JP2010197046A - Biosensor - Google Patents

Abstract

An object of the present invention is to provide an inexpensive biosensor that is simple to measure and manufacture and that can be used for home medical care and the like.
The present invention relates to a molecular recognition component that specifically reacts with a measurement target substance, a stimulus-responsive polymer whose volume changes due to a reaction between the measurement target substance and the molecule recognition component, and a stimulus-responsive polymer. A biosensor comprising a metal particle having an average particle diameter of 0.2 nm to 200 nm in which the intensity of localized surface plasmon resonance changes due to volume change, and the metal particle and the molecular recognition component are fixed to the stimulus-responsive polymer About. The biosensor of the present invention is simple to manufacture and measure, and can also detect a target substance with high sensitivity. Furthermore, since a special material such as a labeled molecule is not required, a biosensor that is less expensive than the conventional one can be obtained.
[Selection figure] None

Description

  The present invention relates to a biosensor, and relates to a biosensor that can be used in fields such as medicine, food, pharmaceuticals, and the environment.

  A biosensor is a chemical sensor that detects a target substance by using the recognition ability of a biomolecule, and is used for detecting a target biological substance in the medical, food, pharmaceutical, and environmental fields. Conventionally, detection methods using ELISA, immunochromatography, and the like are known. However, these methods require an operation of labeling a biological substance with a fluorescent substance or the like. Sensor development is expected.

  Biosensors that do not require labeling include a molecular recognition component that reacts specifically with a target substance in a stimulus-responsive polymer that changes in volume due to environmental changes such as pH, temperature, and ionic strength, A biosensor containing a crystalline colloid is described in Patent Document 1. The crystalline colloid is formed by self-assembly in which charged particles such as polystyrene, polymethyl methacrylate, silicon dioxide and the like naturally gather in a solvent such as water to form a regular array. When a measurement target substance is added to this biosensor, the solvent environment around the polymer changes due to a specific reaction between the target substance and the molecular recognition component, resulting in a volume change and a change in the lattice spacing of the crystalline colloid. It becomes. For this reason, the absorption spectrum before and after the addition of the substance to be measured can be measured, and the target substance can be detected by the wavelength shift of the absorption peak.

JP-T-2001-505236

  However, since the biosensor described in Cited Document 1 requires observation of a wavelength shift, an analyzer having a fine wavelength resolution is required for quantification of the target substance, which is easily used in inspection processes such as home medical care and food factories. It was not available. In addition, in order to form a crystalline colloid, self-assembly of charged particles required a long-time reaction and complicated work such as purification, and thus a biosensor that can be manufactured more simply has been desired. .

  Accordingly, an object of the present invention is to provide an inexpensive biosensor that is simple to measure and manufacture and can be used for home medical care and the like.

  In order to solve the above-mentioned problems, the present inventors have intensively studied a biosensor using a stimulus-responsive polymer. As a result, it has been found that a biosensor using localized surface plasmon resonance of metal particles can easily measure a target substance by changing the intensity of an absorption peak at a specific wavelength, and has arrived at the present invention. .

  That is, the present invention includes a molecular recognition component that specifically reacts with a measurement target substance, a stimulus-responsive polymer that changes in volume due to a reaction between the measurement target substance and the molecule recognition component, and a volume change of the stimulus-responsive polymer. A metal particle having an average particle diameter of 0.2 nm to 200 nm in which the intensity of localized surface plasmon resonance is changed, and the metal particle and the molecular recognition component are fixed to the stimulus-responsive polymer. Related to biosensors.

  In the case of a biosensor that changes the intensity of localized surface plasmon resonance as in the present invention, the target substance can be detected by changing the intensity of absorbance at a specific wavelength without creating an absorption spectrum as in the case of measuring the wavelength shift. Since it can be detected and quantified, measurement with only a simple device is possible. In addition, the biosensor of the present invention is one in which metal particles and molecular recognition components are immobilized on a stimulus-responsive polymer. However, as described in detail below, the labeling operation and the like are complicated. It is possible to manufacture simply without requiring a simple process. The method of fixing the metal particles and the molecular recognition component is preferably based on comprehensive fixing. Inclusive fixation refers to a state in which a metal particle or molecular recognition component is included in a mesh-like enclosure in a stimulus-responsive polymer having a three-dimensional network structure, and can be fixed by a simple method. it can. In this case, the stimulus-responsive polymer may be a gel having a three-dimensional network structure.

