MXPA97002517A - Procedure for debiomaterial immobilization on a b - Google Patents

Abstract

The present invention relates to immobilize biomaterial, such as enzymes, microorganisms, cells, organics and the like, on a substrate with a surface of Si3N4 with NH2 groups active for the link, using a heterobifunctional interleaver with a coupling function of biomaterial on the one hand and with a reactive group before NH2 on the other. The immobilization substrate preferably assumes the form of a layer of Si2N4 with a thickness of 10 to 1000 mm, which is separated from the SiN3 by the CVD system and coated with active NH2 groups for the bond by a surface cleaning by hydrolysis, especially with diluted acid. Advantageously, first a heterobifunctional crosslinking agent is caused between the reaction with an aldehyde reactive with NH 2, or with a group, halide, epoxide, imine or isocyanate and after removing the unbound superfluous interlacing agent is coupled to the biomaterial by means of the group bioreacti

Description

PROCEDURE FOR THE IMMOBILIZATION OF BIOMATERIAL ON A BA8E The invention relates to a process for the immobilization of a biomaterial on a base with a SÍ3N4 surface, to which the biomaterial is covalently bound through binding mediators. The immobilization of biomaterial is of great relevance for the use of bioactivity, particularly in the case of liquid contact. It plays an important role both in the procedural technique and also in the analytical one in the case of separation operations or for the recurrent use of the biofunction. Other areas of interest are pharmacological and medical, as well as environmental technology. A large number of assays are known to immobilize the biomaterial on various surfaces, also of the mineral type such as glass, which is first silanized. E. Tamiya et al. Are described in J. Mol. Catal. 43. (1988) 293-301 an immobilization of urease on a quartz crystal provided with a thin layer of silver, on the surface of which a layer of silicon nitride is ionically bombarded. The glass treated in this way was stored 24 h in air, washed and dried in an air bath. Then an application was made by vaporization with α-amino-propyl-triethoxysilane, followed by application by vaporization with glutanaldehyde. The thin film of organic surface thus created of approx. 100A thick is called low porous. A silane-aldehyde bond could not be observed well. The enzyme of aqueous solution positioned in the film had a relative activity with respect to the free urease of only 2.25%. This concept of immobilization does not seem entirely satisfactory. The object of the invention is a new type of biomaterial fixation which is achieved with a low number of treatment steps and reproducibly leads to relatively resistant products. The method according to the invention for the immobilization of biomaterial on a base of the type mentioned at the start is essentially characterized in that in the base of immobilization a surface SÍ3N4 is envisaged with NHX groups active in connection, with which a linker is reacted cross-linked heterobifunctional on the one hand with an aldehyde, ester, halide, epoxy, imino or isocyanate group reactive to NH2 and on the other with a group reactive to the biomaterial, and then the biomaterial is coupled. Other features of the invention result from the claims.

