TW200521229A - Electro-driven protein immobilized module and operation method thereof - Google Patents

Abstract

The present invention provides an electro-driven protein immobilized module and an operation method thereof. According to the invention, protein is rapidly and efficiently immobilized on the surface of a selected substrate. The present invention uses the property where a protein/enzyme carries a weak negative electric charge in a neutral solution, and an externally applied electric field in order to drive the protein/enzyme to be absorbed and immobilized on a selected substrate in a specific orientation in order to improve the situation where a portion of the activity of the protein is masked when the protein is immobilized by a conventional method, such as absorption or bonding. The present invention increases the activity of the immobilized protein/enzyme and improves over the disadvantages of time consuming associated with a still standing method and enzyme loss associated with a vacuum suction method.

Description

200521229_ Five 'invention description (1) ^ a ^ β [Technical Field] The present invention relates to a protein immobilization module and operation method, especially a protein / enzyme driven by an external electric field to shorten the protein / enzyme diffusion time Equipment and methods. [Prior technology] Proteins are mainly composed of amino acids. Basically, all amino acids contain two functional groups, amino and carboxylic acid. Therefore, protein fixation usually uses its own An amine group (-N%) and a carboxyl group (—CO 0H) are bonded to the substrate. Generally speaking, the method of fixing proteins can be divided into three types according to their types: the first type is carrier-biruHng, which fixes proteins on insoluble substrates (that is, solid substrates) ), It can be divided into three types according to the way of bonding: a. Physical absorption (physical absorption) is mainly by physical van der waals, hydrogen bridges (hydrogen bridges) or affinity (Af fini ty) and so on to adsorb proteins, the advantage is that it is convenient and simple, and it has little effect on the configuration of the protein Wu (con f or mat i on), the disadvantage is that the adsorption binding force is very weak, and it is easy to The change in ion concentration in the solution caused the protein to fall off. b. Ionic bonding is a method in which proteins are ionized and electrostatically attracted to the substrate in the form of ionic bonds. The advantage is that it is simple and easy to operate, and has little effect on the configuration of the protein. However, the type, threshold value, ion concentration, temperature, etc. of the protein reaction buffer solution will greatly affect the fixation effect, but it is stronger than physical adsorption. Binding force 0

200521229 V. Description of the invention (2) 'c. Covalent bonding proteins contain reactive free radicals (such as carboxyl groups and amine groups) that are not directly related to bitterness. They can also be used. The covalent bonding of the functional groups occurs. This bonding form 1 has strong binding properties, so the fixed protein is not easily affected by the external environment. It has detachment from the substrate, but the disadvantage is that the substrate cannot be reused. 3 The second type is the cross-linking method (cross-1 i n ng), which uses two or more ^ energy-base bridging agents to crosslink and combine with proteins to achieve fixation and g-resistance, but the disadvantage is that proteins are easy Inactivated. < Effect The third type is the embedding method (Entrapment) to embed proteins in dense or porous polymers, which can be divided into the following two types. ~ Caiye a. A lattice-type method of burying an egg-stalk tiger using a lattice with a mesh structure of molecular colloids or a cross-locking polymer. B. microcapsule type (micro-capsule-type) mainly contains protein in small particles or capsules. Bei Ji, but these protein immobilization techniques have a common deficiency: random (non-directional) attachment or covalent bonding: k = on the substrate. Since the structure of a protein will affect its activity and function, = temperature, pH value, steric disorder, etc. will affect the protein's conformation, making its activity impaired, or even completely in the process of finalization The method is widely used by most people. The method of attacking the sample is mainly through the diffusion of the protein from the liquid phase to the substrate. The number of jk is used to make the protein efficient. Optimal fixation: some substrates (such as filter paper and semi-permeable membrane), standing method

Page 6 200521229 V. Description of the invention (3) It may be possible that the Cong method exists when the substrate enzyme is sucked on the substrate by vacuum suction and pore-based suction. It also affects its orientation and the substrate. Protein / enzyme regression. [Content] The protein fixation of the present invention accelerates the protein / enzyme and shortens the protein. The present invention sets a plurality of cells at the bottom of the tank, places an electrode on the contact surface, and makes a solution of the protein and the enzyme / enzyme solution. The module with the main purpose of charge receiving is a module for mass / enzyme absorption active site receiving / enzyme diffusion. A plurality of sample holes are placed, and the surrounding is selected to be added to the locking module, and then the sample is dripped into the sample with a small amount of suction. The uneven distribution of the effects of plane diffusion caused by the well-driven electric field on the surface. And the other two] Fang:? The advantage of making protein is time saving and can be diversified. Two = using vacuum pumping material, and the method of making eggs: f will flow directly through the pores of the substrate & shellfish / enzyme in 発 ρ, θ§ Ί疋 A „Lost. In the past, immobilized force proteins and enzymes took too long to adsorb. However, proteins and enzymes 5 were not produced between -phase 3 and '5', as shown in Figure 1. This will make cotton The active site of 5 may be masked, so that its activity is reduced by providing an electric field to control the protein / fermentation to be fixed on the selected substrate, and the activity will be reduced after it is masked. 5 The Xuancai Wu component is the upper and lower tanks. The lower tank is placed on the upper and lower tank elements to fix the sample. The buffer solution is placed on the upper and lower buffers. Pass the current, the protein moves, and select the driving force to determine the direction of the driving force in a specific direction to solve the problem of low eggs. At the same time, the two modules of the corresponding contact surface under the open upper well are set with protein / fermentation-fixed protein / Substrate transfer

