscopy Sciences). The whole was subjected to a irradiation of Br + 7 ions of 25 MeV to a creep of 1 · 10 14 cm -2 to obtain amortization volumetric capable of being completely dissolved until 2200 nm. This process induces a depreciation of the exposed areas and therefore a volumetric expansion of the material creating a topographic elevation of these versus those unaffected of 175 nm. After this process, the mask and substrate were removed was immersed in an aqueous solution of hydrofluoric acid (HF) at 20% by volume, for 25 minutes to dissolve part of the zones affected by irradiation.

Before depositing the oligonucleotide samples, the substrates were sterilized with wells by saturated steam autoclave for 20 minutes at 120 degrees Celsius and 1.0 atmospheres of pressure. Next, a 50 base oligonucleotide solution microliter labeled at the 5 'end labeled with Cy 3 fluorochrome was applied to the corresponding substrates, at 10 micromolar concentration. Four replications were made of each trial. As a nonspecific fluorescence control, another series of four replicas was used in which a microliter of sterile double-distilled water was deposited. The eight samples were incubated at 65 ° for 10 minutes to favor evaporation of the solvent from the solution. Then, the eight samples were treated for 60 minutes with ultraviolet light of 254 nanometers in wavelength to favor the binding of the nucleotide to the substrate. After irradiation, systematic washing of the samples in 8.3 molar urea solution is carried out to favor the removal of oligonucleotide not bound to the substrate. After each wash, an image of the fluorescence emitted by the oligonucleotide that still remains bound to the substrate is taken. Fluorescence measurements were performed on a Typhoon 9210 Variable Mode Imager scanner with specific ImageQuant TL software (Amersham Biosciences). The analysis conditions were optimized to detect the fluorescence emission of the labeled oligonucleotide.

The eight substrate samples were selected treated in which wells had been generated. Four of them were used to deposit a microliter of solution of fluorescently labeled oligonucleotide, at concentration ten micromolar Four other samples were used as control of nonspecific irradiation of the material at the wavelength of the oligonucleotide fluorophore. In this second series it was deposited in each shows a microliter of sterile double-distilled water. The reason for using four replicates for each trial is to rule out the possible variability between the different samples when observing whether effectively the oligonucleotide has been retained on the substrate due to the action of ultraviolet irradiation.

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The first fluorescence image obtained after Sterilizing the samples shows that there is no fluorescent emission significant in the sample, either in the treated area or in the area untreated In the successive images taken after each wash with 8.3 molar urea solution, a solution commonly used in hybridization assays of a different nature as an agent denaturing nucleic acids, it is observed that the treated area specifically retains the oligonucleotide, since it is not lost fluorescence signal after each wash. It is also observed that untreated areas do not retain the oligonucleotide.

In the results obtained when studying the possible oligonucleotide retention by the substrate when there is no irradiation with ultraviolet light, it is observed that the substrate itself alone it is not able to retain the oligonucleotide even in the treated area nor in the untreated area, since the emission is completely lost of fluorescence.

As an exemplary embodiment, it has been applied the method described in the previous example in order to check that the biosensor obtained by the process of the invention has the capacity of support and immobilization of cells on the lithographed surface.

For the generation of wells on the titanium dioxide substrate substrates were employed monocrystalline in rutile phase with orientation <110> and of dimensions 10 x 5 x 0.5 mm 3, with the two largest surfaces polished to optical grade (MTI Corp.). The areas exposed to the radiation were obtained by covering part of one of them by a mask. The mask consisted of a commercial grid type TEM (Spi supplies Cu-400) and was immobilized to the Crystalbond 509 heat-reversible glue surface (Electron Microscopy Sciences). The whole was subjected to a irradiation of Br + 7 ions of 25 MeV to a creep of 1 · 10 14 cm -2. After irradiation, it was moved the mask manually and perform an ion irradiation process Br + 7 of 13 MeV 1 · 10 14 cm -2 for the purpose of generate a structure of different heights and different from a pattern regulate wells. After this process, the mask and the substrate was immersed in an aqueous solution of 20% by volume hydrofluoric acid (HF) for 25 minutes to dissolve part of the areas affected by irradiation. The boss resulting result is a surface of wells superimposed on line and separated by virgin material walls.

