JPWO2016043078A1 - Substrate antigen simultaneous detection biosensor, electrode, substrate antigen simultaneous detection method, and program - Google Patents

Abstract

The present invention detects a change in current value on a conductive polymer based on an enzyme reaction in a conductive polymer to which an enzyme and an antibody are immobilized, and uses a working electrode based on an antigen-antibody reaction in the conductive polymer. A change in reflectance of the reflected light is detected.

Description

  The present invention relates to a substrate antigen simultaneous detection biosensor, an electrode, a substrate antigen simultaneous detection method, and a program.

  Conventionally, techniques related to biosensors have been disclosed.

  In the enzyme-immunochemical test method described in Patent Document 1, it is disclosed that an enzyme reaction and an immune reaction are detected by an enzyme-linked immunosorbent detection method of an analyte using an endogenous calibrator. .

  Moreover, in the flow cell described in Patent Document 2, it is disclosed that surface plasmon resonance measurement and electrochemical measurement are simultaneously performed on the same metal surface.

  Further, the capillary sensor described in Non-Patent Document 1 discloses that an enzyme and an antigen are simultaneously detected by fluorescence using an enzyme-linked immunosorbent assay (ELISA).

  In addition, in the electrochemical biosensor described in Non-Patent Document 2, it is disclosed that an antigen and glucose are detected using a conductive polymer.

Special table 2009-536023 JP 2010-025681 A

Single-step ELISA coupled sensor based on surface-bonded glucose oxidase, anti-body, and physically-adsorbed PEG membrane-contained peroxide-. Terence G. Henares, Erina Tsusumumi, Hiromi Yoshimura, Kunio Kawamura, Toshio Yao, Hideaki Hisamoto, Sensors and Actuators B, Vol. 149, pp. 319-324, 2010 Highly sensitive electrochemical biosensor for glucose, DNA and protein using gold-polyaniline nanocomposites as a common matrix. Ankan Dutta Chowdhury, Rupali Gangopadhyay, Amitabha De, Sensors and Actuators B, Vol. 190, pp. 348-356, 2014

  However, the conventional test method described in Patent Document 1 requires a process such as sample preparation in advance, and thus has a problem that it takes time for measurement and labor.

  In addition, the flow cell described in Patent Document 2 has problems that it is impossible to simultaneously detect an antigen and an enzyme substrate and to immobilize an antibody and an enzyme on a conductive polymer at the same time. It was.

  In addition, the conventional capillary sensor described in Non-Patent Document 1 has a problem that it is difficult to quantify both by one type of measurement because it tries to obtain two pieces of information only from fluorescence information. It was.

  In addition, in the conventional electrochemical biosensor described in Non-Patent Document 2, since the detection of antigen and glucose is separate electrochemical measurements, it is difficult to quantitatively detect each information at the same time. It had the problem that.

  The present invention has been made in view of the above problems, and is a substrate antigen simultaneous detection bio that can simultaneously and quantitatively and easily measure a substrate such as a sugar and an antigen such as a protein in a liquid such as a body fluid. An object is to provide a sensor, an electrode, a method for simultaneously detecting a substrate antigen, and a program.

  In order to achieve such an object, the substrate antigen simultaneous detection biosensor of the present invention includes an electrode on which a conductive polymer on which an enzyme and an antibody are fixed is deposited, and an enzyme reaction in the conductive polymer. Based on the current value detection means for detecting a change in the current value on the conductive polymer, and detects a change in the reflectance of the light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer. And a reflectance detection means.

  The substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, further comprising a solution bottle filled with a liquid in contact with the conductive polymer.

  Moreover, the substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the liquid contains a body fluid.

  The substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the body fluid is urine, biological fluid, or blood.

  The substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the enzyme is glucose oxidase or creatinase.

  The substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the antigen is albumin, a protein other than albumin, or a peptide that is a degradation product of the protein. To do.

  The substrate antigen simultaneous detection biosensor of the present invention is characterized in that in the substrate antigen simultaneous detection biosensor described above, the conductive polymer is polypyrrolecarboxylic acid.

  Further, the substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the reflectance detection means uses the surface plasmon resonance method for the antigen-antibody reaction in the conductive polymer. Based on, detecting a change in the reflectivity of the light reflected by the electrode.

  Moreover, the substrate antigen simultaneous detection biosensor of the present invention is the substrate antigen simultaneous detection biosensor described above, wherein the current value detection means is based on the enzyme reaction in the conductive polymer using an electrochemical measurement method. And detecting a change in the current value on the conductive polymer.

  Further, the substrate antigen simultaneous detection biosensor of the present invention is the above-described substrate antigen simultaneous detection biosensor, wherein the analysis means for acquiring the analysis result based on the change in the current value and the change in the reflectance, and the analysis An analysis result output means for outputting the results is further provided.

