CN1381609A - Gas-phase deposition method for preparing TiO2 gel film - Google Patents

Abstract

The invention discloses a gas-phase deposition method for preparing a titanium dioxide gel film. The preparation steps include: dissolving biologically active substances in a buffer solution; drip-coating the above-mentioned solution on the surface of a carrier, and suspending it in a titanium alkoxy compound Above the liquid level, place the system at a constant temperature of 15-35°C and seal it for 4-8 hours; the vapor of the titanium alkoxy compound contacts the solution on the surface of the carrier to form a titanium dioxide solution, which is then gelled to embed the biologically active substance And fixed on the surface of the carrier, the titanium dioxide gel film can be obtained on the carrier and can be made into a biosensor. The advantage of the present invention is that the hydrolysis rate of the titanium alkoxy compound can be controlled, and precipitation caused by strong hydrolysis caused by direct mixing with water can be avoided; acid and surfactant do not need to be added in the process of sol preparation, and the acidity environment can be controlled at will, which facilitates the maximum Maintain the activity of biologically active substances to the maximum extent. Moreover, the prepared film does not crack and does not fall off from the surface of the carrier.

Description

A kind of vapor deposition method for preparing titanium dioxide gel film

1. Technical field

The invention relates to a method for immobilizing biologically active substances on a carrier, in particular to a gas phase deposition method for preparing a titanium dioxide gel film.

2. Background technology

Biosensor is one of the most interesting frontier research topics. Due to the highly selective molecular recognition function of bioactive substances, it is often used in the preparation of biosensors. The most important technology in the preparation and application of biosensors is the immobilization of biorecognition molecules on the carrier surface. This immobilization process must maintain the activity of biorecognition molecules, so that certain substrates or biological components can be selectively recognized. And the recognition result is converted into a measurable signal, and the content of the substrate or biological component is determined.

Common methods for the immobilization of bioactive substances on the surface of carriers include:

(1) Adsorption method: physical adsorption is a relatively simple immobilization technology, and biologically active substances are adsorbed on the surface of the carrier by electrostatic attraction. Physical adsorption is relatively simple. Compared with other chemical methods, it has less impact on biological activity, but the fixed molecules are easy to fall off the surface of the carrier and have a shorter lifespan.

(2) Covalent bonding method: biologically active substances are bonded to groups on the surface of the carrier through covalent bonds to immobilize them on the surface of the carrier. Covalent bonding on the surface is more difficult than adsorption, the activity of biological substances is easily affected, and it is easy to lose activity during use. When covalently bonding biomolecules on the carrier surface, many factors need to be considered, and there are many operating steps, which often include the activation of the carrier substrate surface and the coupling of biomolecules.

(3) Cross-linking method: the cross-linking between bioactive substance molecules and between the molecules and the substrate is formed by a bifunctional reagent to fix it on the surface of the carrier. The limitation of this method is that the formation conditions of the membrane are not easy to determine, and the pH, ionic strength, temperature and reaction time must be carefully controlled. The thickness of the cross-linked film and the content of bifunctional reagents have important effects on the response of the sensor.

(4) Embedding method: The immobilization technology of embedding bioactive substances in polymers has been widely used, which can fix biomolecules on the surface of the carrier through the three-dimensional network structure of polymers. The technique can adopt mild experimental conditions, and most biologically active substances can be easily incorporated into polymer membranes, and the pore size and geometry of the membrane can also be controlled. But it also has certain limitations, such as the need to control many experimental factors, the free radicals generated during the formation of the polymer and the spatial structure of the polymer will affect the activity of biomolecules, etc.

Using sol/gel porous glass instead of polymer membranes to immobilize biomolecules such as enzymes, proteins, antigens and antibodies, DNA, cells and even biological tissues is a method developed in recent years. Sol/gel glass materials have very attractive characteristics: the preparation method is simple, the porous structure with adjustable pore size can embed bioactive molecules at a lower temperature, thereby maintaining the structure, activity and function of biomolecules. Moreover, the identification object can be contacted with it through the channel of the gel glass to generate an identification signal. The sol/gel itself is also chemically inert, has low swelling and good mechanical stability.

The sol/gel process includes several steps such as the formation of sol (Sol), the gelation of sol into wet gel (Gel) and the drying and aging of wet gel:

(1) Hydrolysis and condensation to form a sol: Most of the methods reported so far use low molecular weight siloxy compounds as precursors to react with water under strong acid catalysis to form a sol. Since siloxanes and water are immiscible, the corresponding alcohols are usually added as co-solvents. The hydrolyzate was condensed to obtain a stable sol.

