WO2012017116A2

WO2012017116A2 - Method for producing biosensors

Info

Publication number: WO2012017116A2
Application number: PCT/ES2011/070536
Authority: WO
Inventors: Ruy SANZ GONZÁLEZ; Pérez Girón JOSÉ VICENTE; Miriam Jaafar Ruiz-Castellanos; Oscar DE LUIS JIMÉNEZ; José Antonio MAS GUTIÉRREZ; José Luis SANZ MONTAÑA; Vicente PÉREZ BOTO; Agustina Asenjo Barahona; Manuel De La Concepción HERNÁNDEZ VÉLEZ; Mercedes SALAICES SÁNCHEZ; Manuel ROS PÉREZ; Antonio Coloma Jerez; María Jesús ALONSO GORDO; Jorge Puente Prieto; Jens Jensen
Original assignee: Nanoate, S.L.
Priority date: 2010-08-05
Filing date: 2011-07-21
Publication date: 2012-02-09
Also published as: ES2351495B1; WO2012017116A3; ES2351495A1

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

BIOSENSOR OBTAINING PROCEDURE

Cam or of the invention

The present invention falls within the field of manufacturing devices on a substrate, more specifically, to the definition and preparation of structured surfaces with the aim of being applicable in chemical analysis and biotechnology analysis processes.

In this last aspect, and more specifically the invention relates, in general, to a method of obtaining and applying a biosensor based on the immobilization of biological molecules or materials on Ti0 ₂ surfaces, and can be applied in the detection and characterization of nucleic acid molecules in general and / or other substances or compounds, such as prokaryotic cells (bacteria), eukaryotic cells and viruses of biotechnological, biosanitary, clinical, veterinary, environmental, agricultural or food interest.

Background of the invention

In the field of biotechnology, the recent development of microarray technology of different biological elements such as those presented above, among others, has been an important development. These microarrays are also called chips or microchips. According to this technology, thousands of molecular probes capable of specifically recognizing target molecules of different nature can be covalently fixed. to a solid support (glass, nitrocellulose, nylon etc.). By means of these microarrays, for example gene expression experiments, nucleotide polymorphism studies (SNPs), micro-sequencing and genotyping of microorganisms and eukaryotic genes can be performed. 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 important limitations such as their resolution (which prevents obtaining very high density arrays or nanoarrays) and the number of recognition points that can be manufactured simultaneously. Such circumstances are mainly due to the difficulty of immobilizing biological materials, usually present in the form of a liquid dispersion, without these liquid deposits overlapping each other. To this difficulty is added the deposit of small volumes of liquid accurately at high speed. In these techniques, robots are used with needles, modified atomic force microscopes (AFM) or elastomeric seals that are impregnated with solutions that They 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 it is possible to resort to substrates provided with three-dimensional patterns or wells where the material can be deposited [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 production. These substrates present serious difficulties or limitations for their use and their reuse that is: optical signal, chemical and physical stability. In many cases the surface of these materials is covered with others for anchoring the biological material on its surface or improving its reflectance.

Titanium dioxide is a biocompatible material, which has been studied as a support for the 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 currently that 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 an ion beam as an active element 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. How consequently, these ions are implanted tens of nanometers of the created structures and can cause unwanted phenomena in them.

The use of masks in ion implantation techniques is very common in the semiconductor industry. One of the most recent alternatives is the so-called Ion Projection Lithography (IPL) [F. Watt et al., 2005], in which lithographic masks of non-contact (stencil masks) are used ΓΤ. Shibata et al., 2002] and a reducing electromagnetic optic that focuses the ion beam. However, this technique presents difficulties due to the fact that it must maintain the alignment between the ion beam, the mask and the substrate [A. A. Tseng. 2005).], For which it relies on external systems of alignment and vibration reduction. This lithographic system has been used with ions in the energy range of hundreds of keV. We have no knowledge, so far, of its use with higher energy ions.

On the other hand it is possible to use ions with an energy range of MeV to generate modifications as they pass through the materials, in which it generates areas with different characteristics than those obtained by ions of lower energy. These modifications are due to the favor of electronic interactions of the ions with the electrons of the atoms of the material. These energy-range ions of MeV, referred to in the scientific literature as fast heavy ions (R. Spohr, "Ion tracks and microtechnoiogy. Basic principies and applications", Vieweg, Wiesbaden, (1990)) present a deviation through the material minimum lateral at the beginning of its path through the material, therefore including the section of interest to be used in the laographic processes, which is of the order of one third of the scope of the ion in the material. The maximum depth at which the ions are detained is high, so that their scope constitutes another advantage of the use of heavy ions with MeV energy over structures created with less energy ions. The ions implanted in this way remain at distances of the order of the micrometer from the surfaces of the generated structures. These structures, in practice, are therefore exempt from the inclusion of the ions used. The irradiation of fast heavy ions has been used successfully in recent decades to induce isolated modifications in sensitive materials. When an ion passes through matter !, it induces a trace of modified material or latent traces. These modifications, when eliminated by chemical attacks, generate pores on materials such as polymers, alloys and crystals. Irradiation with fast heavy ions has also been used for the generation of 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 mine can be resolved without resorting to ion beam targeting using solid lithographic masks that restrict the areas exposed to irradiation.