  The electrode of the present invention is characterized by depositing a conductive polymer in which an enzyme and an antibody are immobilized.

  Further, the substrate antigen simultaneous detection method of the present invention is based on the enzyme reaction in the conductive polymer on the electrode on which the conductive polymer on which the enzyme and the antibody are immobilized is deposited. A current value detecting step for detecting a change in the current value in the electrode, and a reflectance detecting step for detecting a change in the reflectance of the light reflected by the electrode based on an antigen-antibody reaction in the conductive polymer. It is characterized by that.

  Further, the program of the present invention provides a current value on the conductive polymer based on an enzyme reaction in the conductive polymer on the electrode on which the conductive polymer on which the enzyme and the antibody are fixed is deposited. A current value detecting step for detecting a change in the light intensity and a reflectance detecting step for detecting a change in the reflectance of the light reflected by the electrode based on an antigen-antibody reaction in the conductive polymer. And

  According to this invention, the change in the current value on the conductive polymer based on the enzyme reaction in the conductive polymer is detected, and the reflection of light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer is detected. Because it detects the change in rate, it can simultaneously measure quantitatively the enzyme substrate and antigen, which are different information that had to be measured separately, and shorten the measurement time. There is an effect.

  According to this invention, since the solution bottle filled with the liquid in contact with the conductive polymer is provided, there is an effect that simple measurement can be performed.

  According to the present invention, since the liquid contains body fluid, it is possible to simultaneously measure the main components in the body fluid that have been separately measured so far, and it can be applied to future simple body fluid sensors. Play.

  According to the present invention, since the body fluid is urine, biological fluid, or blood, it can be applied to a biosensor that matches the purpose of use such as a simple urine sensor, biological fluid sensor, or blood sensor in the future. There is an effect that can be done.

  According to the present invention, since the enzyme is glucose oxidase or creatinase, the amount of sugar such as urine sugar which is a main component in urine can be quantitatively measured.

  According to this invention, since the antigen is albumin, a protein other than albumin, or a peptide that is a degradation product of protein, the amount of protein such as urine protein that is a major component of urine can be quantitatively measured. There is an effect.

  According to this invention, since the conductive polymer is polypyrrole carboxylic acid, it not only has stability, but also has the effect of facilitating the electrochemical measurement and facilitating quantitative measurement.

  According to the present invention, the surface plasmon resonance method is used to detect a change in the reflectance of light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer, so that the optical measurement can be easily performed. Play.

  According to the present invention, the electrochemical measurement method is used to detect a change in the current value on the conductive polymer based on the enzyme reaction in the conductive polymer, so that the electrochemical measurement can be easily performed. Play.

  According to the present invention, since the analysis result based on the change in the current value and the change in the reflectance is acquired and the analysis result is output, it is possible to promote the quick utilization of the disease diagnosis and sensing. There is an effect.

FIG. 1 is a flowchart showing the basic principle of this embodiment. FIG. 2 is a block diagram showing an example of the configuration of the substrate antigen simultaneous detection biosensor in the present embodiment. FIG. 3 is a block diagram illustrating an example of the configuration of the control unit in the present embodiment. FIG. 4 is a flowchart showing an example of processing of the substrate antigen simultaneous detection biosensor of the present embodiment. FIG. 5 is a diagram showing an example of antibody / glucose oxidase immobilization in the present embodiment. FIG. 6 is a diagram showing an example of the surface plasmon dispersion relationship at the prism / gold thin film boundary / dielectric 10 nm interface in this embodiment, and an example of a reflectance theoretical calculation curve using the Fresnel equation. FIG. 7 is a diagram illustrating an example of simultaneous antigen / glucose detection in the present embodiment. FIG. 8 is a diagram illustrating an example of current value changes detected simultaneously in the present embodiment. FIG. 9 is a diagram showing an example of the reflectance change detected simultaneously in the present embodiment.

  Embodiments of a substrate antigen simultaneous detection biosensor, an electrode, a substrate antigen simultaneous detection method, and a program according to the present invention will be described below in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

[Outline of Embodiment of the Present Invention]
Hereinafter, the outline of the embodiment of the present invention will be described with reference to FIG. 1, and then the configuration and processing of the present embodiment will be described in detail. FIG. 1 is a flowchart showing the basic principle of this embodiment. The present embodiment schematically has the following basic features.

  That is, in this embodiment, as shown in FIG. 1, the user injects (sets) a liquid to be a specimen (sample) into the substrate antigen simultaneous detection biosensor (step SA-1).