  The measurement principle using the biosensor of the present invention will be described. When a measurement target substance is added to the biosensor of the present invention, a specific reaction such as an enzyme reaction or an antigen-antibody reaction proceeds between the target substance and a molecular recognition component. When pH, ionic strength, etc. change due to the influence of a product or the like due to this reaction, the stimulus-responsive polymer expands or contracts, resulting in a volume change. With this volume change, the distance between the metal particles fixed in the polymer also changes, and the absorption peak intensity derived from the localized surface plasmon resonance between the metal particles changes.

  Therefore, when the target substance is added to the biosensor, the absorption peak intensity increases and decreases, and simple measurement is possible. In addition, since changes in peak intensity are caused by changes in pH, etc. due to the reaction between the target substance and molecular recognition components, a highly sensitive biosensor that detects only the target substance using specific reactivity should be used. Can do. Incidentally, the above-mentioned localized surface plasmon resonance refers to a characteristic in which when a metal particle is irradiated with light, free electrons on the particle surface vibrate to generate an electromagnetic field, and only light of a specific wavelength is absorbed or scattered.

  The biosensor of the present invention is a metal particle having an average particle diameter of 0.2 nm to 200 nm in order to develop localized surface plasmon resonance. When the average particle size is less than 0.2 nm, localized surface plasmon resonance tends to hardly occur, and when it exceeds 200 nm, metallic luster tends to occur, and localized surface plasmon resonance tends to be difficult to observe.

  As the metal particles in the present invention, a metal that causes localized surface plasmon resonance can be used, and those composed of at least one of Ag, Au, Pt, Pd, Rh, Ir, Ru, and Os are applied. It is preferable. According to these metal particles, a biosensor capable of observing localized surface plasmon resonance in the wavelength region of visible light can be obtained, and measurement with a simple device is possible. In order to obtain a biosensor with high measurement sensitivity, it is preferable to use metal particles having a uniform particle size and a sharp particle size distribution.

  The metal particles are more preferably made of Ag among the above-described ones. This is because, according to the present inventors, it has been found that when Ag is used, a biosensor having an absolutely large absorption peak intensity derived from localized surface plasmon resonance is obtained. And according to the biosensor having a large peak intensity, it is possible to accurately detect a change in intensity when the target substance is added, and improve the measurement sensitivity.

  When the metal particles are made of Au, the average particle size is preferably 10 to 150 nm, and more preferably 40 to 100 nm. Moreover, it is preferable to select metal particles having a suitable average particle diameter depending on the type of molecular recognition component used. If the average particle diameter is 10 to 150 nm, it is possible to observe a change in absorption peak intensity. If it is 40 to 100 nm, a biosensor exhibiting sufficient absorption peak intensity for measurement of the target substance can do. Moreover, it is preferable to use a particle having a standard deviation of particle size within 10%. This is because the magnitude of the absorption peak intensity can be improved to make it sufficient for measurement of the target substance.

  In the biosensor of the present invention, the metal particles described above are fixed to the stimulus-responsive polymer and can suppress aggregation of the metal particles to a certain extent. However, in order to further improve the stability, the metal particles are protected on the surface of the metal particles. It is preferable to provide an agent. As the protective agent, one or more of polyvinyl pyrrolidone, polyvinyl alcohol, citric acid, polyethylene glycol, tannic acid, and ascorbic acid can be used. The protective agent is a compound that is chemically or physically adsorbed around the metal particles, and can suppress the aggregation of the metal particles and can stabilize the particle size distribution within an appropriate range.

  The concentration of the metal particles in the biosensor is preferably in the range of 0.001 wt% to 0.10 wt%. If the concentration of the metal particles is too high, the detection limit of the measuring device may be exceeded, and if it is too low, the absorption peak intensity may not be detected well. The concentration is preferably adjusted as appropriate depending on the type of metal. As described above, the biosensor using Ag has a large absolute value of the absorption peak intensity, and thus can be set to a relatively low concentration. % To 0.05 wt%.