The invention is suitable for the immobilization of enzymes, microorganisms, cells, antibodies, antigens, organelles, tissue sections, etc. on bases such as semiconductor substrates, films, wall surfaces, granulates, building components, in particular of the mineral type, and useful in areas of application such as those mentioned at the beginning. The silicon nitride surfaces can be precipitated from a SiH4 / NH3 mixture, inter alia, by the CVD technique (see A. Garde et al. ESSDERC 1994 -11-15 September 1994, Ed. C. Hill &P. Ashburn). They take oxygen from the air and, under the effect of humidity, tend to hydrolysis forming Si-OH, Si-NH and Si-NH2 groups. These groups can be used as reactive functions for the coupling of biomaterial through a cross linker on the nitride surface. In the process according to the invention, a SiS3N4 surface free of oxides is particularly conveniently used for the treatment with dilute hydrofluoric acid and a covalent coupling of the enzyme is procured in two steps, selecting a cross linker whose first function reacts first with the NH2 groups or also NH groups on the nitride surface, and whose second function is then reacted with the protein. Alternatively, the cross linker can also first react with the protein, and then the product is reacted with the S3N4 surface. The aldehyde, halide, epoxide, imide or isocyanate functional groups are particularly suitable as the crosslinker NH2-reactive group. A large number of reaction possibilities with amino groups can be found in the rest, for example in US-PS 5 234 820. For practice, various compounds of the Pierce company are already offered in the ^ Immuno Technology Catalog &; Handbook "of 1992/3. For the reaction with the biomaterial cross linker functions are exploited, which are able to create a covalent bond through functional groups of the enzyme, in particular with carboxy terminal groups or side chain groups, as groups -SH, -COOH or -OH or aromatic rings According to the invention, the binding of the biomaterial to the nitride surface is sought under the use of a heterobifunctional crosslinker, hereunder also being understood under a crosslinker heterobifunctional a crosslinker which has two basically chemical functions of the same type, but with different reactivity with respect to the various reaction partners.The heterobifunctional crosslinkers are reacted in steps with the surface SÍ3N4 and then with the protein.The following amino-specific reactions, indicated individually, They develop at room temperature at a neutral pH until to slightly alkaline. The increase in temperature and pH value increases the speed of the reaction, but also the hydrolysis rate of the cross linker. The buffer used must not contain amines or other compounds with which the crosslinker functional groups could react. N-hydroxysuccinimidester reacts specifically with primary amines. Under dissociation of N-hydroxysuccimide an amide bond is obtained between the primary amine and the radical group of the ester used. If no water-soluble analogue is used, a crosslinker that possesses these functional groups must first be dissolved in a small amount of an organic solvent (for example DMSO) and then only diluted to the final concentration in aqueous buffer. The ion strength of the shock absorber should not be too high to avoid salting effects. A slightly alkaline pH (7-9) guarantees that the primary amines are in a non-proportioned state. The aldehydes have a strongly reducing carbonyl group. This reacts with primary amines under dissociation of water. The reaction of the primary amines with imidoesters takes place in the pH range between 8 and 9. The ester dissociates and the primary amine forms a guanidino compound with the imido group. The link with the protein takes place with the help of the second functional group still free of the cross linker. This can react specifically or not specifically with thiol, carboxyl or carbohydrate groups of the protein. If the crosslinker functionally ends as a specific thiol group such as maleicide, an activated halide or pyridyl bisulfide, then the protein to be linked must possess a free sulfhydryl group (usually a cysteine radical). If it is not available, it can be generated by reduction of protein sulfides. Alternatively, the primary amines of the protein can be modified in such a way that the sulfhydryl groups are available (Reagent Trauts). To prevent oxidation of these groups, the absorber used must be degassed. The addition of the EDTA complex former prevents oxidation by possible metals present in the solution. Maleimide reacts in slightly acidic to neutral media (pH 6.5-7.5), while for halides and pyridyl bisulfide, pH values greater than or equal to 7 are recommended. Glycolized proteins can be crosslinked through the hydroxy group in the side chains of sugar. If a crosslinker activated with carbohydrate is used, which has as a functional group for example hydrazide, then the carbohydrate group of the protein must first be oxidized to an aldehyde (for example with NaI04). The carbonyl group that is formed then reacts with the hydrazide to give semicarbazon. One possibility of direct binding between carboxy and amino groups is the reaction with carbodiimides. In the acid range of pH (4-5), the carbodiimides transform the carboxy group into an activated ester bond. This reacts with primary amines under formation of an amide bond and urea cleavage. In the case of using a cross linkerwhose non-specific functional end of amino has a photo-reactive group (for example azidophenyl), all immobilization must be carried out in a dark room under red light. The azidophenyl azido group is activated by light of the wavelength 265-275 nm. The invention can be used for the linking of the most diverse biomaterials to bases or carriers of all kinds. It was especially tested in the example of penicillin sensors, so the following description refers to them. For this, it refers to the attached notations. These show schematically: Figure 1 a sensor principle (measurement arrangement 5). Figure 2 shows a typical measurement curve for a concentration range of 10 -4 to 10-1 mol / l of penicillin. And Figure 3 the calibration curve of a sensor 10 according to the invention.