  The substrate antigen simultaneous detection biosensor is a current value on the conductive polymer based on an enzyme reaction in the conductive polymer on the electrode on which the conductive polymer on which the enzyme and the antibody are fixed is deposited. Is detected (electrochemical detection) (step SA-2).

  The substrate antigen simultaneous detection biosensor detects (optical detection) a change in the reflectance of light reflected by the electrode based on the antigen-antibody reaction in the conductive polymer (step SA-3), and ends the process. To do. Note that step SA-2 and step SA-3 may be executed simultaneously, and either may be executed first.

  This is the end of the description of the outline of the present embodiment.

[Configuration of Substrate Antigen Simultaneous Detection Biosensor 100]
Next, details of the configuration of the substrate antigen simultaneous detection biosensor 100 in the present embodiment will be described below with reference to FIGS. 2 and 3. FIG. 2 is a block diagram showing an example of the configuration of the substrate antigen simultaneous detection biosensor 100 in the present embodiment, and conceptually shows only the portion related to the present invention in the configuration.

  As shown in FIG. 2, the substrate antigen simultaneous detection biosensor 100 schematically includes a substrate 10, a working electrode 20, a potentiostat 30, a counter electrode 31, a reference electrode 32, a solution tank 40, a glass prism 50, and a laser oscillator 51. , A polarizer 52, and a detector 53.

  Here, the substrate 10 forms the working electrode 20 on the surface and is made of metal, glass, plastic, metal oxide, or the like.

  The working electrode 20 is an electrode on which a conductive polymer 21 on which an enzyme 22 and an antibody 23 are fixed is deposited (see FIG. 5). Here, the enzyme 22 may be glucose oxidase, creatinase, or the like. The antigen acting on the antibody 23 may be albumin, a protein other than albumin, a peptide that is a degradation product of protein, creatinine, or the like. The conductive polymer 21 may be polypyrrole carboxylic acid (PP3C) or the like. The working electrode 20 may be composed of a gold thin film or the like. The conductive polymer 21 on the working electrode 20 may be in contact with the liquid in the solution tank 40.

  The potentiostat 30 is a current detector that is connected to the working electrode 20, the counter electrode 31, and the reference electrode 32 and measures a current (current value) between the working electrode 20 and the counter electrode 31. Here, the potentiostat 30 may make the potential of the working electrode 20 constant with respect to the reference electrode 32. Further, the potentiostat 30 may be configured so that no current flows through the reference electrode 32.

  The counter electrode 31 may be platinum, gold, carbon, mercury, or the like.

  The reference electrode 32 may be a hydrogen electrode or a silver-silver chloride electrode.

  The solution tank 40 is a container filled with a liquid in contact with the working electrode 20. Here, the liquid may include a body fluid. The body fluid may be urine, biological fluid, or blood. Further, the liquid in the solution tank 40 may be in contact with the counter electrode 31 and the reference electrode 32.

  The glass prism 50 is in contact with the surface of the substrate 10 on which the working electrode 20 is not provided. Here, as shown in FIG. 2, the dielectric constant (εp) of the glass prism 50 may be larger than the dielectric constant (εd) of the liquid in the solution tank 40.

  The laser oscillator 51 is a device that generates laser light. Here, the laser oscillator 51 may irradiate the working electrode 20 with a laser.

  The polarizer 52 creates linearly polarized light from natural light or circularly polarized light. Here, the polarizer 52 may be installed between the laser oscillator 51 and the glass prism 50.

  The detector 53 is a reflected light detector that detects the light reflected by the working electrode 20 and / or the reflectance of the light.

  Although omitted in FIG. 2, the substrate antigen simultaneous detection biosensor 100 may further include a control unit 102, a storage unit 106, an input / output unit 112, an input / output interface unit, and a communication interface unit. Good. Each part of these substrate antigen simultaneous detection biosensors 100 is communicably connected via an arbitrary communication path.

  Here, with reference to FIG. 3, the structure of the control part 102 in this embodiment is demonstrated. FIG. 3 is a block diagram illustrating an example of the configuration of the control unit 102 in the present embodiment.

  The control unit 102 includes an internal memory for storing a control program such as an OS (Operating System), a program that defines various processing procedures, and necessary data. And the control part 102 performs the information processing for performing various processes by these programs. The control unit 102 may control the input / output unit 112, the input / output interface unit, and the communication interface unit.

  As illustrated in FIG. 3, the control unit 102 includes a current value detection unit 102a, a reflectance detection unit 102b, an analysis unit 102c, and an analysis result output unit 102d in terms of functional concept.

  Among these, the current value detection unit 102 a is current value detection means for detecting a change in current value on the conductive polymer 21 based on an enzyme reaction in the conductive polymer 21. Here, the current value detection unit 102a may detect a change in the current value on the conductive polymer 21 based on an enzyme reaction in the conductive polymer 21 using an electrochemical measurement method. The current value detection unit 102a may be provided in the potentiostat 30.