  The stimulus-responsive polymer is preferably composed of at least one of polyacrylamide, polydimethylsiloxane, polyacrylic acid, polyvinyl methyl ether, polyvinyl alcohol, and polyethylene glycol. It is known that volume changes occur due to environmental changes such as pH, temperature, ionic strength, and the like, and it can be polymerized easily.

  In the present invention, the molecular recognition component specifically reacts with the substance to be measured, and may be one or more of enzymes, antigens, antibodies, DNA, RNA, aptamers, peptide nucleic acids, and sugar chains. preferable. Examples of the enzyme include glucose oxidase and glucose dehydrogenase when the substance to be measured is glucose, lactate oxidase and the like when lactic acid is used, and acetaldehyde dehydrogenase and the like when acetaldehyde is used. be able to.

  As a specific reaction between a molecular recognition component and a substance to be measured, an antigen-antibody reaction or an enzyme reaction is known, and the biosensor of the present invention can be used regardless of whether an antigen-antibody reaction or an enzyme reaction is used. The target substance can be detected, and a wide variety of substances can be measured. Here, the antigen-antibody reaction stably forms an antigen-antibody complex and is maintained in the state of the complex after the reaction. However, in the enzyme reaction, the enzyme and the target substance are once combined to cause an enzyme reaction. However, since it is separated after completion of the reaction, it is difficult to use it for the method of measuring the state change caused by the complex.

  For example, biosensors in which molecular recognition components are immobilized on a substrate have been conventionally known, but these biosensors measure state changes caused by the formation of a complex between the substance to be measured and the molecular recognition components. Therefore, measurement using an enzyme reaction has been difficult. On the other hand, since the biosensor of the present invention uses a stimulus-responsive polymer, even when the enzyme and the target substance are separated, the volume of the polymer changes due to changes in pH, ionic strength, etc., and the enzyme reaction is utilized. Measurement can be performed.

  The biosensor of the present invention is polymerized by adding metal particles having a particle size of 0.2 nm to 200 nm to a solvent containing a monomer for forming a stimulus responsive polymer, and then polymerizing the polymer. It can be produced by a method of fixing a molecular recognition component by fixing a molecular recognition element. For the formation of stimuli-responsive polymers, general polymerization methods can be used. However, when polyacrylamide or the like is used, stimuli-responsive polymers can be generated with only a simple light source by photopolymerization. Can be easily manufactured.

  The molecular recognition element is fixed by introducing a carboxyl group into the polymerized stimulus-responsive polymer, dehydrating and condensing it, and then binding N-hydroxysuccinimide or a compound thereof to bind N-hydroxysuccinimide and the molecular recognition component. It is preferably performed by bonding with an amino group. When polyacrylamide is used as the stimulus-responsive polymer, a carboxyl group can be introduced by adding an alkaline reagent, and the molecular recognition component can be fixed by a simple method.

  In addition, as a fixing method other than the method for introducing the carboxyl group, for example, for a stimulus-responsive polymer having an amino group, the molecular recognition component may be chemically bonded and fixed using glutaraldehyde or the like. it can. Alternatively, the molecular recognition element may be directly included and fixed without using the functional group of the stimulus-responsive polymer.

  As described above, the metal particles used in the production method of the present invention preferably have a protective agent on the surface, and are composed of one or more metal salts of Ag, Au, Pt, Pd, Rh, Ir, Ru, and Os. It can be prepared by adding one or more protective agents of polyvinyl pyrrolidone, polyvinyl alcohol, citric acid, polyethylene glycol, tannic acid, and ascorbic acid to the solution, followed by reduction.

  Moreover, as a monomer for forming the stimuli-responsive polymer, acrylamide, dimethylsiloxane, acrylic acid, vinyl methyl ether, vinyl alcohol, ethylene glycol and the like can be used, and the monomer concentration in the solvent is 10 wt% to It is preferable that it is 30 wt%. This is because, within this range, the substance to be measured is sufficiently diffused to improve the detection sensitivity. If the monomer concentration is too low, the strength of the polymerized polymer may not be sufficient as a biosensor. If the monomer concentration is too high, the substance to be measured will not easily diffuse in the polymer and the reaction with the molecular recognition element will be delayed. It is difficult to become a biosensor with good response.

