JP4819640B2 - Cellulosic material film, biosensor using the same, and manufacturing method thereof - Google Patents

Description

  The present invention relates to a cellulosic material having both a fixing function and a liquid feeding function of a biomolecule recognition material for a biosensor, and a biosensor including the material.

  A sensor system using a total reflection optical system typified by a surface plasmon resonance (SPR) method is a system that selectively detects a refractive index change that occurs in the vicinity of a substrate surface (total reflection surface). By immobilizing a molecule selective substance near the substrate surface, this system can detect the refractive index change near the substrate surface due to the adsorption reaction of the measurement target substance contained in the sample with high sensitivity. It is highly useful as a biosensor system. In addition, although there are some differences depending on the wavelength of light used and the design of the optical system, in many cases, the sensing area is an area within a distance of 1 μm from the surface of the substrate. Is possible.

  In a biosensor using a total reflection optical system typified by SPR, measurement is generally performed using a replaceable chip having a sensor film having a biomolecule-selective material immobilized on a substrate. In order to obtain a stable and sensitive response in a biosensor, a function of stably immobilizing a biological molecule-selective material in a larger amount and stably feeding a liquid sample as a sample is required.

  The material of the sensor membrane of the biosensor chip is desirably low density from the viewpoint of improving the sensitivity of the sensor membrane and securing a space for immobilizing the biomolecule selective material. In addition, it is desired to have a function of immobilizing a biomolecule selective material. However, in the case of a membrane mainly composed of cellulose acetate, which is a material capable of forming a low-density membrane among cellulosic materials, it does not have a functional group that contributes to the covalent immobilization of the biomolecule-selective material. . Therefore, cellulose acetate without any treatment is not suitable for chemical immobilization. In addition, it is also desired that the material of the sensor film has a function of suppressing adsorption of contaminants (for example, proteins derived from serum) contained in the sample.

  Moreover, in the current biosensor, it is common to feed a sample using a pump. However, for the purpose of cost reduction and simplification of measurement, and maintenance-free biosensors, it has been studied to provide a sensor film with a liquid feeding function. As one of such mechanisms, the use of the effect of an aqueous solution soaking into a filter paper by capillary action has been studied, and a measurement chip in which the filter paper is put in a plastic channel has been announced (see Non-Patent Document 1).

J-E. Kim et al., Anal. Chem., 2005, 77, 7901-7907 Journal of Membrane, Science, 161 (1-2), 31-40 (1999)

  To provide a novel material for a sensor membrane of a biosensor capable of forming a low-density membrane, immobilizing a biomolecule-selective material by covalent bonding, and suppressing the adsorption of impurities, and further having a liquid feeding function Is the subject of the present invention. Another object of the present invention is to provide a biosensor using the novel material.

  The cellulosic material according to the first aspect of the present invention includes a long-chain surfactant polymer having an immobilized functional group that contributes to immobilization of a biological material, and a cellulose derivative. Here, it is desirable that the immobilized functional group is an amino group. In addition, the cellulose derivative can have a hydroxyl group or a carboxyl group. Furthermore, it is desirable that the long-chain surface-active polymer has a viscosity average molecular weight of 100,000 to 2,000,000, more preferably 500,000 to 1,000,000.

  A biosensor chip according to a second aspect of the present invention includes a substrate and a film containing the cellulosic material according to the first aspect. Here, a metal thin film may be further included between the substrate and the film containing the cellulosic material.

  By mixing a long-chain surface active polymer into which an immobilized functional group has been introduced in advance and a cellulose derivative, biomolecule-selective materials represented by antibodies and enzymes are immobilized by covalent bonds to inexpensive cellulosic materials. And a function of suppressing the adsorption of foreign substances such as proteins. The cellulosic material of the present invention is useful as a sensor membrane material for biosensors because it can form a low-density film and also has a liquid feeding function.

  The biosensor chip produced using the cellulose-based material of the present invention is inexpensive, can stably immobilize the biomolecule-selective material, and at the same time suppress the generation of noise due to contaminants. Can do.