  The reflectance detector 102 b is a reflectance detector that detects a change in reflectance of light reflected by the working electrode 20 based on an antigen-antibody reaction in the conductive polymer 21. Here, the reflectance detection unit 102b may detect a change in reflectance of light reflected by the working electrode 20 based on an antigen-antibody reaction in the conductive polymer 21 by using a surface plasmon resonance method. The reflectance detection unit 102b may be provided in the detector 53.

  The analysis unit 102c is an analysis unit that acquires an analysis result based on a change in current value and a change in reflectance. Here, the analysis unit 102c may acquire an analysis result regarding the substrate of the enzyme 22 based on the change in the current value and the amount of the antigen based on the change in the reflectance.

  The analysis result output unit 102d is an analysis result output unit that outputs an analysis result. Here, the analysis result output unit 102 d may output the analysis result via the input / output unit 112.

  The storage unit 106 is a device that stores various databases and tables.

  Various databases and tables stored in the storage unit 106 are storage means such as a fixed disk device. For example, the storage unit 106 stores various programs, tables, files, databases, web pages, and the like used for various processes.

  Here, the storage unit 106 may store a current value, a change in the current value, a light reflectance, a change in the light reflectance, and / or an analysis result.

  The input / output unit 112 performs data input / output (I / O). Here, the input / output unit 112 may be, for example, a key input unit, a touch panel, a control pad (for example, a touch pad and a game pad), a mouse, a keyboard, and a microphone. The input / output unit 112 may be a display unit that displays a display screen of an application or the like (for example, a display, a monitor, a touch panel, or the like including a liquid crystal or an organic EL). The input / output unit 112 may be an audio output unit (for example, a speaker) that outputs audio information as audio.

  The input / output interface unit is an interface connected to the input / output unit 112. The input / output interface unit connects the control unit 102 and the input / output unit 112 and controls the input / output unit 112.

  The communication interface unit is an interface connected to a communication device such as a router connected to a communication line or the like. The communication interface unit performs communication control between the substrate antigen simultaneous detection biosensor 100 and a network (or a communication device such as a router). That is, the communication interface unit has a function of communicating data with an external system and other terminals via a communication line. Furthermore, the substrate antigen simultaneous detection biosensor 100 is communicably connected to a network (such as the Internet) via a communication device such as a router and a wired or wireless communication line such as a dedicated line.

  Above, description of an example of a structure of the substrate antigen simultaneous detection biosensor 100 in this embodiment is finished.

[Processing of substrate antigen simultaneous detection biosensor 100]
Next, details of the processing of the substrate antigen simultaneous detection biosensor 100 according to the present embodiment configured as described above will be described in detail with reference to FIGS. FIG. 4 is a flowchart showing an example of processing of the substrate antigen simultaneous detection biosensor 100 in the present embodiment.

  First, as shown in FIG. 4, the user puts a specimen (sample) in the solution tank 40 of the substrate antigen simultaneous detection biosensor 100 filled with a liquid (buffer solution) in contact with the conductive polymer 21 on the working electrode 20. A bodily fluid is injected (step SB-1). Here, the body fluid may be urine, biological fluid, or blood.

  Here, an example of antibody / glucose oxidase immobilization in the present embodiment will be described with reference to FIG. FIG. 5 is a diagram showing an example of antibody / glucose oxidase immobilization in the present embodiment.

  As shown in FIG. 5, in the preparation of a sample for simultaneous detection of antigen and glucose in this embodiment, a conductive polymer (polypyrrolecarboxylic acid (PP3C) thin film is formed on the working electrode (gold thin film) 20 by electrolytic polymerization. ) 21 was deposited (step SC-1). That is, in this sample preparation, the conductive polymer 21 having a carboxyl group is deposited on the working electrode 20 by an electrolytic polymerization method.

  Here, the electropolymerization may be performed by a cyclic voltammetry method (for example, 0.1 M pyrrolecarboxylic acid (P3C) in a 0.5 M sulfuric acid aqueous solution is subjected to a potential range of 0 V to 0.8 V and a sweep rate of 20 mV / 5 cycles may be performed with s).

  And in this sample preparation, after activating the carboxyl group on PP3C thin film 21 in PBS solution using 0.4 MEDC / 0.1MNHS, 1 mg / mL enzyme (glucose oxidase (GOx)) 22 Reaction adsorption (immobilization) is performed on the surface of the activated PP3C thin film 21 (step SC-2). That is, in this sample preparation, glucose oxidase 22 is immobilized for glucose detection.

