JPH0678985B2 - Electrode for detecting electrochemical optical property and method for detecting electrochemical optical property - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrode for detecting an electrochemical optical characteristic for detecting an electrochemical optical characteristic, particularly, an electrochemical chemiluminescence in a solution system. The present invention relates to an electrode for detecting an electrochemical optical characteristic and a method for detecting an electrochemical optical characteristic using the electrode.

2. Description of the Related Art In recent years, electrochemical optical properties such as electrochemical luminescence have been measured in various fields. For example, in the quantitative measurement of an antigen-antibody reaction, a substance such as luminol and pyrene that emits electrochemiluminescence is immobilized in advance on the antigen, the antibody is reacted with the antigen, and the substance is immobilized on the antigen after the reaction with the antibody. It is used to quantify antibodies by measuring the amount of electrochemiluminescence at this time by utilizing the fact that the emission of electrochemiluminescent substances is suppressed (BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATION Vol.12
8, No.2,1985 April 30,1985, `` ELECTRO CHEMICAL LUMI
NE-SCENCE-BASED HOMOGENEOUS IMMUNOASSAY '' Yoshihi
to Ikariyama, Hideyuki Kunoh and Masuo Aizaw
a).

Conventionally, in order to detect the electrochemiluminescence of luminol or the like in such a solution system, a photodetector in which the electrode is immersed in the solution and the electrochemiluminescence generated by the applied voltage is placed outside the solution container. It is measured by.

However, when the electrode and the photodetector are independent in this way,
From the viewpoint of reproducibility of measured values, the positional relationship between the working electrode and the counter electrode needs to be constant, and the positional relationship between the electrode, the photodetector, and the container must be constant, which is complicated in operation. There is a problem that the device becomes complicated and large in size.

In addition, since the distance from the electrode to the photodetector is inevitably long, the detection sensitivity of electrochemiluminescence decreases.

II Object of the Invention The object of the present invention is a small electrode for detecting electrochemical optical characteristics such as high sensitivity of detection of electrochemical optical characteristics such as electrochemical luminescence, reproducibility of measured values, and easy handling. Another object of the present invention is to provide an electrochemical optical property detection method.

III DISCLOSURE OF THE INVENTION Such an object is achieved by the present invention described below.

That is, the first invention has a light-transmissive working electrode, a counter electrode, and an optical fiber integrally, and the working electrode is integrally formed on a light incident surface of the optical fiber. And an electrode for detecting electrochemical optical characteristics.

A second aspect of the invention uses an electrode that integrally has a light-transmissive working electrode, a counter electrode, and an optical fiber, and the working electrode is integrally formed on a light incident surface of the optical fiber, The electrochemical optical property detection method is characterized by immersing this electrode in a test solution and applying a voltage between the working electrode and the counter electrode to detect the electrochemical optical property.

IV Specific Structure of the Invention Hereinafter, the specific structure of the present invention will be described in detail.

A preferred embodiment of the electrode for detecting an electrochemical optical characteristic of the present invention is shown in FIG.

The electrochemical optical characteristic detection electrode 1 of the present invention integrally has a light-transmissive working electrode 2, a counter electrode 3 and an optical fiber 4, and the working electrode 2 is provided on the light incident surface of the optical fiber 4. It is integrally formed.

The side of the optical fiber 4 opposite to the side on which the working electrode 2 is formed, that is, the light emitting surface side of the optical fiber 4 is connected to a photodetector (not shown). The working electrode 2 and the counter electrode 3 are connected to the lead wire 5 via a conductive joint portion 6, and the opposite side of the working wire 2 and the counter electrode 3 of the lead wire 5 is a voltage pulse generator, for example, a potentiometer. It is connected to a voltage pulse generator consisting of an ostat and a function generator.

Since the working electrode 2 is formed on the light incident surface of the optical fiber 4, it needs to be transparent to the electrochemiluminescent light to be detected. In this case, the transmittance is preferably 50% or more.

