JP3020428B2 - Ultra-thin protein film formation by alternate adsorption method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a protein ultrathin film whose structure is controlled at a molecular level by an alternate adsorption method.

[0002]

2. Description of the Related Art Conventionally, methods for immobilizing a protein such as an enzyme include a carrier immobilization method in which the enzyme is bound to a water-insoluble carrier, a cross-linking method in which enzymes are cross-linked with a cross-linking reagent to make them insoluble, and the like. Inclusion methods, such as encapsulation in fine particles of a molecular gel, are widely used. In any of these methods, proteins are immobilized in a region having a size far exceeding the molecular size with a random orientation. However, some of the immobilization methods involve a chemical reaction and may cause protein denaturation.

There is also known a method in which a solid substrate such as mica is immersed in a protein solution and the protein is immobilized by adsorption on the surface. However, this method also has a problem that the surface of the protein is denatured when the substrate is dried, and the thickness of the protein layer to be immobilized is limited to the extent that the interaction with the solid substrate can be performed. It does not necessarily result in a thin protein film.

[0004] More recently, a technique has been proposed in which a protein is immobilized between bilayers with good orientation by casting the protein together with a bilayer membrane-forming lipid. Is cast directly, so that a film having a macroscopic thickness can be obtained, and an ultrathin film having a controlled molecular level thickness cannot be obtained. As described above, the existing technology such as the simple adsorption method and the casting method cannot control the structure such as the thickness of the thin film at the molecular level.

On the other hand, the LB method is known as a method for forming a thin film in which molecules are arranged with the precision of molecular size. This involves dissolving an amphipathic substance such as lipid in an organic solvent, developing it on an aqueous solution, and transferring the resulting monomolecular film onto a solid substrate, thereby laminating a thin film of any thickness. This is a method of laminating while controlling the order. This LB
Attempts have been made to apply the method to proteins to create ultrathin films at the molecular level of proteins.However, many proteins lose their activity due to surface modification at the air-water interface. It is not widely applied to general proteins. In addition, this method has a disadvantage that the protein is dissipated in the aqueous phase. Recently, a method has been devised in which an enzyme coated with lipids or the like is dissolved in an organic solvent and the protein is spread on the water surface with minimal denaturation by a required amount. It is necessary to take time and effort to perform a multi-step process of developing the LB film on the surface of the water and the subsequent production of the LB film, which is not a highly productive method. Generally speaking, the LB method is inferior in productivity, and the equipment used is expensive and the handling is not easy.
It has not yet been widely adopted as a protein immobilization method.

[0006] Furthermore, a method has been proposed in which a protein is spread on mercury instead of an aqueous solution to form an arrayed protein layer without denaturation. In addition, it is necessary to use an expensive device, and it has not been widely adopted.
As described above, in the method of developing a protein on an aqueous solution or mercury, safety, surface denaturation, low productivity, and expensive equipment become problems.

[0007] Recently, several methods for alternately adsorbing a protein with other components have been reported, one of which is based on the interaction between a special protein called biotin-avidin. Has to be chemically modified with great effort, and this method is not easily applicable to any protein. In addition, methods of alternately adsorbing proteins by zirconium phosphate and biphasic lipids (bolaamphiphiles) by electrostatic interaction have been proposed. For some reason, lamination is limited to about several layers, and is applied only to a limited type of protein.

As described above, when an ultra-thin protein film is prepared by the conventional method, it is difficult to control the structure of the protein during formation, such as denaturation of the protein, the film thickness, etc., and the generality that it is used only for a specific protein. Lack of equipment, expensive equipment,
One of the problems is the difficulty of the preparation technology. Therefore,
The present invention solves the above-mentioned drawbacks of the prior art, and does not involve denaturation of the protein, and can be performed by any protein.
It is an object of the present invention to provide a new method that can easily form an ultrathin film of a protein by controlling the structure at a molecular level for an arbitrary number of layers and stacking it.

[0009]

The present invention solves the above-mentioned problems by alternately immersing a solid substrate in a solution of a single protein and a solution of an organic polymer ion having an opposite charge thereto. Forming an ultra-thin protein film having a structure controlled at a molecular level on a solid substrate by electrostatic interaction without denaturation of the protein thin film by means of an alternate adsorption method. )I will provide a.