The effects of irradiation of monocrystalline titanium dioxide in the rutile phase and other materials with heavy ions in the MeV range for the generation of latent traces, both isolated and overlapping, as well as their subsequent attack to generate micro and nanostructures with depths of several micrometers and with aspect ratios (depth-lateral dimension) greater than 25, have been studied in depth by various authors and collected by. Sanz, "Nanostructures based on Ti0 ₂ and ZnO obtained by ionic irradiation", Doctoral Thesis, Autonomous University of Madrid, March 2009 (http://hdl.handle.net/10261/22699).

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

On the other hand, the substrate described in US 2006157873 allows the deposition and fixation of multiple substances after the chemical functionalization of its Si-based surface, but its lack of transparency hinders its use in microscopy examinations. Added to this capacity is the one related to the non-emission of titanium oxide fluorescence in the range of wavelengths normally used by the usual biological markers.

Likewise, substrates equipped with wells obtained by optical lithography (WO20100 1939) do not have the ability to fix biological substances and emit fluorescence.

Description of the invention

The present invention tries to solve the problems derived from the deposition of materials on substrates by means of a lithography process with ions on titanium dioxide substrates that generates three-dimensional structures with different physical-chemical characteristics. The. areas exposed to irradiation have the capacity themselves to be functional for immobilization or support of chemical or biological materials, avoiding their overlapping and facilitating their examination through the use of optical microscopy, fluorescence and / or AF techniques.

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

The irradiated Ti0 ₂ substrate of the invention gives rise to the amorphous structure of Ti0 ₂ with a hydrophilic behavior, as opposed to the hydrophobic behavior of the non-irradiated material, as well as with a great capacity for the support of biological material, and the immobilization of chemical material and biological These characteristics and capacities are not evident given the variations presented by the amorphous structure compared to those previously obtained through other chemical and physical processes. In addition to this, the optical properties of the material, transparency in wavelengths of the visible spectrum and the fact of not emitting fluorescence in the wavelengths in which they emit the fluorescent markers commonly used for the marking of biological material, make the oxide of Titanium more suitable than other materials used as support such as glass, Si, Si0 ₂ and polymers. As an example to illustrate these advantages, we can indicate the possibility of illuminating the surface opposite the surface where the material deposit was made (lighting in transmission). This greatly simplifies the process of illumination and detection by being the light sources and the detectors located in opposite places, for example but not limiting in examinations with confocal microscopy.

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

The biosensor obtained by the process of the invention is a titanium oxide substrate totally or partially exposed from its surface to at least one process of heavy ion irradiation in the energy range of MeV, hereinafter ionic irradiation, which sustains or to which is adhered one or more deposits of chemical material or biological that may be performed both on regions of the substrate oxide ^'virgin titanium, and those which have been modified both surface volumetrically by ion irradiation and exposed or not the effect of an attack chemical that eliminates part of these volumes, ¹ The procedure comprises the following stages:

1) Ionic irradiation

A first stage of ionic irradiation on certain areas of a rutile phase monocrystalline titanium oxide substrate, giving rise to amorphous T0 ₂ in the areas of the irradiated substrate.

The type of irradiation can be of any accelerated heavy ion, heavy ion being understood as any mass greater than that of H, which deposits by means of ialastic interactions, with the electron cloud of atoms, at least on the surface, an energy greater than 5 , 1 KeV per nm route.

Irradiation, as it passes through a substrate of monocrystalline Ti0 ₂ in the 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 a general case it can be associated with a cylindrical shape, whose. radius and depth will depend on the total energy deposited by the ion. If the ion irradiation passes a threshold fluence, this is the number of ions passing through an area, the superposition of these traces is achieved in singular principle, forming a connected volume capable of being dissolved by etching consisting of an aqueous solution of HF

The value of the required threshold creep depends on the energy of the ion used. For example, for irradiation with Br ions of energies ranging from 9 MeV to 50 eV, the threshold creep must be equal to or greater than 8-10 ¹³ cm ^'z .

However experiments performed with higher energy ions, for example ions

Cu of 84.5 MeV, show threshold fluences of 5-10 ¹³ cm ^"2 .