  And in this sample preparation, the PP3C thin film 21 was once again deposited on it by the electropolymerization method on the same conditions (step SC-3). That is, in this sample preparation, the conductive polymer 21 having a carboxyl group is further deposited by electrolytic polymerization.

  In this sample preparation, the carboxyl group of the newly deposited PP3C thin film 21 is activated using 0.4 MEDC / 0.1 MNHS, and the antibody (Anti-IgG) 23 is immobilized on the activated site. (Step SC-4). That is, in this sample preparation, the antibody 23 is immobilized for antigen detection.

  Furthermore, in this sample preparation, in order to prevent the antigen (IgG) from adsorbing to the activated site that remains without the antibody 23 being immobilized, the remaining activated site is inactivated using ethanolamine, A sample for simultaneous detection of antigen and glucose may be used.

  Thus, in the present sample preparation, the antibody 23 that causes an adsorption reaction specifically with the antigen and the glucose oxidase 22 necessary for the detection of glucose are simultaneously immobilized in the conductive polymer 21.

  That is, in the immobilization method of glucose oxidase 22 and antibody 23 to conductive polymer 21 on working electrode (gold electrode) 20 in this embodiment, pyrrole having a carboxyl group is electropolymerized in an electrolyte solution, A polypyrrole dielectric may be deposited on the gold electrode. Thereafter, in the immobilization method in this embodiment, the antibody 23 may be immobilized after the carboxyl group is activated, glucose oxidase is immobilized, and the polypyrrole dielectric is deposited and activated.

  Returning to FIG. 4, the current value detection unit 102 a uses the current (current value) between the working electrode 20 and the counter electrode 31 measured by the potentiostat 30 by using an electrochemical measurement method to detect the antibody 23. A change in the current value on the conductive polymer 21 based on the enzyme reaction in the conductive polymer 21 to which is fixed is detected (electrochemical detection) (step SB-2).

  The reflectance detection unit 102b irradiates the working electrode 20 from the laser oscillator 51 through the polarizer 52 through the glass prism 50 using the surface plasmon resonance method, and is reflected by the working electrode 20 to be reflected by the glass prism 50. Of the light reflected by the working electrode 20 based on the antigen-antibody reaction in the conductive polymer 21 to which the enzyme 22 is immobilized, using the reflectance of the light detected by the detector 53 based on the light reaching through A change in reflectance is detected (optical detection) (step SB-3).

  Here, the enzyme 22 may be glucose oxidase, creatinase, or the like. Note that step SB-2 and step SB-3 may be executed simultaneously, and either may be executed first.

  Here, with reference to FIG. 2 and FIG. 6, an example of the surface plasmon resonance excitation measurement method in the present embodiment will be described. FIG. 6 is a diagram showing an example of the surface plasmon dispersion relationship at the prism / gold thin film boundary / dielectric 10 nm interface in this embodiment, and an example of a reflectance theoretical calculation curve using the Fresnel equation.

  The surface plasmon resonance excitation measurement method (surface plasmon resonance method) is a measurement method capable of obtaining optical information of a metal thin film / dielectric interface with high sensitivity. And the surface plasmon resonance method combined with the electrochemical measurement in this embodiment is that the metal thin film used for surface plasmon excitation can be used as a working electrode in the electrochemical measurement at the same time, so that the surface of the metal thin film Optical information and electrochemical information can be obtained simultaneously.

  The surface plasmon resonance method combined with the electrochemical measurement method in this embodiment is a very effective measurement that can detect both light and electrochemical signals with high sensitivity in sensing mediated by a conductive polymer. It is also a law.

As shown in FIG. 2, in this embodiment, light is incident on a medium having a small refractive index from a glass prism (prism) 50, which is a medium having a large refractive index, at an incident angle θ ₀ = critical angle θc or more. If so, the light is totally reflected. At this time, an electromagnetic wave and an evanescent wave (surface plasmon wave) 1 that attenuates exponentially in the z direction and propagates in the x direction exist near the surface of the prism 50.

  The evanescent wave 1 can be detected experimentally using a near-field microscope and can be derived theoretically from Maxwell's equations. Here, when a sufficiently thin noble metal thin film (45 to 50 nm) exists on the bottom surface of the prism 50, the evanescent wave 1 oozes out to the metal / liquid interface.

  Although the wave vector obtained from the dispersion relation of surface plasmons in the metal is always larger than the wave vector of incident light from the air, the wave vector of incident light can be increased by passing through the prism 50 having a large refractive index. It becomes possible.

  Here, in the present embodiment, when the incident angle is changed and the x component of the wave vector of incident light matches the wave vector of the surface plasmon localized in the metal, the surface plasmon is resonantly excited.

  At this time, since the energy of the incident light is taken as the energy of the surface plasmon, when the reflected light is detected by the detector (photodetector) 53, it is observed as a sharp attenuation of the reflectance as shown in FIG.