  The stimuli-responsive polymer can be formed by adding metal particles to a solvent containing the above-described monomer and performing photopolymerization by ultraviolet irradiation. As described above, a colloidal solution in which metal particles are dispersed in a solvent can be used as the metal particles. The final concentration of the metal particles in the biosensor is preferably in the range of 0.005% to 0.025%. This is because the absorption intensity is too low and the detection limit of the measuring instrument is unlikely to occur, and the change in absorption intensity can be observed stably. The initiator for photopolymerization includes 2,2-diethoxyacetophenone, 1- [4- (2-hydroxyethoxy) -phenyl] -2-hydroxy-2-methyl-1-propan-1-one Alkylphenone photopolymerization initiators such as (IRGACURE 2959, manufactured by Ciba Specialty Chemicals Co., Ltd.) can be used. Moreover, it is preferable to add a crosslinking agent to the solvent containing a monomer, for example, N, N'-methylenebisacrylamide can be used.

  When a carboxyl group is introduced to immobilize the molecular recognition component on the stimulus-responsive polymer, it is particularly preferable to use sodium hydroxide as the alkaline reagent to be used. In addition, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-hydroxybenzotriazole and the like can be used for dehydration condensation. As N-hydroxysuccinimide (NHS) or a compound thereof, N-hydroxysuccinimide and the like can be used. Introduction of a carboxyl group and dehydration condensation are performed to bind N-hydroxysuccinimide, and N-hydroxysuccinimide easily binds to an amino group in a protein, so that a molecular recognition component can be immobilized.

  As described above, as a method for measuring a target substance using the biosensor of the present invention, the target substance is added to the biosensor, and the change in the intensity of the localized surface plasmon resonance is detected by the change in the intensity of the absorption peak. be able to.

  The absorption peak can be measured by a method of detecting transmitted light or reflected light of light irradiated on the biosensor with an ultraviolet-visible light spectrophotometer. For example, when measuring transmitted light, it can be measured by placing a biosensor in a quartz cell, adding a solution to be measured, and observing the change in intensity of the absorption peak at a wavelength of 300 to 800 nm.

  As described above, the biosensor of the present invention is simple to manufacture and measure, and can also detect a target substance with high sensitivity. Furthermore, since a special material such as a labeled molecule is not required, a biosensor that is less expensive than the conventional one can be obtained.

  Hereinafter, the best embodiment of the present invention will be described.

First Embodiment : A biosensor was manufactured by the following method using glucose as a measurement target substance, metal particles consisting of Ag, a polyacrylamide as a stimulus-responsive polymer, and glucose oxidase as a molecular recognition component. .

[Production of metal particles]
Metal particles made of Ag were prepared using polyvinylpyrrolidone (hereinafter referred to as PVP) as a protective agent. To a solution of 25 g of PVP dissolved in 125 ml of ethylene glycol, 1.0 g of silver nitrate (Ag concentration: 63.5 wt%) was added as crystals, stirred, and reduced by heating to obtain metal particles. The obtained metal particles were washed with water, then added with acetone, stirred and allowed to stand, and the supernatant was removed to recover the precipitate. This precipitate was dried, dissolved in water, filtered and concentrated to obtain an aqueous solution.

  It was 20 nm when the average particle diameter of the metal particle obtained by the above was measured. In addition, the average particle diameter was calculated from the particle size distribution by measuring the particle size of 100 specimens in the photograph using a photograph taken by TEM (manufactured by JEOL Ltd., JEM-2010) ( The average particle size in the following was also measured by the same method).

[Preparation of stimuli-responsive polymer gel by photopolymerization]
In a 10 wt% acrylamide solution (a solution containing 9.7% acrylamide as a monomer and 0.3% N, N′-methylenebisacrylamide as a cross-linking agent) to a final concentration of 0.001% The obtained metal particles were added and stirred. Next, 2,2-dimethoxyacetophenone as a photopolymerization initiator was added and developed on a glass substrate, irradiated with ultraviolet rays for 5 minutes with a transilluminator (ultraviolet irradiation device), and photopolymerized to gel. The obtained gel was washed with ultrapure water.