Such a working electrode 2 may be formed on the entire light incident surface of the optical fiber 4 or may be formed partially. When partially formed, the shape may be a donut shape, a lattice shape, a honeycomb shape, or any other shape capable of applying a voltage in the vicinity of the light incident surface of the optical fiber 4 in combination with the counter electrode 3.

Alternatively, the working electrode 2 and the counter electrode 3 may be opposed to each other and formed on the light incident surface of the optical fiber 4. In this case, the working electrode 2 and the counter electrode 3 are preferably comb-shaped.

Although the counter electrode 3 is formed in the circumferential direction of the outer peripheral side surface of the optical fiber 4 in the example shown in FIG. 1, the present invention is not limited to this example, and the working electrode 2 is formed on the light incident surface of the optical fiber 4 as described above. It may be formed so as to face.

However, it is necessary to make the area of the counter electrode 3 larger than that of the working electrode 2 because of the ease of manufacturing.
Considering that the end face diameter is small, the counter electrode 3 is preferably formed on the outer peripheral side surface of the optical fiber 4.

The area of the counter electrode 3 is at least twice the area of the working electrode 2,
It is more preferably 3 to 10 times. This facilitates control of the applied potential.

In each of the above examples, the working electrode 2 and the counter electrode 3
Is not directly connected to the lead wire 5, but the lead portions integrally formed with the working electrode 2 and the counter electrode 3 are connected to the optical fiber 4
Extending in the axial direction of the outer peripheral side of, notch a part of the counter electrode,
The lead wire 5 may be connected to the lead portion at a portion above the liquid surface of the test solution during use.

As the material of the working electrode 2, it is preferable to use a metal having a low ionization tendency such as platinum, gold and copper or a metal compound such as ITO and SnO ₂ : Sb.

To form such a working electrode 2 on the light incident surface of the optical fiber 4, a sputtering method, a vapor deposition method, a coating method, or the like may be used. At this time, if appropriate masking is performed, the counter electrode 3 made of the above metal or metal compound is simultaneously formed on the light incident surface of the optical fiber 4 facing the working electrode 2 or on the outer peripheral side surface of the optical fiber 4. can do. In addition, the lead portion described above can be formed in the same manner.

The thickness of the working electrode 2 thus formed is generally
About 100Å to 300Å is preferable. If this thickness is less than 100Å, the strength of the electrode will be insufficient, and the electrical conductivity as an electrode will be insufficient (electrical resistance will increase). When it exceeds, the light transmittance of the working electrode 2 becomes insufficient.

When the counter electrode 3 is formed on the light incident surface of the optical fiber 4 so as to face the working electrode 2 as described above, the counter electrode 3 needs to have light transmissivity similarly to the working electrode 2 described above. It is preferable to use the above-mentioned metal or metal compound used for the working electrode 2, but in other cases, it is not necessary to have light transmittance, and thus various conductive substances can be used.

The counter electrode 3 may be formed by applying a conductive paint by printing or the like in addition to the above-mentioned sputtering method, vapor deposition method, coating method, or the like.
A thin metal plate or the like may be wound around the outer peripheral side surface of the optical fiber 4.

By integrally forming the optical fiber 4, the working electrode 2, and the counter electrode 3 as described above, the distance between the electrodes and the distance between the electrode and the light detecting portion are constant, and there is no deviation thereof, and the handling is easy. Become.

Since the joint portion 6 has conductivity, it is formed by adhering a conductive material such as a conductive adhesive or solder. In order to impart insulation, durability, corrosion resistance and the like to the joint 6, it is preferable to cover the vicinity of the joint 6 with a resin adhesive or the like having non-conductivity, water resistance and corrosion resistance. Even when the lead portion as described above is provided, it is preferable to provide such a coating in order to impart insulation to the lead portion.