200521229 V. Description of the invention (4) f. Before selecting the substrate fixing module, pre-treatment is performed to make the surface have a charge 'the charge of the protein / enzyme itself is opposite to the surface charge of the selected substrate 2 in a specific direction' Combined with the surface charge of the selected substrate. [Implementation method] With regard to the detailed description and technical content of the present invention, the following is described with reference to the drawings: ° Please refer to "Figure 2" for the module structure of the present invention. The module can be divided into upper slot 1 and lower slot. The upper tank 1 is a closed body surrounded by two sides, and the upper and lower sides can communicate with each other. The lower tank 2 is a closed tank, only one end communicates with the upper tank 1, and the upper tank 1 is provided with The first electrode li, the electrode 11 is a cathode, and the first electrode 11 is connected to a metal name line 1 1 0. The metal platinum line 1 1 0 is mainly an electric field generating element, and the metal platinum line 1 i 0 may also be a A conductive metal plate is used instead; a second electrode 2 1 is provided in the lower tank 2, and the second electrode 21 is an anode. A silicon pad 6 is placed between the two modules. The silicon pad 6 has a leak-proof effect. The selected base The material 3 is placed on the surface of the silicone pad 6. The selected substrate 3 may be a porous film, a porous powder, or a porous granular body. The selected substrate 3 in this embodiment is a porous film. The material is one or more selected from nitrocellulose (ce 1 1 u 1 〇se nitrate), Composition of nylon, Polyvinylidene fluoride (PVDF), cellulose (ceiiuiose); and the material of the porous powder is one or more selected from ceramics, oxidation (Alumina), silica (silica), graphite (graphite); the material of the porous particles is one or more selected from ceramics (glass), alumina (aiumina), two oxygen

200521229 ___ V. Description of the invention (5) ^ ----- 1 ^ A combination of S ^ 1Ca and graphite (graPhlte). The contact surface of the upper tank body and the selected substrate 3 is provided with a plurality of sample wells 2, while the contact surface of the lower tank body 2 and the selected substrate 3 is provided with a plurality of holes 2 4 'Silicon pad 6 is also provided with a plurality of holes corresponding to the sample well'. The upper and lower tanks 1 and 2 are fixed by a plurality of locking elements 4. The holes of the sample well 12 of the upper tank and the hole of the Shixi rubber pad 6 and the hole 2 4 of the lower tank 2 (see "Figure 3" ”) Are aligned up and down. : As shown in "Figure 3" of green tea, the fixed substrate 3 has a charge, and its surface charge is opposite to that of proteins / enzymes. The selected substrate 3 is placed on a stone mat 6 Then, the buffer is dissolved into a wet environment from the Jintongguan 2 through 3, and the height of the buffer solution overwhelms the height of the electrode. The buffer solution is selected from a phosphate buffer solution ( ph〇sphate buffer), Tris (hydroxymethyl) aminomethane buffer (Tris buffer), N-hydroxyethyl-N-2-ethanesulfonic acid buffer solution (N-2-hydroxyethylpiperazine —N , —2 —ethane sUlf0nic acid buffer; HEPES buffer), and the pH value of the buffer solution is controlled between 7 and 11. The isoelectric point p I of the protein / enzyme to be immobilized is lower than 7, which makes the protein / enzyme show weak negative electric property in the buffer solution, which is favorable for electric field oriented immobilization. The energizing voltage is 80-200 volts, and its current density is controlled at 38 mA-113 mA. After energizing for 15 minutes, the buffer solution is discharged from the outlet 22. Please refer to "Figure 4", through the present invention, the protein / enzyme 5 can be combined with the selected substrate 3 in a specific direction, and

200521229 V. Description of the invention (6) 5 is arranged according to a sequence rule, and its active sites 50 will not interfere with each other or be reduced in activity due to stereo masking. Please refer to "Figure 5". Using the module operation of the present invention and the general vacuum suction immobilization method, Acetylcholinesterase (AchE), pH value 7.2, buffer solution The injury was a 50 mM Tris (hydroxymethyl) aminomethane-hydrochloride buffer (Tris-HCl buffer), which contained 0.2 μm acetic acid acetam !; acetyl thiochol ine iodide; Ach I) and 0.4mM 5,5'-dithio-bis- (2-nitrobenzoic acid) (5,5'-Dithio-bis (2-nitrobenzoic acid); DTNB) were reacted at 25 ° C. 1 50 seconds. Among them, an electric field intensity of 100 volts and an electric field intensity of 50 volts were used to solidify the test 'Acetylcholinesterase · A ch E' after being fixed on the substrate and passed through a light beam having a wavelength of 4.05 nm. Irradiate and measure the light absorption value of the light. The former has an activity of Acetyl cholinesterase (Acetyl cholinesterase; AchE) with an electric field strength of 100 volts, which is much higher than that of Acetylcholinesterase (50 volts). ;