  The cellulosic material of the present invention contains a long-chain surface active polymer having an immobilized functional group and a cellulose derivative having a functional group.

  The long-chain surface-active polymer that can be used in the present invention is a polymer having surface activity, and includes polyalkylene glycols such as polyethylene glycol (PEG), polypropylene glycol, and polybutylene glycol. The long-chain surface-active polymer has a viscosity average molecular weight of 100,000 to 2,000,000, preferably 500,000 to 1,000,000. By having a viscosity average molecular weight of 100,000 or more, it is possible to prevent the long-chain surface-active polymer from flowing out of the sensor film by washing. In fact, it is known that in a membrane made from a mixture of PEG having a viscosity average molecular weight of 20,000 and a cellulosic material, only about 4% of PEG remains in the membrane after washing (non-patent literature). 2). Moreover, the solubility (for example, solubility with respect to acetone etc.) required at the time of mixing with a cellulosic material can be obtained by making a viscosity average molecular weight into 1 million or less.

  The immobilized functional group that can be introduced into the long-chain surface active polymer includes an amino group, a maleimide group, a succinimidyl group, an iodoacetamide group, and the like, and is preferably an amino group. When polyalkylene glycols are used, for example, the hydroxyl group at the end of polyalkylene glycol is activated using methanesulfonylation or toluenesulfonylation, followed by the action of an amine compound as a nucleophile, thereby causing an amino group. Can be introduced. Alternatively, as a reactive agent for introducing an immobilized functional group to polyalkylene glycols, hexamethylene diisocyanate, N, N′-polymethylene bisiodoacetamide, N, N′-ethylene bismaleimide, ethylene glycol bis Succinidyl succinate, bisdiasobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridylthiopropionate), N-succinimidyl 4- (N-maleimidomethyl) Cyclohexane-1-carboxylate, N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl 4- (N-iodoacetyl) aminobenzoate, N-succinimidyl 4- (1 -Ma Imidophenyl) butyrate, iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate, N -Succinimidyl-S-acetylmercaptoacetate can be used.

  The cellulose derivative that can be used in the cellulosic material of the present invention is a material having both a hydrophobic portion and a hydrophilic portion, cellulose; cellulose esters such as cellulose acetate, cellulose acetate butyrate, and nitrocellulose; and methylcellulose, Cellulose ethers such as ethyl cellulose, hydroxyethyl cellulose and carboxymethyl cellulose are included. Cellulose esters and cellulose ethers that can be used may be those in which only some hydroxyl groups are esterified or etherified and still have hydroxyl groups as functional groups.

  A cellulose-based material solution can be obtained by pulverizing, mixing, and dissolving a solid cellulose derivative and a long-chain surfactant polymer having an immobilized functional group in an organic solvent. In the present invention, 0.5 to 20 parts by weight, preferably 1 to 3 parts by weight of a long-chain surfactant polymer having an immobilized functional group is mixed with 100 parts by weight of the cellulose derivative. By this process, the cellulose derivative and the long-chain surface active polymer are complexed at the molecular level, and the long-chain surface active polymer is entangled with the cellulose derivative, and the cellulose functional group that becomes a site causing nonspecific adsorption is added to the above-described functional group. The immobilized functional group acts as a mask. In addition, when the film is formed, the long-chain surface-active polymer can be prevented from being detached from the cellulose derivative and flowing out or lost from the cellulose-based material film. In particular, when carboxymethyl cellulose having a carboxyl group as a functional group is used as a cellulose derivative, a stronger interaction works between the carboxyl group and an amino group introduced as an immobilized functional group into the long-chain surfactant polymer, It is considered that the entanglement between the cellulose derivative and the long-chain surface-active polymer becomes strong.