  Further, as shown in FIG. 6, when the conductive polymer (dielectric thin film) 21 is present on the working electrode (metal thin film) 20, the dispersion relation of the surface plasmon changes, and the dip angle corresponding to this changes. Shift to high angle.

  Furthermore, as shown in FIG. 2, when a three-electrode electrochemical cell is attached, the metal thin film serves as the working electrode 20. And the thin film of the conductive polymer 21 is also actively applied to a biosensor or a chemical sensor.

  In addition, reversible doping / dedoping of the conductive polymer 21 is performed electrochemically, and a metal-insulator transition is caused by transfer of electrons to and from the electrode, resulting in a large dielectric constant. In many cases, the optical characteristics change greatly.

  Thus, the working electrode 20 in this embodiment can also be used as a sensing sensitizing material for surface plasmon resonance characteristics by utilizing characteristic changes, and can be used for detection of both electrical (chemical) signals and optical signals. It can be used.

  An example of simultaneous antigen / glucose detection in the present embodiment will be described with reference to FIG. FIG. 7 is a diagram illustrating an example of simultaneous antigen / glucose detection in the present embodiment.

  As shown in FIG. 7, in this embodiment, an antibody 23 and an enzyme (glucose oxidase) 22 are immobilized on a gold electrode on which a conductive polymer 21 having a carboxyl group is deposited. It is used as a working electrode 20 for electrochemical measurement and at the same time as a substrate for surface plasmon resonance (SPR).

  In this embodiment, two phenomena that occur on the working electrode 20 and the gold electrode that is the surface plasmon resonance substrate are detected as a current value and a change in light reflectance.

  In addition, an example of measured values that are simultaneously detected in the present embodiment will be described with reference to FIGS. 8 and 9. FIG. 8 is a diagram illustrating an example of current value changes detected simultaneously in the present embodiment. FIG. 9 is a diagram showing an example of the reflectance change detected simultaneously in the present embodiment.

  In the measurement shown in FIG. 8 and FIG. 9, first, an antigen (5 μg / mL) and glucose (20 mM) are simultaneously injected into a solution tank (cell) 40 filled with PBS (Phosphor Buffer Buffered Saline). The surface plasmon resonance reflectance is measured.

  Subsequently, in this measurement, after rinsing with PBS, only glucose (20 mM) is injected into a solution tank (cell) 40 filled with PBS, and the current value and the surface plasmon resonance reflectance are measured.

  Subsequently, in this measurement, after rinsing with PBS, only the antigen (5 μg / mL) is injected into the solution tank (cell) 40 filled with PBS, and the current value and the surface plasmon resonance reflectance are measured. Yes.

  As shown in FIG. 8, when glucose and antigen are injected at the same time, or when only glucose is injected, the current value changes, and when only antigen is injected, the current value does not react. . Thereby, it was confirmed that the current value change is due to glucose (oxidation-reduction reaction with glucose oxidase).

  On the other hand, as shown in FIG. 9, there is an increase in the baseline due to the shape change of the conductive polymer, but when glucose and an antigen are injected simultaneously, the reflectance increases. Here, after rinsing with PBS, a decrease in reflectivity due to desorption of the adsorbed material is observed, but since the reflectivity is increased compared to before adsorption, the antigen remains without being rinsed. Was confirmed.

  Next, as shown in FIG. 9, when only glucose is injected, the reflectivity is once increased, but the reflectivity is restored after rinsing with PBS. That is, it was confirmed that glucose and glucose oxidase were once physically adsorbed, but were completely washed away by rinsing and did not remain on the surface.

  And as shown in FIG. 9, when only an antigen is put, the reflectance increases. That is, even after rinsing with PBS, it remained almost unwashed, and it was confirmed that the antigen was adsorbed specifically to the antibody. From these results, it was confirmed that after rinsing with PBS, only the antigen can be detected by optical detection (SPR).

  That is, as shown in FIGS. 8 and 9, it was measured that both current (electrochemical detection) and SPR (optical detection) increased in the case of simultaneous injection of glucose and antigen into the cell 40.

  Further, as shown in FIG. 8 and FIG. 9, in the case of glucose injection into the cell 40, it is measured that the current (electrochemical detection) increases and the SPR (optical detection) does not change. It was done.

  Further, as shown in FIG. 8 and FIG. 9, in the case of injection of an antigen into the cell 40, it is measured that the current (electrochemical detection) does not change, and the SPR (optical detection) increases. It was done.

  Thus, in this embodiment, it was shown that each information regarding glucose detection by current change and antigen detection by SPR reflectance can be detected separately.