In some cases, differently from the above, the working electrode 2 is made of a transparent or opaque rigid material to form these structures, or the working electrode 2 and the counter electrode 3 are made of a transparent or opaque rigid material. Alternatively, the structures may be formed and these structures may be bonded to the end face of the optical fiber.

As the optical fiber 4, a normal optical fiber made of glass, resin, or the like may be used, and its diameter may be about 1 mm or more, depending on the application.

The length of the optical fiber 4 depends on the application but is 5 to 100c.
It is about m.

V. Specific Action of the Invention An electrochemical luminescence measuring device is shown in FIG. 2 as one structural example of the electrochemical optical property measuring device using the electrode for detecting the electrochemical optical property of the present invention.

In the electrochemical optical characteristic detecting electrode 1 of the present invention, the working electrode 2 and the counter electrode 3 are connected by a lead wire 5 to a pulse voltage generator including a function generator 21 and a potentiostat 22, for example.

When a step-like pulse voltage is applied between the working electrode 2 and the counter electrode 3 by the voltage generator, electrochemical emission of luminol or the like occurs in the test solution in the sample container 11, and this emitted light is emitted from the optical fiber. 4 enters the optical fiber 4 from the light incident surface side and enters the photodetector 23 such as a photomultiplier or a photodiode connected to the light emitting surface side of the optical fiber 4,
Photoelectric conversion is performed. The converted electric signal is sent to a signal output device 26 such as an oscillograph or a recorder via a preamplifier 24 and a pulse counter 25, for example. The above-mentioned stepwise pulse voltage is sent from the pulse voltage generator to the signal output device 26, and the electrochemical luminescence with respect to the voltage applied to the working electrode 2 and the counter electrode 3 can be measured.

In this case, the applied pulse may be various depending on the light emission mechanism of the test substance. For example, in Luminor,
It is preferable to first apply a predetermined voltage in pulses and then apply a reverse voltage in pulses. As the first applied voltage, for example, in the case of luminol, a negative voltage is applied to the working electrode 2. At this time, excited active species are formed. After that, the active species generated by applying a voltage opposite to the initial applied voltage,
Luminescence is generated by reacting with the excited active species.

In this case, the first applied voltage is 3 V or more in absolute value with respect to the counter electrode in Luminol, and the second applied voltage is absolute value in the next.
1.2V or more. The pulse width of the initial voltage is 3 to 20se.
c, and the next reverse voltage is about 6 to 60 seconds.

Then, this step-like pulse voltage application, for example, 15 ~
By repeating the cycle for about 90 seconds, stable light emission is repeated after the second time.

Alternatively, for example, in the case of aminopyrene, the stepwise pulse voltage application is not performed in this way, and the negative voltage may simply be repeatedly applied in the pulse form.

When such pulse-like excitation is performed, a significantly higher luminous efficiency can be obtained as compared with the case where the applied voltage is scanned from positive to negative or from negative to positive according to so-called cyclic volt luminometry.

As the luminescent substance, luminol, pyrene, anthracene, rubrene, fluorescein and their derivatives can be used. The solvent may be either aqueous or non-aqueous depending on the luminescent substance used.

Further, the electrode of the present invention is particularly effective when used for detecting an antigen-antibody reaction.

In this case, first, the above-mentioned luminescent substance is immobilized on the antigen and labeled. As the antigen, any of various proteins, polypeptides, polysaccharides, lipids, nucleic acids and the like can be used.

Further, a cross-linking agent may be usually used for fixing the luminescent substance.

An antibody is reacted with this labeled antigen. The reaction is carried out in an aqueous solution containing a suitable buffer.

Thereafter, this solution is usually used as it is, and its luminescence may be measured using the electrode of the present invention.

The electrochemical optical characteristics in the solution system to be detected in the present invention include electrochemical emission, color change such as optical spectrum change, transmittance and reflectance change, and fluorescence change.