Further, in the present invention is the above method, the alternate lamination of the protein and an organic polymer ion repeated required number of times a method of forming an ultra-thin film (claim 2), furthermore,
A solid substrate having a charge or immobilized charge is immersed in a solution of an organic polymer ion or protein having a net opposite charge to the solid substrate to neutralize the charge on the solid substrate and to overcharge. The process of performing surface charge reversal due to saturation, and further immersing in a solution of a protein or organic polymer ion having the opposite net charge to neutralize the surface charge and reverse the surface charge due to saturation of excess charge. The present invention also provides a method for obtaining a protein ultrathin film whose structure is controlled at a molecular level by repeating a required number of times using a solution of a single protein and a solution of an organic polymer ion alternately.

[0011]

According to the present invention, an arbitrary number of ultrathin protein films having a film thickness at the molecular level and maintaining the activity are formed by the above method. The formation of such a protein ultrathin film is based on the following principle. That is, first, when the surface of a solid substrate having a charge opposite to that of a protein is immersed in an aqueous solution of the protein, the protein is adsorbed by electrostatic interaction. At that time, the protein not only neutralizes the charge on the substrate surface, but also adsorbs excessively, and a new charge appears on the surface.
When this is immersed in an aqueous solution of an organic polymer ion having a charge opposite to that of the protein, a new opposite charge appears on the surface due to neutralization and excessive adsorption of the charge by the organic polymer ion. By repeating this process, alternate adsorption of proteins and organic polymer ions is performed substantially indefinitely. In addition, the amount of excess adsorption in each step is limited by the saturation of the charge, and the same amount of protein and organic polymer ions is immobilized.

As can be seen from the above principle of preparation, in the method of the present invention, since an aqueous solution of a protein can be used as it is, denaturation of the protein can be avoided. Also, considering that protein adsorption is based on electrostatic interaction and that all proteins have some surface charge, the method of the present invention is a method widely applied to water-soluble proteins. It can be said that. In other words, since the surface charge of the protein can be controlled by the pH in the solution, a wide variety of proteins can be immobilized by appropriately selecting the pH of the aqueous protein solution and the partner organic polymer ion. .

In the method of the present invention, since a flexible organic polymer ion whose conformation can be freely changed according to the shape of the protein is used for immobilizing the protein, zirconium phosphate or double-headed It is applied to a wider range of proteins than when rigid components such as lipids (bolaamphiphiles) are used. In addition, this method is performed in a short time using a very simple method of merely immersing the substrate in an aqueous solution of a protein or an aqueous solution of an organic polymer ion, and requires almost no special equipment.

Therefore, the method of the present invention will be described more specifically. First, the organic polymer ion used in the present invention refers to a polymer having a charged functional group in a main chain or a side chain. In this case, the polyanion generally has a negatively charged functional group such as sulfonic acid, sulfuric acid, and carboxylic acid. Examples thereof include polystyrenesulfonic acid (PSS), polyvinyl sulfate (PVS), and dextran. Sulfuric acid, chondroitin sulfate, polyacrylic acid (PAA), polymethacrylic acid (PMA), polymaleic acid, polyfumaric acid and the like are used. The polycation generally has a positively charged functional group such as a quaternary ammonium group or an amino group, such as polyethyleneimine (PEI), polyallylamine hydrochloride (PAH), or polydiallyldimethyl. Ammonium chloride (PDD
A), polyvinylpyridine (PVP), polylysine and the like can be used. These organic polymer ions are
Both are water-soluble or soluble in a mixture of water and an organic solvent.

Further, a conductive polymer, poly (aniline-N
-Charged biopolymers such as functional polymer ions such as -propanesulfonic acid) (PAN), charged polysaccharides such as various deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), and pectin can also be used. it can. Since proteins generally have a charge on the surface if they are water-soluble,
Any type can be used. For example, cytochrome (Cyt; molecular weight 12400, isoelectric point 10.1),
Lysozyme (Lys; molecular weight 14000, isoelectric point 1
1), histone f3 (His; molecular weight 15300, isoelectric point 11), myoglobin (Mb; molecular weight 17800, isoelectric point 7.0), hemoglobin (Hb; molecular weight 6400)
0, isoelectric point 6.8), glucose oxidase (GO
D; molecular weight 186000, isoelectric point 4.2), glucoamylase (GA; molecular weight 100,000, isoelectric point 4.2),
Alcohol dehydrogenase (ADH; molecular weight 100,000;
Isoelectric point 9), diaphorase (DA; molecular weight 70000)
0, isoelectric point 4) and the like. As a solid substrate, a substrate having a surface charge such as silver (surface anionic), glass or quartz (surface anion), a polymer film having a surface charge, or gold (a surface has
Charge can be introduced by the adsorption of mercaptopropionic acid or the like), or any electrode that can introduce charge such as various electrodes can be used.