  Returning to FIG. 4, the analysis unit 102c acquires the analysis result regarding the substrate of the enzyme 22 based on the change in the current value and the amount of the antigen based on the change in the reflectance (step SB-4). Here, the substrate may be glucose or creatinine. Further, the antigen may be albumin, a protein other than albumin, a peptide that is a degradation product of protein, creatinine, or the like.

  Then, the analysis result output unit 102d outputs the analysis result via the input / output unit 112 (step SB-5), and ends the process. In the present embodiment, a doctor (or veterinarian) or the like refers to the analysis result that is output, so that various diseases or the like (for example, diabetes, gout, pregnancy) with respect to the human (or animal) that provided the specimen Diagnosis of heart disease, various lifestyle-related diseases, etc.) or health condition.

  As described above, in this embodiment, in order to solve the problem of the conventional technique in which protein and sugar are separately measured, an antibody and glucose for detecting an antigen on a conductive polymer are detected. Glucose oxidase is simultaneously immobilized.

  Here, the reaction between glucose and glucose oxidase does not change the refractive index on the surface of the conductive polymer, but involves movement of electrons. In contrast, the antigen-antibody reaction is not accompanied by electron transfer, but is accompanied by a change in refractive index because it is chemically bonded to the surface of the conductive polymer.

  Therefore, in the present embodiment, using these different phenomena, electrochemical information and optical information are obtained by electrochemical-surface plasmon resonance spectroscopy combining electrochemical measurement and surface plasmon resonance spectroscopy. Each is detected separately at the same time.

  That is, by using electrochemical-surface plasmon resonance spectroscopy, the glucose-glucose oxidase reaction promoted by the conductive polymer is performed by current detection, and the antigen-antibody reaction is performed by light detection by the surface plasmon resonance method. , It is possible to obtain information separately.

  Thus, in the present embodiment, it is possible to simultaneously detect the antigen and glucose on the same substrate. This can be applied to a urine sensor that separately detects information on urine sugar, albumin, and creatinine, which are main substances in urine.

  Above, description of an example of a process of the substrate antigen simultaneous detection biosensor 100 in this embodiment is finished.

[Other embodiments]
Although the embodiments of the present invention have been described so far, the present invention is not limited to the above-described embodiments, but can be applied to various different embodiments within the scope of the technical idea described in the claims. It may be implemented.

  For example, although the case where the substrate antigen simultaneous detection biosensor 100 performs processing in a stand-alone form has been described as an example, the substrate antigen simultaneous detection biosensor 100 is a client terminal (a separate housing from the substrate antigen simultaneous detection biosensor 100). The processing result may be returned to the client terminal.

  In addition, among the processes described in the embodiment, all or part of the processes described as being automatically performed can be performed manually, or the processes described as being performed manually can be performed. All or a part can be automatically performed by a known method.

  In addition, unless otherwise specified, the processing procedures, control procedures, specific names, information including registration data for each processing, parameters such as search conditions, screen examples, and database configurations shown in the above documents and drawings Can be changed arbitrarily.

  Moreover, regarding the substrate antigen simultaneous detection biosensor 100, each illustrated component is functionally conceptual and does not necessarily need to be physically configured as illustrated.

  For example, the processing functions provided in each device of the substrate antigen simultaneous detection biosensor 100, particularly the processing functions performed by the control unit 102, are all or any part of them are assigned to a CPU (Central Processing Unit) and the CPU. It may be realized by a program that is interpreted and executed, or may be realized as hardware by wired logic. The program is recorded on a non-transitory computer-readable recording medium including programmed instructions for causing a computer to execute the method according to the present invention, which will be described later. It is mechanically read by the detection biosensor 100. That is, in the storage unit 106 such as a ROM or an HDD (Hard Disk Drive), a computer program for giving instructions to the CPU and performing various processes in cooperation with the OS (Operating System) is recorded. This computer program is executed by being loaded into the RAM, and constitutes a control unit in cooperation with the CPU.

  The computer program may be stored in an application program server connected to the substrate antigen simultaneous detection biosensor 100 via an arbitrary network, and may be downloaded in whole or in part as necessary. Is also possible.

  In addition, the program according to the present invention may be stored in a computer-readable recording medium, and may be configured as a program product. Here, the “recording medium” means a memory card, USB memory, SD card, flexible disk, magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, MO, DVD, and Blu-ray (registered trademark). It includes any “portable physical medium” such as Disc.

  The “program” is a data processing method described in an arbitrary language or description method, and may be in any format such as source code or binary code. The “program” is not necessarily limited to a single configuration, but is distributed in the form of a plurality of modules and libraries, or in cooperation with a separate program represented by an OS (Operating System). Including those that achieve the function. Note that a well-known configuration and procedure can be used for a specific configuration for reading a recording medium, a reading procedure, an installation procedure after reading, and the like in each device described in the embodiment.