(2) Gelation: The colloidal particles produced by hydrolysis are stable due to the repulsion between the negative charges on their surfaces. However, with the progress of hydrolysis and polymerization, water is consumed, the solvent evaporates, and the concentration of colloidal particles increases, which weakens this stabilizing effect and causes gelation.

(3) Drying and aging: In the final stage of gelation, with the volatilization of water and solvent, the capillary force causes the gel network to gather and shrink. Drying and cracking of the gel tends to occur at this time. It has been suggested to add some surfactants to the Sol-Gel process to prevent this from happening.

The common method for the preparation of sol/gel biosensors is to disperse the bioactive substance in the sol, and then drop-coat or dip-coat it on the surface of the flat matrix carrier, and then the sol gels on the surface of the carrier at an appropriate temperature, so that the biological activity The substance is immobilized on the surface of the carrier. Since the silica sol needs to be prepared under the condition of strong acid catalysis, the activity of biological substances will be reduced or even lost if the acidity is too high. During the production process of the sensor, the silica sol drip-coated on the surface of the carrier is prone to cracking when it is gelled into a film, which makes the film fall off from the surface of the carrier and cannot effectively fix the biologically active substance. In order to prevent the cracking and falling off of the film, a surface active drying control agent is usually added in advance during the process of hydrolysis and condensation to form the sol to control the volatilization speed of the water in the sol and prevent the formed gel film from cracking. However, surface-active dryness control agents are all organic compounds, which will affect the activity of biologically active substances to varying degrees, and ultimately affect the performance of biosensors.

3. Contents of the invention

1. Purpose of the invention: the purpose of the invention is to provide a method for forming a titanium dioxide sol/gel film on the surface of a carrier by vapor deposition, so that it can be used for fixing biologically active substances, thereby developing a novel biosensor.

2. Technical solution: In order to achieve the above object, a vapor phase deposition method for preparing titanium dioxide gel film according to the present invention is to use the gelation process of titanium sol for the embedding of biologically active substances on the surface of the carrier, and to construct a new type of titanium dioxide gel film. Biomimetic catalytic interfaces (biofunctional membranes) and biosensors. The specific steps are:

(1) The biologically active substance is dissolved in a buffer solution. The pH value of the selected buffer solution varies with the type of biomolecules, and the standard is to maximize the activity of the biological substance.

(2) Drop-coat a certain concentration of the above-mentioned mixed solution on the surface of the carrier, suspend it above the liquid level of the titanium alkoxy compound, and then seal the system.

(3) Place the above closed system in a constant temperature of 15-35°C for 4-8 hours.

(4) In a closed system, the titanium alkoxy compound vapor on the liquid surface contacts the solution on the surface of the carrier, and a slow hydrolysis reaction occurs to form titanium dioxide sol, which embeds and fixes the biologically active substance on the surface of the carrier after gelation , TiO2 gel film can be obtained on the carrier and can be made into a biosensor.

In the present invention, there are four main factors affecting the obtained biofunctional film and biosensor performance:

(1) The pH value of the buffer solution: Only at a certain acidity, the biological substance has the best activity. If the acidity of the buffer solution deviates from this value, dissolving biomolecules in it will damage its activity, thereby affecting the performance of biofunctional membranes and biosensors.

(2) Concentration of biologically active substances: After the sol on the surface of the carrier is gelled, the capacity for embedding biologically active substances is limited. If the concentration is too high, some biological substances will not be embedded in the gel membrane; if the concentration of biological substances is too low, the capacity of the membrane will not reach saturation, which will eventually affect the performance of biological functional membranes and biosensors.

(3) Temperature: When the carrier that is drip-coated with the biological substance solution is suspended above the tetraisopropoxytitanium liquid level, two processes will occur simultaneously. One is that tetraisopropoxytitanium vapor is deposited on the carrier surface, The other is the volatilization of water in the solution on the surface of the carrier. If the temperature is too high, the vapor pressure of tetraisopropoxytitanium is too large, and the speed of hydrolysis is too fast, and what is formed at this time is not a sol, but a particle precipitation of titanium dioxide; if the temperature is too low, the tetraisopropoxytitanium If the vapor pressure is too small, the volatilization rate of water will be faster than that of vapor deposition, so that there is not enough water on the surface of the carrier to undergo hydrolysis reaction with the vapor of titanium tetraisopropoxide to form the desired sol.