Although the protein and the organic polymer ion are basically made into an aqueous solution, an organic solvent can be mixed depending on the solubility. Although the concentration of each solution depends on the solubility of the protein or the organic polymer ion, it is not necessary to set a particularly strict concentration because the adsorption process is based on the neutralization and resaturation of the immobilized charge. Examples of standard concentrations include a protein solution of 1 mg / ml and an organic polymer ion of 1-3 mg / ml, but are not limited to this concentration range. If necessary, the pH of the solution is adjusted by adding an acid or an alkali or using a buffer so that the protein and the organic polymer ion are sufficiently charged.

The method of the present invention will be described as an operation procedure. For example, first, a solid substrate having a charged surface is alternately immersed in two kinds of organic polymer ion solutions (polycation and polyanion) to form a flexible organic substrate. A thin film of a polymer ion is formed on a solid substrate. Finally, an organic polymer ion having a charge opposite to that of the target protein is immobilized on the outermost layer. Thereafter, a process of immersing in a protein aqueous solution, washing with water, immersing in an organic polymer ion aqueous solution, and washing with water is repeated to form a multilayer ultrathin protein film. Of course, the organic polymer ion is not limited to one kind as long as it meets an appropriate charge condition, and an ultrathin film having different organic polymer ion layers can be obtained. Further, as the solution of the organic polymer ion, a polymer having the same charge can be mixed and used. For example, a clay compound such as montmorillonite may be mixed with an organic polymer ion to form a composite thin film to form a thin film.

The present invention will be described in more detail with reference to the following examples.

[0019]

EXAMPLE 1-3 According to the method of the present invention, protein was immobilized on a quartz crystal covered with a silver electrode to show that the protein layer was repeatedly immobilized by a fixed amount. . A crystal oscillator is known as a microbalance, and is a device that can measure the amount of a substance fixed on its surface with an accuracy of 10 ⁻⁹ g from a change in frequency. The surface of the silver electrode (area about 0.2 cm ² ) is anionic due to partial oxidation. First, a polycation or polyanion was immobilized on a silver electrode crystal oscillator, and the protein adsorption rate on the surface was determined. Example 1 shows the adsorption of Mb on PSS (polystyrene sulfonic acid), Example 2 shows the adsorption of Lys on PSS, and Example 3 shows PEI.
(Polyethyleneimine) GOD adsorption was measured.

FIG. 1 shows a change in the frequency of a crystal oscillator based on the adsorption of Mb (myoglobin). In this case, the time constant of the adsorption is approximately 3 minutes, and the adsorption reaches equilibrium within 10 minutes. This example shows that the adsorption has reached equilibrium and a certain amount of Mb has been adsorbed, and that
This indicates that the processing is completed within a short time of less than 0 minutes. Table 1 shows the time constant of protein adsorption including this case. In either case, the adsorption process requires only about 10-15 minutes. This is an extremely short operation time compared to conventional methods such as the LB method created through various processes.

[0021]

[Table 1]

Example 4-13 Next, multilayer lamination of various proteins was examined.
Since the procedure is almost common to proteins, Mb
The case of is shown as an example. First, a silver electrode crystal oscillator was used for 1.
It was immersed in a 5 mg / ml PEI aqueous solution to adsorb cationic organic polymer ions. After washing with water, 3mg / ml
The substrate was immersed in a PSS aqueous solution, and the substrate surface was covered with an anionic flexible organic polymer ion. Next, this substrate is
1 and alternately immersed in a 1 mg / ml aqueous solution of cationic Mb adjusted to pH 4 to 5 and a 3 mg / ml aqueous solution of PSS with a washing process with water interposed therebetween, and by repeating this process, an alternate laminated film of Mb and PSS was obtained. . The time required for one adsorption process is about 10 to 20 minutes, and no special equipment is required other than ordinary laboratory equipment such as a beaker. FIG. 2 shows the frequency change in the adsorption process in this case (Example 4). It can be seen that a constant amount of adsorption is repeatedly performed.