For the cell immobilization process, seeded vascular smooth muscle cells (CMLV), isolated from aorta rat at a density of 40,000-60,000, in the wells superimposed in line and separated by walls of material Virgin of the biosensor manufactured. Crops were grown in supports using a DMEM culture medium with low glucose supplemented with 10% fetal bovine serum by volume. Up to date Next they were fixed with 4% paraformaldehyde by volume, prepared in phosphate buffer saline (Phosphate buffer saline PBS) for 10 minutes and after different washings with PBS they were mounted on slides, visualized by optical microscopy using different increases. Immobilized and individualized cells may be used to carry out the analysis simultaneously quantitative and qualitative of biochemical, genetic and / or parameters molecular in a single biosensor. Among these applications stands out the quantification of oxygen consumption of a cell type in different experimental and / or pathophysiological situations or the determination of expression patterns and location of different proteins or nucleic acids. Thus, these biosensors will allow analyze the effect on oxygen consumption and expression and / or location of a protein or nucleic acid of so many treatments as wells with immobilized cells have been used in the test. Similarly, the biosensor, understood as support with cell immobilization capacity, will allow to analyze the effect of a treatment on the expression and / or location of a number of proteins or genes equal to the number of wells with cells immobilized inside that the user has decided to use.

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<110> NANOATE, S.L.

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<120> PROCEDURE FOR OBTAINING BIOSENSORS

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<130> P655 / 2010

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<160> 1

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<170> PatentIn version 3.3

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<210> 1

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<211> 50

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<212> DNA

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<213> artificial

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<220>

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<223> cDNA derived from mRNA of rat beta-actin

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<400> 1

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Claims (12)

1. Procedure for obtaining biosensors (1) comprising a TiO2 substrate, characterized by comprising:

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a first stage of ionic irradiation over certain areas (3) of a monocrystalline titanium oxide substrate (2) in the rutile phase, giving rise to amorphous TiO_ {2} in the areas of the substrate irradiated

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a second stage of deposit of a material selected from chemical or biological (4), on at least one of the zones of amorphous TiO_ {2} (5) obtained in the previous stage.

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2. Method according to claim 1 characterized in that it comprises an intermediate stage between the first and the second stage, of acid attack on the irradiated surface giving rise to a plurality of wells (6). 3. Method according to claim 2 characterized in that the acid attack is carried out with an aqueous solution of hydrofluoric acid. 4. Method according to claims 1-3 characterized in that it comprises a third stage of fixing with ultraviolet light of the chemical or biological material (4) deposited in the second stage. Method according to the preceding claims, characterized in that it comprises a fixing step on the titanium oxide substrate of at least one mask material, prior to ionic irradiation. Method according to claim 5, characterized in that the mask material is a solid material that resists ionic flow without suffering high deformations, selected from Au, Cu, Cu / Ni, Al 2 O 3, SiO 2 } or combinations thereof. 7. Method according to previous claims characterized in that in the second stage selected biological material is deposited among cells, oligonucleotides, fatty acids or proteins. 8. Method according to claim 7 characterized in that the proteins are enzymes or antibodies. 9. Method according to claims 1-6 characterized in that in the second stage a solution of organic biomolecules is deposited as chemical material. 10. Method according to previous claims characterized in that the ionic irradiation is an irradiation of any accelerated ion, of a mass greater than that of Hydrogen, which deposits, by means of inelastic interactions, an energy greater than 5.1 KeV per nm traveled. 11. Method according to claim 10 characterized in that the ionic irradiation is an irradiation with Br ions of energies ranging from 9 MeV to 50 MeV. 12. Biosensor (1) characterized by comprising a substrate of rutile phase monocrystalline titanium oxide with a plurality of areas of amorphous titanium oxide obtained by ionic radiation, with one or several deposits of chemical or biological material adhered on the oxide zones of amorphous titanium.