AchE) 'compared with the general vacuum suction method, the same results were obtained, so it can be proved that the present invention significantly shortens the standing time. The above-mentioned ones are only the preferred embodiments of the present invention. When the scope of implementation of the present invention cannot be limited, that is, all equal changes and modifications made in accordance with the scope of the patent application for the present invention shall still belong to the invention patent. Covered 0

Page 10 200521229____ 、 Simple illustration of the drawing [Simplified illustration of the drawing] Figure 1 shows the schematic diagram of protein / enzyme fixation. FIG. 2 is a schematic structural diagram of a preferred embodiment of the present invention. FIG. 3 is a schematic cross-sectional view of a preferred embodiment of the present invention. FIG. 4 is a schematic diagram of protein / enzyme immobilization according to a preferred embodiment of the present invention. Figure 5. Comparison of the absorbance of enzymes with different electric field intensities and conventional pumping. [Explanation of Symbols] 1 ......... Upper groove 11 ........ First electrode _ 110 ........ Metal platinum wire · 12 .... .... Sample well 2 ......... Generating plasma 21 ........ Second electrode 22 ........ Exit 23 ........ Liquid inlet switch 2 4 ........ hole 3 ......... selected substrate 4 ........ locking element 5 ........ protein / Zyme # 50 ........ active site 6 ........ silicone layer

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Claims (1)

200521229 6. Scope of patent application 1 · An electrically driven protein immobilization I The isoelectric point pI of white matter / enzyme is different from that of the following: in a wet environment, the egg enzyme has a thunder # in the buffer solution. The pH of the liquid makes the protein / a charge exactly the same as that of the protein / enzyme. The substrate has been treated. The surface contains a field to drive the protein / enzyme to the opposite side. Through-plus electrical white matter / enzyme, it generates a bond with the selected substrate. Evil γ = fixed substrate accelerates movement, egg 2. If the scope of patent application is the first! Item: ′ on the selected substrate. Methods: # 中 Λ # Α electric-driven proteins were immobilized in ， zhong, and the applied electric field strength in the middle 5h was 80-200 volts. 3. As described in item 1 of the scope of the patent application for Shenzhong, # 中 此 这-an electrically driven protein immobilization 4: The current density of the electric field from i I is 38 ~ 1—. 4. · As described in item [L] in the scope of the patent application_ Method, bean dipper,-, six / this kind of electrically driven protein immobilization (Phosphate buffer), Sanjing Jiameiqi Qitian Erqichong / healing liquid hllffA, M ψ-based wind-based oxane buffer solution (Tris n_2 monoethoxylic acid buffer solution (hepes 5 square = Shen = profit range η) described in an electric drive proteinization method I, wherein the pH value of the buffer solution It is 7-11. 6 into; type protein immobilization module, the protein / enzyme solution is dripped into the plate group, and then a current is formed to form a path, wherein the module includes. Kaizhi: ί tank, where the upper The slot system is a closed electrode with two electrodes at the upper and lower ends. The upper slot is provided with two lower slots at the bottom. The lower slot is a closed slot with only one end communicating with the body. A separate electrode is provided in the lower tank body, corresponding to the sample well Page 12 200521229___ Sixth, the patent application scope is provided with corresponding holes in the tank; an electric field generating element is connected to the upper tank and the electrode in the lower tank respectively; a silicon pad, the silicone A pad is disposed between the upper slot body and the lower slot body, and the silicone pad is provided with a plurality of holes aligned with the holes of the sample well and the lower slot body; a selected substrate, the selected substrate is fixed to the stone On the rubber pad; a buffer solution is added to the container in which the upper tank body and the lower tank body are combined to form a wet environment, and the height of the buffer solution floods the height of the electrode of the upper tank body. 7. An electrically-driven protein immobilization module as described in item 6 of the patent application scope, wherein the electric field generating element is a metal wire. 8. The electrically-driven protein immobilization module according to item 6 of the patent application scope, wherein the electric field generating element is a metal guide. 9. An electrically-driven protein immobilization module according to item 6 of the patent application scope, wherein the selected substrate is a thin film. 10. An electrically driven protein immobilization module as described in item 6 of the scope of patent application, wherein the selected substrate is a powder. 11. An electrically driven protein immobilization module according to item 6 of the scope of the patent application, wherein the selected substrate is granular. Page 13