For which the ionic radiation is in specific areas of the substrate of ^" ΠΟ2, a previous stage of fixation is carried out on the titanium oxide substrate, of at least one element that acts as a mask whose density difference and thickness of motifs allow selective braking of ions.

Any material capable of slowing an ion flow can be used to compose the mask to be used, depending on the desired braking capacity. Solid materials that resist ionic flow without high deformations are preferred, for example metals such as Au, Cu, Cu / Ni, oxides such as Al ₂ 0 ₃₎

S1O2. It is possible to use spatially heterogeneous masks of different materials to take advantage of the different braking capabilities of each material. Among the materials, metals or masks whose first layer exposed to the flow of ions are of a metallic nature are preferred, to more easily diffuse the generated heat. The Fixation of the mask to the titanium oxide substrate can be by physical or chemical elements.

2) Deposit of chemical or biological substance or material.

A second stage of deposit of a substance or 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 less in one of the three-dimensional structures generated from the amorphous Ti0 ₂ obtained.

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 the adhesion of, for example, oligonucleotides.

4) Intermediate acid attack between the first and second stage

The zones subjected to ionic irradiation can be subsequently exposed to the effect of at least one chemical attack that eliminates part of the volumes corresponding to those zones to generate a three-dimensional pattern in depth, so, optionally, an intermediate stage can be performed between the first and the second, in which, after the ionic irradiation process, the substrate is subjected to a selective acid attack in order to eliminate part of the exposed material, being able to apply as many ionic irradiation processes and acid attacks as necessary with the removal or replacement of different masks if required, until obtaining the three-dimensional structure with the desired topographic profiles on the Ti0 ₂ substrate. The time required for the dissolution of the affected Ti0 ₂ will depend on the concentration of the acid used. For example, a 20% volume HF solution will attack the entire volume susceptible to dissolution in 25 minutes at room temperature.

The depths obtained after the acid attack, for creep equal to or greater than 8-10 ¹³ cnrf ² of Br ions with different energies on substrates of monocrystalline Ti0 ₂ in the rutile phase regardless of their surface orientation are shown in the following table:

Energy (MeV) Depth

9 10 nm

13 700 nm

25 2000 nm

50 4000 nm 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 rates obtained after at least some of the ionic irradiation and subsequent chemical attack processes such as channels or wells, certain type procedures can be carried out or facilitated Physical, chemical, biological, biophysical, biochemical, etc. These processes can be carried out directly on the structures themselves or on functionalized structures by deposition of other materials. As examples we can indicate the adhesion of oligonucleotides to at least one of these structures and the cell support on them.

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

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

A particular aspect of the invention is that the biosensor obtained by said method allows it to be illuminated from the bottom base of the substrate. This aspect is a crucial advantage for its compatibility with usual detection techniques by means of optical microscopy, such as a cell proliferation examination or analysis with confocai microscopy, and which other substrates do not possess.

Another particular aspect of the biosensor obtained by the process of the invention is given by its resistance to high temperatures, less than 450 ° C and chemical stability, which gives it the ability to reuse by cleaning it with organic solvents, such as acetone, with solutions. aggressive, such as H ₂ S0 _4: H ₂ 0 ₂ (1: 1), also allowing autoclaving processes. These properties of resistance to sterilization and cleaning processes confer reuse capabilities, which reduces the cost of operation. ,

Brief description of the drawings

A series of drawings will be described very briefly below _. help to better understand the invention and that expressly relate to an embodiment of said invention presented as a non-limiting example thereof.

Figure 1 shows a diagram of the procedure for obtaining the biosensor with an intermediate stage of acid attack.

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

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

1. - biosensor

2. - Ti0 ₂ substrate lithographed with heavy ions

3. - areas exposed to ionic irradiation

4. - biological deposit,

5. - Ti0 ₂ amorphous

6. - well

Detailed description of one embodiment

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

a first stage (A) in which an ionic irradiation is applied on certain areas (3) of a substrate-of mor2 moriocrystalline in rutile phase giving rise to amorphous T1O2 (5),

- a second stage (B) of deposition of biological material (4) on the amorphous Ti0 ₂ (5) obtained in the stage _. first, selected from oligonucleotides, enzymes; organic proteins or molecules

Optionally an intermediate stage (C) can be carried out between the first and the second stage, as shown in Figure 1, in which an acid attack is carried out on the substrate of ^" ΠΟ2 irradiated ionically (2), giving rise to a plurality of wells (7) in which the acid attack itself does not eliminate all the ^" amorphous ΠΟ2 (5) obtained from the irradiation of the first stage, as can be seen in Figure 1. In this way, the biological material that it is deposited in the second stage, it is still deposited on the amorphous Ti0 ₂ (5) obtained from the irradiation.