  Since the cellulosic material of the present invention has an immobilized functional group that can be covalently bonded to a biomolecule-selective material such as an antibody or an enzyme, the biomolecule-selective material can be stably immobilized. . Accordingly, it is possible to prevent the biomolecule-selective material from flowing out of the cellulosic material during liquid feeding such as a liquid sample or a buffer solution. In addition, due to the masking effect of the long-chain surface active polymer in the cellulosic material, impurities (proteins, etc.) that inhibit the reaction between the substance to be measured and the biomolecule-selective material are not functional groups of the cellulose derivative. It can be prevented from being adsorbed in the cellulosic material by specific adsorption. These synergistic actions enable stable biosensing of the target substance based on the reaction between the target substance and the biomolecule-selective material.

  A first configuration example of a biosensor chip using the cellulose-based material of the present invention is shown in FIG. FIG. 1A is a top view, and FIG. 1B is a cross-sectional view taken along a cutting line bb. The biosensor chip having this configuration includes a substrate 10, a metal thin film 20 provided on the substrate 10, and a sensor film 30 provided in direct contact with the metal thin film 20. The biosensor chip having this configuration is particularly useful as a biosensor chip for SPR measurement. The sample is dropped onto the sensor film at the sample dropping point 60 and flows in the sample flow direction by capillary action. In the measurement region 70, various optical measurements (especially SPR) are performed to measure the target substance contained in the sample.

  The substrate 10 can be formed using any material known in the art such as glass and plastic resin (including acrylic resin, polyester resin, silicone resin, polyimide resin, and the like). The substrate 10 is preferably transparent at the wavelengths of excitation light (incident light) 110 and reflected light used for measurement.

  The metal thin film 20 is a metal thin film for exciting surface plasmons present on the metal surface. The metal thin film 20 can be produced using a metal such as gold, silver, or copper. Further, in order to efficiently combine the evanescent wave and the surface plasmon in the sensor film 30, it is desirable to have a film thickness of about 40 to 50 nm. The metal thin film 20 can be formed using any method known in the art such as vapor deposition, sputtering, ion plating, laser ablation. The metal thin film 20 can be omitted in a biosensor chip that performs fluorescence analysis using a biomolecule-selective material having a fluorescent group. In a biosensor chip using a total reflection optical system represented by the surface plasmon resonance (SPR) method, the metal thin film 20 provides a total reflection surface.

  The sensor film 30 is a film that includes the cellulose material of the present invention and has a function of immobilizing a biomolecule-selective material and a function as a sample channel. The sensor film 30 can have various shapes, but preferably has a rectangular shape. The sensor film 30 can be formed by applying a solution or dispersion of the cellulose material of the present invention on the metal thin film 20 and then evaporating the solvent. As a solvent for forming a solution or dispersion used for coating, acetone, ethanol, isopropanol or the like can be used, and it is preferable to use a mixed solvent of acetone and ethanol or isopropanol. As a coating method, any method known in the art can be used, but a spin coating method can be preferably used. Further, the removal of the solvent by evaporation may be performed under a temperature condition at room temperature and a pressure condition at atmospheric pressure. The sensor film 30 has pores ranging from several hundred nm to 1 μm or more. The size of the pores can be controlled by changing the blending ratio of the coating solution containing the cellulose derivative.

Before the sensor film 30 is formed, a double-sided seal member or a resist (not shown) may be disposed on the metal thin film 20 to define the formation position and shape of the sensor film 30. As a material for forming the seal member 40, a material that can be patterned can be used. Examples of such a material include inorganic oxides such as SiO ₂ , Al ₂ O ₃ , Si ₃ N ₄ , and AlN, or nitriding. Products, metals, semiconductors, etc. The resist may be a polymer material such as a photoresist. The double-sided seal member and the resist can be formed using any method known in the art. Preferably, a double-sided sealing member or a resist is provided to form a sensor film, and then the double-sided sealing member or the resist and the sensor film formed thereon are removed to define the formation position and shape. 30 can be obtained.

  The biosensor chip of the present invention may have a plurality of independent sensor films 30. FIG. 1A shows a configuration example in which three independent sensor films 30 are provided. By immobilizing different types of biological system molecule-selective materials for each of a plurality of independent sensor membranes 30, a plurality of types of target substances can be measured using one biosensor chip. It becomes possible.