Example: In silicon wafers with p ~, or p (1 mOhmcm - 30 Ohmcm) was first created by thermal oxidation dry between 700 and 12002C (here at 10002C) in a diffusion oven, an electrically non-conductive layer of silicon dioxide with a thickness of 5-100 nm. On top of this, a nitride was placed by chemical precipitation in the gas phase (PECVD) // - of non-conductive silicon with a thickness of 10-100 nm. The The proportion of SÍH4 / NH3 in the reaction gas was 2/1, the temperature of the substrate 200-5002C (here 3002C) and the pressure during precipitation 1 - 3 Torr (here 1.5 Torr). It followed an annealing step under N2 atmosphere (5-60 minutes at 700-10002C). Finally the unpolished part of the The substrate was provided with an ohm contact (for example 10-1000 nm Al, Au). The material used was placed by thermal vaporization under vacuum at a base pressure < 10 -5. The precipitation rate was between 0.1 and 10 nm / s. The insert was then annealed in an RTA oven at 150-5002C (here 4002C) in an N2 atmosphere. Immediately before the start of the enzyme immobilization process, the plates were cleaned in acetone, 2-propanol and distilled water in an ultrasound bath and corroded 10-60 s (here 30 s) in dilute hydrofluoric acid (1-10%). HF). Using the heterobifunctional cross linker ANB-NOS (N-5-azido-2-nitrobenzoyloxysuccinimide), it was first dissolved in a small amount of DMSO and then diluted with a 0.2 M triethanolamine buffer (pH 5-9) to a final concentration 0.5-10 mM. This solution was applied on the SÍ3N4 surface and incubated between 5 and 40 minutes at room temperature. At low temperature, incubate longer. Molecules not bonded to the silicon nitride surface were removed by rinsing with triethanolamine buffer (TEA). Then, the enzyme (penicillinase type 1 from Bacillus Cereus, Sigma P 0389) was dissolved (1000-5000 units / ml) in a buffer containing no amino groups (for example TEA, in particular not in TRIS buffer or glycine) and added to the surface of silicon nitride pretreated with cross linker. After an incubation time of 1-240 min (here 15 min) at temperatures between 4 and 60 ° C, in particular at room temperature, the binding of the enzyme molecules to the still free functional groups of the cross linker was induced by light in the wavelength range of 320-350 nm. After completion of the immobilization procedure, ready-to-use penicillin sensors were rinsed with 0.1 M TRIS buffer (pH 7-8) and distilled water and dried at least 10 minutes in air, or under N or inert gas. With the field effect sensors made in this way, measurements were made to determine the concentration of penicillin in aqueous solutions. In FIG. 1, the measurement arrangement is shown schematically. The field effect sensors produced in accordance with the invention, consisting of the silicon substrate 1, the insulating layer 2 (silicon dioxide and silicon nitride), the crosslinker layer 3 and the enzyme layer (penicillin layer) 4, were integrated into a measurement cell. This was filled with an aqueous measurement solution, which contained penicillin G in a concentration between 10 -5 and 1 mol / l. A reference electrode (for example Ag / AgCl) is immersed in the measuring solution 6. The potentials are taken through the reference electrode 7 and a contact electrode 8 on the silicon substrate. Figure 2 shows a typical measurement curve, which was recorded in the CONCAP (CONstant CAPacitance) mode in the concentration range between 10-4 and 10-1 mol / l of penicillin. The sodium salt penicillin G (Sigma P 3032) was dissolved in 10 mM TRIS-HCl buffer, pH 7. With the increasing concentration of penicillin the concentration of the penicillin acid formed increases and with it the concentration of the hydrogen ions near of the surface of silicon nitride that acts as a pH transducer. This results in a displacement of the potential to the silicon nitride / electrolyte boundary surface at more positive voltage values, or, negative. The application over time allows an observation of the course of the potential dependent on the concentration of the reaction of the enzyme. At the indicated times the change of the measurement solution took place. Figure 3 shows the chemical transmission characteristic line obtained from Figure 2. It represents the calibration curve of the field effect sensor developed according to the invention. The data on the sensitivity of a chemosensor or potentium-electric biosensor are given in relation to the Nernstsch relation in that they are based on the linear range of this curve, that is, in the range in which there is a logarithmic relationship between the concentration of penicillin and the potential in contact. This range is in the sensor prepared according to the invention between pPenicillin 2.3 and 3.3, which corresponds to 0.5 and 5 mM. The sensitivity is 50 mV per decade. The exact position of the linear measurement range and the absolute sensitivity depend essentially on the choice of the composition of the shock absorber, its concentration, that is, the capacity of the shock absorber and the pH value. By an appropriate choice of these parameters, the measurement range necessary for a measurement can be intentionally adjusted. For example, the linear measurement range when using an IMIDAZOL buffer (pH 7) is between 2 and 20 mM penicillin, for a HEPES buffer approximately between 1 and 10 mM. Increasing the pH value shifts the position of the linear range of the calibration curve to higher concentrations of penicillin; when decreasing, at lower concentrations. The sensors produced according to the invention have a high long-term stability of more than 140 days. The sensitivity is 50 mV per decade of penicillin. It is possible to produce a field effect transistor which, in the gate range of a construction of the same type as that described in the invention, has a capacitive layer structure.

Claims (6)

NOVELTY OF THE INVENTION Having described the foregoing invention, the content of the following is claimed as property: CLAIMS

1. A method for the immobilization of biomaterial on a base with surface SÍ3N4, to which the biomaterial is covalently linked by means of binding mediators, characterized in that a surface SÍ3N4 with active NHX groups in connection with the binding is foreseen in the immobilization base. that a crosslinker is reacted with aldehyde, ester, halide, epoxide, imino or isocyanate groups NH2 ~ reactants on the one hand, and a reactive group on biomaterial on the other, in which the biomaterial is coupled.

2. A method according to claim 1, characterized in that the group or function reactive to biomaterial of the crosslinker is a reactive group with terminal or side chain functions of proteins.

3. A process according to claim 1 or 2, characterized in that a crosslinker is selected with a group that reacts with carboxyl, sulfhydryl or hydroxy groups or that is coupled to aromatic rings, active as regards biomaterial.

4. A process according to one of the preceding claims, characterized in that on the basis of immobilization, a SIS3N4 layer of 10-1000 nm thickness is precipitated by CVD of a mixture of SÍH4 / NH3, and is provided by surface hydrolyzing with groups NHX assets in terms of link. A method according to one of the preceding claims, characterized in that enzymes, microorganisms, cells, antibodies, antigens, organelles or tissue sections are fixed on semi-conductor substrates, films, wall surfaces or granules, in particular of the mineral type, as a biomaterial. . 6. A process in variation of claim 1, characterized in that for the link through the cross linker, first it is reacted with the biomaterial and then the bonding is made with the surface SÍ3N4.