  Various databases and the like stored in the storage unit 106 are storage devices such as a memory device such as a RAM and a ROM, a fixed disk device such as a hard disk, a flexible disk, and an optical disk. Programs, tables, databases, web page files, and the like.

  The substrate antigen simultaneous detection biosensor 100 may be configured by connecting an arbitrary peripheral device to a known device. The substrate antigen simultaneous detection biosensor 100 may be realized by installing software (including a program, data, and the like) that realizes the method of the present invention in a known apparatus.

  Furthermore, the specific form of distribution / integration of the devices is not limited to that shown in the figure, and all or a part of them may be functional or physical in arbitrary units according to various additions or according to functional loads. Can be distributed and integrated. In other words, the above-described embodiments may be arbitrarily combined and may be selectively implemented.

  As described in detail above, according to the present invention, a substrate antigen simultaneous detection bio that can simultaneously and quantitatively and easily measure a substrate such as a sugar and an antigen such as a protein in a fluid such as a body fluid. Since a sensor, an electrode, a substrate antigen simultaneous detection method, and a program can be provided, it is extremely useful particularly in various fields such as medicine, pharmaceuticals, drug discovery, and biological research.

DESCRIPTION OF SYMBOLS 1 Evanescent wave 10 Substrate 20 Working electrode 21 Conductive polymer 22 Enzyme 23 Antibody 30 Potentiostat 31 Counter electrode 32 Reference electrode 40 Solution tank 50 Glass prism 51 Laser oscillator 52 Polarizer 53 Detector 100 Substrate antigen simultaneous detection biosensor 102 Control part 102a Current value detection unit 102b Reflectance detection unit 102c Analysis unit 102d Analysis result output unit 106 Storage unit 112 Input / output unit

Claims (13)

A working electrode on which a conductive polymer having an enzyme and an antibody immobilized thereon is deposited;
Current value detection means for detecting a change in current value on the conductive polymer based on an enzymatic reaction in the conductive polymer;
A reflectance detection means for detecting a change in reflectance of light reflected by the working electrode based on an antigen-antibody reaction in the conductive polymer;
A biosensor for simultaneous detection of substrate antigens, comprising: A solution tank filled with a liquid in contact with the conductive polymer;
The substrate antigen simultaneous detection biosensor according to claim 1, further comprising: The liquid is
The biosensor for simultaneous detection of substrate antigens according to claim 2, comprising a body fluid. The body fluid is
The biosensor for simultaneous detection of substrate antigens according to claim 3, wherein the biosensor is urine, biological fluid, or blood. The enzyme is
5. The substrate antigen simultaneous detection biosensor according to claim 1, wherein the biosensor is glucose oxidase or creatinase. The antigen is
6. The substrate antigen simultaneous detection biosensor according to claim 1, which is albumin, a protein other than albumin, or a peptide that is a degradation product of the protein. The conductive polymer is
7. The substrate antigen simultaneous detection biosensor according to claim 1, wherein the biosensor is a polypyrrole carboxylic acid. The reflectance detection means includes
8. A change in the reflectance of the light reflected by the working electrode based on the antigen-antibody reaction in the conductive polymer is detected using a surface plasmon resonance method. The biosensor for simultaneous detection of substrate antigens according to any one of the above. The current value detecting means includes
9. The change in the current value on the conductive polymer based on the enzyme reaction in the conductive polymer is detected using an electrochemical measurement method. The biosensor for simultaneous detection of substrate antigens according to one. Analysis means for obtaining an analysis result based on the change in the current value and the change in the reflectance;
Analysis result output means for outputting the analysis result;
The substrate antigen simultaneous detection biosensor according to any one of claims 1 to 9, further comprising:   An electrode characterized by depositing a conductive polymer on which an enzyme and an antibody are immobilized. A substrate antigen simultaneous detection method executed in a substrate antigen simultaneous detection biosensor,
A current value detecting step for detecting a change in current value on the conductive polymer based on an enzyme reaction in the conductive polymer on the working electrode on which the conductive polymer on which the enzyme and the antibody are immobilized is deposited; ,
A reflectance detection step for detecting a change in reflectance of light reflected by the working electrode based on an antigen-antibody reaction in the conductive polymer;
A method for simultaneously detecting a substrate antigen, comprising: A program for causing a biosensor for simultaneous detection of substrate antigens,
A current value detecting step for detecting a change in current value on the conductive polymer based on an enzyme reaction in the conductive polymer on the working electrode on which the conductive polymer on which the enzyme and the antibody are immobilized is deposited; ,
A reflectance detection step for detecting a change in reflectance of light reflected by the working electrode based on an antigen-antibody reaction in the conductive polymer;
A program for running

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