Table 2 shows the results for other proteins. Despite the random choice of protein, reproducible frequency change values were obtained with approximately 5% accuracy in each case. This is a result showing that the method of the present invention is applied to a wide range of proteins. In addition, the change in frequency due to adsorption had a good correlation with the molecular weight of the protein, and the protein with a higher molecular weight showed a larger change in frequency. Calculations taking into account the actual surface area of the crystal oscillator reveal that these proteins form a tightly packed monolayer.

[0024]

[Table 2]

As can be seen from Examples 8 and 9 shown in Table 2, it is also possible to laminate the same protein with the oppositely charged organic polymer ion by changing the pH of the solution. is there. FIG. 3 (25,000 times) and FIG. 4 (80,000)
5) and FIG. 5 (90,000 times) are for the protein ultra-thin film prepared in Example 10, and it can be seen that a structure having a smooth surface is obtained.

Further, as can be seen from Examples 11 to 13 shown in Table 2, the organic polymer ion component can be laminated using PAN which is a conductive polymer or DNA which is a biopolymer. These examples show that the method of the present invention can provide a highly functionalized laminated film. Example 14 Next, a similar thin film was formed on a quartz substrate having an anionic surface, and the activity of the laminated protein layer was examined spectroscopically.

As Example 14, an alternately laminated film of Mb and PSS was formed on a quartz substrate under the same conditions as in Example 4. FIG. 6 shows UV spectrum data at each number of layers. The absorption peak near 400 nm is the Soret absorption band of porphyrin present in the active center of Mb. Although this absorption peak reflects that Mb retains its natural activity without denaturation, the absorption intensity increases in proportion to the number of layers. This proves that the activity of Mb is maintained even in the laminated film. Example 15 As Example 15, an alternately laminated film of GOD and PEI was formed on a quartz substrate under the same conditions as in Example 10. This membrane is treated with glucose, phenazine methylsulfate, iodonitr
When immersed in a solution containing otetrazolium violet,
Within 30 minutes, it was observed that the membrane was colored purple based on iodonitroformazane. The result is shown in FIG. 7 in the form of a UV spectrum. The absorption peak near 600 nm indicating the activity of GOD increased with the number of layers (1 in the graph).
Indicates the spectrum of the GOD single-layer film, and 2 indicates the spectrum of the two-layer film. This result indicates that GOD has the original activity in the laminated film, and it can be seen that not only the outer GOD but also the inner GOD function.

[0028]

As described above in detail, in the present invention, various protein thin films can be formed in any number of layers and in any combination without denaturation by alternate adsorption using organic polymer ions. Created. The protein thin film prepared by the method of the present invention is used in combination with an electronic device or the like to develop a molecular element having a high function of a protein and a quick response capability based on an ultrathin film.

[Brief description of the drawings]

FIG. 1 is a diagram showing a change in the frequency of a crystal oscillator based on a time change in a protein adsorption process in Example 1.

FIG. 2 is a diagram showing a change in the crystal oscillator frequency of a thin film formed in Example 4.

FIG. 3 is a scanning electron micrograph (25,000 times) of a drawing of a thin film formed in Example 10 in place of a drawing.

FIG. 4 is a photograph replacing the 80,000-fold drawing corresponding to FIG. 3;

FIG. 5 is a photograph replacing the 90,000-fold drawing corresponding to FIG. 3;

FIG. 6 is a UV spectrum diagram of a thin film formed in Example 14.

FIG. 7 is a UV spectrum chart based on the GOD activity test of Example 15.

Continuation of front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C08J 5/00-5/24 B01J 19/00

Claims (3)

(57) [Claims] [Claim 1] A protein <br/> protein solution Toko single protein solids substrate was immersed alternately in a solution of organic polymer ions having a charge opposite to the solid substrate by the molecular-level structure A method for producing a protein ultrathin film by an alternate adsorption method, which comprises obtaining a controlled protein ultrathin film. 2. The method according to claim 1, wherein the alternate lamination of proteins and organic polymer ions is repeated a required number of times to obtain an ultrathin film. 3. A method for neutralizing a charge on a solid substrate by immersing a charged or immobilized solid substrate in a solution of an organic polymer ion or protein having a net opposite charge to the solid substrate. And reverses the surface charge by saturation of the excess charge, and then immerses it in a solution of a protein or organic polymer ion having the opposite net charge to neutralize the surface charge and reverse the surface charge by saturation of the excess charge. An alternate adsorption method characterized in that the process is repeated a required number of times using a solution of a single protein and a solution of an organic polymer ion alternately to obtain an ultrathin protein film whose structure is controlled at the molecular level on a solid substrate. Of ultra-thin protein film