  In the part located in the measurement region 70 of the sensor membrane 30, the biological system molecule selective material is immobilized. First, an activator is dropped on the sensor film 30 to activate the immobilized functional group in the sensor film 30. When the immobilized functional group is an amino group, an aqueous glutaraldehyde solution or the like can be used as an activator. Next, a solution of the biomolecule-selective material is dropped and allowed to stand for a time sufficient for immobilization. Finally, washing is performed using water to remove excess biomolecule-selective material that has not been immobilized. Since the biomolecule-selective material immobilized by this method is bonded to the long-chain surface active polymer by a covalent bond, it does not flow out from the sensor membrane 30 in the measurement region 70 by a sample or a buffer solution. .

  A second configuration example of the biosensor chip of the present invention is shown in FIG. 2A is a top view, and FIG. 2B is a cross-sectional view taken along the cutting line bb. The biosensor chip of this configuration is configured such that the oil removal film 40 in the region including the sample dropping point 60 and the sensor film 30 in the region including the measurement region 70 are connected in fluid communication with each other. It has the same configuration as the biosensor chip shown in FIG. The biosensor chip having this configuration is also particularly useful as a biosensor chip for SPR measurement.

  In food-related industries, identification of substances in samples containing a large amount of fats and oils, for example, identification of toxins and antigens in milk may be required. As with contaminants derived from proteins, fats and oils may also interfere with biosensing, and therefore are preferably excluded from the sample.

  In the biosensor chip of this configuration, the region extending from the sample dropping point 60 to the front of the measurement region 70 is constituted by the oil removal film 40. The material for forming the oil removal film 40 has lipophilicity in addition to not adsorbing the substance to be measured consisting of the protein to be measured and having appropriate hydrophilicity to ensure the flow of the sample. Therefore, it is required that oil and fat can be effectively adsorbed. For example, the oil / fat removing film 40 can be formed using a material that has been made lipophilic by conjugating a long-chain surface-active polymer to a cellulose derivative having both hydrophilicity and hydrophobicity. Cellulose derivatives and long-chain surface-active polymers that can be used except that the long-chain surface-active polymer does not have to have an immobilizing functional group for immobilizing a biomolecule-selective material. The same material as described above can be used. For this purpose, it is desirable to mix 0.5 to 20 parts by weight, preferably 1 to 3 parts by weight of the long-chain surfactant polymer with respect to 100 parts by weight of the cellulose derivative.

  The biosensor chip having this configuration can be manufactured by performing patterning using a double-sided seal member or a resist on each of the sensor film 30 and the oil removing film 40 after the metal thin film 20 is formed. In order to ensure fluid communication between the sensor film 30 and the oil removal film 40, a portion where the sensor film 30 and the oil removal film 40 overlap may be provided.

  If the biosensor chip of this configuration is used, the sample dropped at the sample dropping point 60 is freed of oil and fat contained during movement in the oil and fat removal film 40, and the sample not containing fat and oil is removed from the sensor film 30. To be supplied. And in the sensor film | membrane 30, the contaminant which inhibits reaction with the measuring object substance and bio-system molecule selective material adsorb | sucks in a cellulose-type material by the nonspecific adsorption | suction of the functional group of a cellulose derivative. This makes it possible to measure the target substance with high sensitivity. Therefore, when the biosensor chip of this configuration is used, it is possible to reduce the cost by eliminating the necessity of performing a separate process for removing oil and fat, and at the same time, the sample may be altered in the process. Therefore, it is possible to measure the target substance with high reliability.

[Example 1]
A long-chain PEG having a viscosity average molecular weight of 100,000 was added to 0.2 mol·L ⁻¹ of tosyl chloride in pyridine and stirred at room temperature for 15 minutes. Subsequently, trioxatridecanediamine was added and stirred at room temperature for an additional 15 minutes. After irradiating the reaction mixture with 300 W microwave, solvent exchange was performed to prepare an ethanol solution of amino group-introduced PEG (solid content 5%).