[Immobilization of molecular recognition element]
The polymer gel obtained above was hydrolyzed by adding a 0.3N NaOH aqueous solution containing 10% tetramethylethylenediamine to introduce carboxyl groups. Thereafter, a 100 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution was added to dehydrate and condense the carboxyl group, and N-hydroxysuccinimide (NHS) was added to bind to the carboxyl group. Glucose oxidase (EC1.1.3.4), which is a molecular recognition component, was added at 100 U (unit) / ml (addition amount), and bound to the NHS group and immobilized to obtain a biosensor. In glucose oxidase, 1 U (unit) is defined as the amount of enzyme that produces 1 μmol of D-glucono-δ-lactone in 1 minute at 25 ° C.

Second Embodiment : A biosensor was manufactured by the same method as in the first embodiment except that metal particles were made of Au and manufactured using citric acid as a protective agent. A chloroauric acid solution and a citric acid solution were prepared by dissolving 0.17 g of chloroauric acid tetrahydrate and 0.49 g of trisodium citrate dihydrate in 25 ml and 100 ml of ultrapure water, respectively. Next, 6 ml of chloroauric acid solution and 200 ml of pure water were put into a 500 ml three-necked flask and heated to reflux for 30 minutes. After the liquid temperature was stabilized, 50 ml of a citric acid solution was mixed, heated to reflux for 15 minutes, stopped and allowed to cool at room temperature. 26 ml of this solution was placed in a 500 ml three-necked flask and stirred in a thermostatic bath until the liquid temperature reached 30 ° C. When the liquid temperature is stabilized, 252 ml of chloroauric acid solution in which 0.076 g of chloroauric acid tetrahydrate is dissolved and 256 ml of L-ascorbic acid solution in which 0.092 g of sodium L-ascorbate is dissolved are added dropwise simultaneously. Then, the reaction was performed with stirring for 1 hour to form metal particles made of Au. As shown in the TEM observation photograph of FIG. 1, the metal particles were spherical and had a uniform particle size, and the average particle size was 40 nm.

  The absorption spectrum of the biosensor obtained by the above method was measured, and the change in the intensity of the absorption spectrum was measured using glucose as a measurement target by the biosensor of the first embodiment.

Measurement of absorption spectrum of biosensor: The biosensor was placed in a quartz cell having an optical path length of 1.0 cm, and an absorption spectrum at a wavelength of 300 to 800 nm was observed with a spectrophotometer. As shown in FIG. 2, the biosensor of the first embodiment using Ag has an absorption peak at a wavelength of 410.2 nm, and the biosensor of the second embodiment using Au has an absorption peak at a wavelength of 527.6 nm. It was found that the absolute value of the peak intensity was greater when Further, yellow color was confirmed with the naked eye with the biosensor using Ag, and red color was confirmed with the naked eye with the biosensor using Au.

[Measurement of glucose]
Using the biosensor obtained by the above method, glucose as a target substance was measured. In the following measurement, the biosensor of the first embodiment was used. A 100 mM glucose solution (solvent: 20 mM phosphate buffer (pH 7.4)) was dropped onto the biosensor, and the enzyme reaction was carried out at 25 ° C. for 0 to 60 minutes. Then, the change in the intensity of the absorption peak was measured with a spectrophotometer. did. Moreover, it measured similarly also when the 100 mM sucrose solution or the 20 mM phosphate buffer solution (pH 7.4) was added instead of the glucose solution. For the change in intensity of the absorption peak, an absorption spectrum was prepared by the same method as the above-described absorption spectrum measurement, and the change in peak intensity at 410.2 nm was observed.