As an exemplary embodiment, the method described in the previous lines has been applied in order to verify that the invention has the ability to adhere oligonucleotides in irradiated areas against non-adhesion to non-irradiated areas. For this purpose, a 50 base length deoxyoligonucleotide corresponding to the cDNA derived from the mRNA of the? -Actin gene (SEQ, ID. NO: 1) of rat was labeled at the 5 'end with a fluorescent molecule, specifically the fluorochrome Cy3 After resuspending the labeled oligonucleotide in sterile nuclease-free water, aliquots of a microliter at a concentration of ten micromolar were deposited on structured substrates in the form of wells.

For the generation of the wells on the titanium dioxide substrate, monocrystalline substrates were used in the rutile phase with orientation <100> and dimensions 10 x 5 x 0.5 mm ³ , with the two major surfaces polished to optical grade (MTI Corp .). The areas exposed to irradiation were obtained by covering part of one of them by means of a mask '. The mask consisted of a TEM type grid (Spi supplies Cu-400) and was immobilized to the surface by Crystaibond 509 thermo-reversible glue (Electron Microscopy. Sciences). The whole was subjected to an irradiation of Br ^{+ r} ions of 25 MeV to a fluence of 1-10 ¹⁴ cm ^"2 to obtain a volumetric amorphization capable of being completely dissolved until 2200 nm. This process induces an amorphization of the exposed areas and therefore a volumetric expansion of the material creating a topographic elevation of these against the unaffected 175 nm. After this process the mask was removed and the substrate was immersed in a 20% aqueous solution of hydrofluoric acid (HF) in volume, for 25 minutes to dissolve part of the areas 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 ⁰ for 0 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 is still taken. remains attached to the substrate. 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 samples of treated substrate in which wells had been generated were selected. Four of them were used to deposit a microliter of fluorescently labeled oligonucleotide solution, at a concentration of ten micromolar. Four other samples were used as a control for nonspecific irradiation of the material at the fluorophore wavelength of! oligonucleotide In this second series, a microliter of sterile double-distilled water was deposited in each sample. The reason for using four replicates for each test is to rule out the possible variability between the different samples in order to see if the oligonucleotide has been effectively retained on the substrate due to the action of ultraviolet irradiation.

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

In the results obtained when studying the possible retention of oligonucleotide by the substrate when there is no irradiation with ultraviolet light, it is observed that the substrate alone is capable of retaining the oligonucleotide either in the treated area or in the untreated area, since that the fluorescence emission is completely lost.

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

For the generation of the wells on the titanium dioxide substrate, rutile phase monocrystalline substrates with orientation <110> and dimensions 10 x 5 x 0.5 mm ^{3 were used} , with the two major surfaces polished to optical grade (TI Corp .). The areas exposed to radiation were obtained by covering part of, one of them through a mask. The mask consisted of a grid commercial type TEM {Spi supplies Cu-400) and was immobilized to the surface using Crystalbond 509 thermo-reversible glue (Electron IVlicroscopy Sciences). The joint was subjected to an irradiation of Br ⁺⁷ ions of 25 MeV to a creep of 1 - 0 ¹⁴ cm ^"2. After irradiation, the mask was moved manually and a process of irradiation of Br ⁺⁷ ions of 13 MeV 1 -10 ¹⁴ cm ^"2 coh the objective of generating a structure of different heights and different from a regular pattern of wells. After this process, the mask was removed and the substrate was immersed in a 20% aqueous solution of hydrofluoric acid (HF) for 25 minutes to dissolve part of the areas affected by irradiation. The resulting obtained pattern is a surface of wells superimposed in line and separated by walls of virgin material.

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

Claims

REIVINPICATIONS

1- Procedure for obtaining biosensors (1) comprising a Ti0 ₂ substrate, characterized by comprising:

a first stage of ionic irradiation on certain areas (3) of a substrate of titanium oxide (2) monocristalinq in rutile phase, giving rise to amorphous Ti0 ₂ (5) in the areas of the irradiated substrate.

- a second stage of deposit of a material selected from chemical or biological (4), on at least one of the zones of amorphous Ti0 ₂ (5) obtained in the previous stage.

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 fixation with ultraviolet light of the chemical or biological material (4) deposited in the second stage

5. - 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.

6. - Process according to claim 5 wherein the mask material is a solid material which resists ionic character stream without suffering high strains selected from Au, Cu, Cu / Ni, Al ₂ 0 _3, ₂ or combinations S¡0 thereof.

7 - Method according to previous claims characterized in that in the second stage biological material selected from cells, oligonucleotides, fatty acids or proteins is deposited

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 more deposits of chemical or biological material adhered on the areas of amorphous titanium oxide