  A slurry was formed by adding 1.2 g of an amino group-introduced PEG ethanol solution prepared in Example 1 to 2 g of powdered cellulose acetate and grinding with adding acetone. Subsequently, it was diluted with acetone to prepare a complex solution of cellulose acetate / amino group-introduced PEG.

[Example 2]
On the BK7 glass substrate 10 (16 mm × 16 mm × 1 mm), a gold thin film 20 having a film thickness of 50 nm was purified using a vacuum deposition method. Next, the surface of the thin gold film was covered with a double-sided seal member (Duramount PTT25 (manufactured by Kimoto Co., Ltd.)) except for portions (rectangular shape of width 1 mm × length 10 mm, 3 locations) where the sensor film 30 was formed. Next, the cellulose acetate / amino group-introduced PEG complex solution of Example 1 was applied by spin coating and dried. Then, the double-sided sealing member and the cellulose-based material film formed thereon were peeled and removed to obtain a biosensor chip having the configuration of FIG.

  A scanning electron microscope (SEM) photograph of the obtained cellulose material (complex of cellulose acetate / amino group-introduced PEG) film is shown in FIG. The obtained cellulose-based material film was a porous film having pores having a diameter of several hundred nm to 1 μm or more.

  The obtained cellulose-based material film was immersed in water for one night, and observed again by SEM. As a result, there was no change in the film structure before and after the observation. From this, it can be seen that in the cellulose-based material film of the present invention, the outflow of the amino group-introduced PEG accompanying the change in structure does not occur.

[Example 3]
An aqueous glutaraldehyde solution was dropped onto the sensor film in the observation region 70 of the biosensor chip produced in Example 2, and the plate was allowed to stand for 1 minute and then washed with pure water. Next, a solution containing an antibody labeled with a fluorescent substance was dropped, and after standing for 30 minutes, the surface was washed with pure water.

  When the sensor film 30 in the observation region 70 of the treated biosensor chip was observed with a fluorescence microscope, fluorescence could be observed. From this, it was confirmed that the fluorescently labeled antibody was immobilized in the sensor film 30.

  Further, it was washed with pure water for 2 hours and observed again with a fluorescence microscope. As a result, fluorescence could be observed in the same manner as described above. From this, it is determined that the biomolecule-selective material (fluorescently labeled antibody) is immobilized to the amino group-introduced PEG by a covalent bond and that these materials do not flow out of the sensor membrane 30 by washing with pure water. It was done. As described above, a composite membrane made of a cellulose material having an antibody immobilization function can be produced by conjugating a long-chain PEG having an amino group-introduced viscosity average molecular weight of 100,000 or more.

[Example 4]
For each of the buffer solution containing bovine serum albumin as a contaminant model and the buffer solution not containing bovine serum albumin, SPR measurement was performed using the biosensor chip produced in Example 2. Each buffer solution was dropped on the sample dropping point 60, and the SPR resonance angle was measured on the sensor film 30 in the measurement region 70 after the solutions moved in the sensor film 30 and sufficiently developed. As a result, when a buffer solution containing 240 μg · mL ⁻¹ bovine serum albumin was used, the SPR resonance angle was shifted by 16.4 mm (mdeg) compared to a buffer solution containing no bovine serum albumin.

[Comparative Example 1]
A biosensor chip was produced in the same manner as in Example 2 except that a cellulose acetate solution was used instead of the cellulose acetate / amino group-introduced PEG complex solution.

As a result of performing SPR measurement similar to Example 4 using the obtained biosensor chip, when a buffer solution containing 240 μg · mL ⁻¹ bovine serum albumin was used, a buffer solution containing bovine serum albumin was used. In comparison, the SPR resonance angle was shifted by 262.4 millimeters (mdeg).

  In the SPR measurement, the shift amount of the SPR resonance angle corresponds to the concentration change of the substance (including the material of the sensor film 30) existing on the surface of the gold thin film 20. Thus, an increase in shift indicates that more bovine serum albumin has been adsorbed. Comparing Example 4 and Comparative Example 2, the SPR resonance angle shift amount of Example 4 was significantly smaller than the value of Comparative Example 4, and the presence of amino group-introduced PEG effectively suppressed the adsorption of bovine serum albumin. I understand that.