  As shown in FIG. 3, it was confirmed that the change in the optical characteristics in which the peak intensity was decreased by the addition of glucose, and the biosensor using Ag was found to have good measurement sensitivity. Further, FIG. 4 shows that the absorption peak intensity decreases only when glucose is added, and when sucrose or phosphate buffer is added, the absorption peak intensity does not change, and only glucose can be specifically detected. This is because glucose oxidase specifically binds to glucose and causes an enzymatic reaction. When gluconic acid or hydrogen peroxide is generated by the enzymatic reaction, the polymer gel swells due to a change in ionic strength, It is considered that the intensity of the localized surface plasmon resonance of the metal particles is reduced and the intensity of the absorption peak is reduced.

[Measurement of change in peak intensity with concentration of glucose solution]
Changes in absorption peak intensity after reaction for 30 minutes with the concentration of the glucose solution added to the biosensor as 1 × 10 ⁻³ M, 10 ⁻⁶ M, 10 ⁻⁹ M, 10 ⁻¹² M, and 10 ⁻¹⁵ M And a calibration curve was created.

From FIG. 5, it was found that even if the glucose concentration was 10 ⁻¹² M, it could be detected, and it was a highly sensitive biosensor. For this reason, even when saliva other than blood is used as a sample, glucose can be detected.

[Measurement of peak intensity change with acrylamide concentration]
When the concentration of the acrylamide solution used in the polymer gel was 20% and 30%, photopolymerization was performed in the same manner as described above using metal particles made of Ag to produce a biosensor. About each, the 100 mM glucose solution was added and made to react, and the intensity | strength change of the absorption peak was measured.

  From FIG. 6, it was found that the lower the concentration of the acrylamide solution, the better the responsiveness of the biosensor, and the absorbance intensity decreased in a short time. This is thought to be because when the concentration of the polyacrylamide gel is low, the degree of cross-linking of the polymer chain is low, so that glucose as a measurement target substance is freely diffused and easily binds to glucose oxidase.

The TEM observation photograph of the metal particle in 2nd Embodiment. Absorption spectrum of biosensor. Change with time in absorption peak intensity after addition of glucose. Changes in absorbance after addition of glucose, sucrose, and phosphate buffer. Change in absorbance with respect to glucose concentration. Absorbance change when acrylamide concentration is changed.

Claims (9)

A molecular recognition component that reacts specifically with the analyte,
A stimulus-responsive polymer whose volume changes due to a reaction between the substance to be measured and the molecular recognition component;
Metal particles having an average particle diameter of 0.2 nm to 200 nm in which the intensity of localized surface plasmon resonance changes due to a volume change of the stimulus-responsive polymer,
The biosensor, wherein the metal particles and the molecular recognition component are fixed to the stimulus-responsive polymer. The biosensor according to claim 1, wherein the metal particles are made of at least one of Ag, Au, Pt, Pd, Rh, Ir, Ru, and Os. The biosensor according to claim 1, wherein the metal particles are made of Ag. The biosensor according to any one of claims 1 to 3, wherein the metal particles have at least one protective agent selected from polyvinyl pyrrolidone, polyvinyl alcohol, citric acid, polyethylene glycol, tannic acid, and ascorbic acid on the surface. The biosensor according to any one of claims 1 to 4, wherein the stimulus-responsive polymer comprises at least one of polyacrylamide, polydimethylsiloxane, polyacrylic acid, polyvinyl methyl ether, polyvinyl alcohol, and polyethylene glycol. The biosensor according to any one of claims 1 to 5, wherein the molecular recognition component is at least one of an enzyme, an antigen, an antibody, DNA, RNA, an aptamer, a peptide nucleic acid, and a sugar chain. It is a manufacturing method of the biosensor in any one of Claims 1-6,
To a solvent containing a monomer for forming a stimuli-responsive polymer, metal particles having an average particle size of 0.2 nm to 200 nm are added and polymerized,
A method for producing a biosensor in which a molecular recognition element is immobilized on the polymerized stimulus-responsive polymer. The molecular recognition element is fixed by introducing a carboxyl group into a polymerized stimulus-responsive polymer, dehydrating and condensing it, and then binding N-hydroxysuccinimide or a compound thereof to form an amino group in the molecular recognition component. The method for producing a biosensor according to claim 7, wherein the biosensor is bound by binding. A measurement method for adding a measurement target substance to the biosensor according to claim 1, and detecting a change in intensity of localized surface plasmon resonance based on a change in intensity of an absorption peak.