(4) Time: Sufficient time can make the hydrolysis reaction fully, and can also ensure the complete gelation of the sol. If the time is too short, the reaction will be insufficient and the gelation will not be complete; if the time is too long, it will be meaningless after the sol is completely gelled.

3. Beneficial effects: Compared with the prior art, the present invention has the remarkable advantage that it can control the hydrolysis rate of titanium alkoxy compound, avoid its direct mixing with water and produce precipitation due to strong hydrolysis, and no need for sol preparation. By adding acid and surfactant, the acidity environment can be controlled at will, so as to maintain the activity of biologically active substances to the greatest extent. Moreover, the prepared membrane does not crack and does not fall off from the surface of the carrier, and the embedded bioactive substances are not easy to leak due to the porous structure in the gel membrane and the interaction of hydrogen bonds. This technology combines the preparation of sol and the process of film formation into one, and the sol/gel forms a film while embedding biologically active substances and being fixed on the surface of the carrier. This method not only simplifies the preparation steps of sol/gel film formation and biosensor, but also the prepared biosensor is also due to the good biocompatibility of titanium dioxide gel film, the mild conditions for gel film formation, and the difficulty of leakage of biological active substances. The activity will not be damaged, the film is not easy to crack and other advantages, so it can maintain high activity, long life and good stability.

4. The best way to implement

Embodiment 1: select the disk-shaped plane glassy carbon electrode of diameter 4mm to make carrier material, and biologically active substance takes horseradish peroxidase (HRP) as example as the object of embedding and fixation:

(1) First polish the surface of the electrode with metallographic sandpaper, then polish it with 1.0, 0.3, and 0.05 μm γ-alumina slurry on the suede, then rinse it with secondary water, and then wash the surface of the electrode with 1:1 nitric acid , acetone, and secondary water ultrasonic cleaning to obtain a fresh and clean electrode surface, and dry at room temperature.

(2) Dissolve 5 mg of HRP in 1 ml of 0.02M pH7.0 phosphate buffer solution, take 10 microliters of HRP solution, drop-coat it on the surface of the electrode treated in step (1), and then suspend the electrode in four Above the liquid level of titanium isopropoxide, the system was sealed and kept in a constant temperature box at 25°C for 6 hours.

(3) In a closed system, titanium tetraisopropoxide steam contacts the enzyme solution on the surface of the electrode, and a slow hydrolysis reaction occurs to generate titanium dioxide sol. The sol embeds the HRP in the gel film during the gelation process, thereby being fixed on the surface of the electrode to make the HRP biofunctional film and the H ₂ O ₂ biosensor.

Embodiment 2: Use the biologically active substance glucose oxidase (GOx) as the object of embedding and fixing:

(1) Treat the electrode surface with the first step in Example 1.

(2) Dissolve 2 mg of GOx in 1 ml of 0.02M pH7.3 phosphate buffer solution, take 10 microliters of GOx solution and apply it dropwise on the electrode surface treated in step (1), then suspend the electrode surface in four Above the methoxytitanium liquid level, the system was sealed and kept in a water bath at 20° C. for 5 hours.

(3) Similarly, when the enzyme solution on the electrode surface contacts with tetramethoxytitanium vapor, a slow hydrolysis reaction occurs to generate titanium dioxide sol. The sol embeds GOx in the gel film during the gelation process, thereby being immobilized on the electrode surface to make GOx biofunctional film and glucose biosensor.

Claims (2)

1, a kind of vapor deposition method for preparing titania gel film, it is characterized in that this preparation step is: (1) Dissolving the biologically active substance in a buffer solution; (2) drop-coating the above-mentioned mixed solution on the surface of the carrier, and suspending it above the liquid level of the titanium alkoxy compound, and then sealing the system; (3) Place the above closed system in a constant temperature of 15-35°C for 4-8 hours; (4) In a closed system, the titanium alkoxy compound vapor on the liquid surface contacts the solution on the surface of the carrier, and a slow hydrolysis reaction occurs to form titanium dioxide sol, which embeds and fixes the biologically active substance on the surface of the carrier after gelation , TiO2 gel film can be obtained on the carrier and can be made into a biosensor. 2. A vapor deposition method for preparing titanium dioxide gel film according to claim 1, characterized in that said titanium alkoxide compound is tetraisopropoxytitanium or tetramethoxytitanium.