[Example 5]
A slurry was formed by adding 1.2 g of ethanol solution of long-chain PEG having a viscosity average molecular weight of 100,000 (solid content 5%) to 2 g of powdered cellulose acetate and grinding with adding acetone. Subsequently, it was diluted with acetone to prepare a complex solution of cellulose acetate and long chain PEG.

  Subsequently, in place of the cellulose acetate / amino group-introduced PEG complex solution, the biosensor having the structure of FIG. 1 was used in the same manner as in Example 2 using the cellulose acetate / long chain PEG complex solution prepared above. A chip was prepared.

  Milk and a buffer solution as materials containing a large amount of oil and fat were dropped on the sample dropping point 60 of the obtained biosensor chip, and SPR measurement was performed. As a result, when milk was used, the SPR resonance angle was shifted by about 3.28 degrees compared to the buffer solution.

[Comparative Example 2]
Milk and a buffer solution were dropped at the sample dropping point 60 of the biosensor chip produced in Comparative Example 1, and SPR measurement was performed. As a result, when milk was used, the SPR resonance angle was shifted by about 1.476 degrees (about half) compared to the buffer solution.

  From the above results, Example 5 showed a resonance angle shift of about twice that of Comparative Example 2, so that the cellulose acetate / long-chain PEG complex membrane further reduced the fat and oil contained in the milk. It can be seen that many adsorbed. Therefore, it was found that the cellulose acetate / long-chain PEG composite film had a function of removing fats and oils from the sample solution. Therefore, as shown in FIG. 2, the long-chain surface active polymer / cellulose acetate is placed upstream of the sensor membrane 30 of the immobilized functional group-introduced long-chain surface active polymer / cellulose acetate complex for immobilizing the biomolecule selection material. By forming the composite oil removal film 40, a biosensor chip having a function of adsorbing and removing components typified by oil while the sample solution flows through the film can be produced. The SPR resonance angle when milk is dropped at the sample dropping point of this chip and the milk moves through the membrane and reaches the measurement region, the biosensor in which the cellulose acetate / long chain PEG complex is not formed in the previous stage The angle was 1.804 degrees lower than when milk was measured using an industrial chip.

It is a figure which shows the 1st structural example of the chip | tip for biosensors of this invention, (a) is a top view, (b) is sectional drawing cut | disconnected by the cutting line bb. It is a figure which shows the 2nd structural example of the chip | tip for biosensors of this invention, (a) is a top view, (b) is sectional drawing cut | disconnected by the cutting line bb. 2 is a scanning electron micrograph of a long-chain PEG / cellulose acetate composite membrane.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Substrate 20 Metal thin film 30 Sensor film 40 Oil removal film 60 Sample dropping point 70 Measurement area

Claims (9)

In a cellulose-based material film comprising a long-chain surface-active polymer having an immobilized functional group that contributes to immobilization of a biomaterial, and a cellulose derivative ,
A cellulose-based material film characterized by having pores . The cellulose-based material film according to claim 1, wherein the immobilized functional group is an amino group. The cellulose-based material film according to claim 1, wherein the cellulose derivative has a hydroxyl group. The cellulose-based material film according to claim 1, wherein the cellulose derivative has a carboxyl group. The cellulose-based material film according to any one of claims 1 to 4, wherein the long-chain surface-active polymer has a viscosity average molecular weight of 100,000 to 2,000,000. The cellulose-based material film according to claim 5, wherein the long-chain surface-active polymer has a viscosity average molecular weight of 500,000 to 1,000,000. A biosensor chip comprising a substrate and the cellulose-based material film according to claim 1. The biosensor chip according to claim 7, further comprising a metal thin film between the substrate and the cellulosic material film . A step of pulverizing, mixing or dissolving the cellulose derivative and the long-chain surface-active polymer in an organic solvent to form a coating solution;
Applying a coating solution to form a coating film;
Forming a cellulosic material film having pores by evaporating the solvent from the coating film;
A method for producing a cellulose-based material film, comprising:

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