VI Effect of the Invention The electrode for detecting electrochemical optical properties of the present invention integrally has a light-transmissive working electrode, a counter electrode, and an optical fiber, and the working electrode is integrally formed on the light incident surface of the optical fiber. Has been formed.

Since the electrode and the photodetector are connected by an optical fiber, the positional relationship between the working electrode and the counter electrode for ensuring reproducibility and the electrode, the photodetector, and the test solution are ensured when measuring the electrochemical optical characteristics. There is no need for an operation or a device for keeping the positional relationship with the container constant, and there is no problem that the operation is complicated and the device becomes complicated and large. Moreover, the reproducibility of measured values is good.

Moreover, since the electrodes and the photodetector are connected by an optical fiber, there is almost no light attenuation and the measurement sensitivity is high.

VII Specific Examples of the Invention Hereinafter, specific examples of the present invention will be described in detail.

[Example 1] An end face of a polymethylmethacrylate resin-based optical fiber having a diameter of 2.5 mm and a length of 12 cm was smoothly polished, and platinum was sputtered on one end face to a thickness of 200 Å to form a working electrode.
The counter electrode was formed at the same time as the working electrode in a strip shape with a width of 3 mm on the outer peripheral side surface 2 mm above the working electrode formation surface of the optical fiber by masking when forming the working electrode.

In this way, an electrode for detecting an electrochemical optical characteristic as shown in FIG. 1 was prepared, and an aqueous luminol solution was used as a test solution and incorporated into an electrochemical optical characteristic measuring device having the configuration shown in FIG. It is.

Stepped pulse voltage shown in FIG. 3 to the working electrode of the electrochemical optical characteristic detecting electrodes _{(e = + 2V, e 2} = -3V,
(t ₁ −t ₀ = 5 sec, t ₂ −t ₁ = 10 sec) was applied at 30-second intervals, and the relationship between the luminol concentration in the solution and the maximum luminescence amount was measured. The maximum luminescence amount was obtained by measuring the average of 10 times of pulses from the 2nd time to the 11th time when the luminescence was stable, and prepared a standard curve. The voltages shown in FIG. 3 are for the counter electrode.

Further, as a comparison, a working electrode of 10 × 10 mm and a counter electrode of 10 × 30 mm are provided at right angles to each other, the optical fiber and the photodetector are the same as those of the present invention, and a stepwise pulse voltage is similarly used. The maximum amount of light emission was measured by applying, and a standard curve was prepared.

The working electrode was opposed to the light incident surface of the optical fiber, and the distance was 2 mm.

Results are shown in FIG.

As shown in FIG. 4, the electrode for detecting electrochemical optical characteristics of the present invention provided a measurement sensitivity 100 times or more that of the conventional electrode.

In the above, the applied voltage was not pulsed, but was measured by scanning from −3 V to +2 V according to so-called cyclic volt luminometry. The scan speed is
It was set to 100 mV / sec. Compared to this case, the maximum light emission amount was improved 100 times when the stepwise pulse voltage was applied.

Example 2 Human IgG antibody was quantified using the electrochemiluminescence measurement device produced in Example 1.

A commercially available human IgG and luminol were cross-linked with glutaraldehyde to prepare a human IgG solution labeled with luminol. The molar ratio was human IgG: luminol: glutaraldehyde = 1: 100: 100. A phosphate buffer solution was used as the solvent.

Next, a commercially available anti-human IgG antibody was dissolved in a phosphate buffer to prepare anti-human IgG antibody solutions having various concentrations. These were added to the above-mentioned luminol-labeled human IgG solution to cause an antigen-antibody reaction to prepare a calibration curve.

The incubation was carried out at 37 ° C for 60 minutes.

When such a calibration curve was repeatedly prepared, it was confirmed that there was little deviation between the calibration curves, and that the reproducibility of the measured values of the electrochemical optical characteristic detection electrode of the present invention was high.

[Example 3] In the same manner as in Examples 1 and 2, luminol was changed to aminopyrene, and a pulse was repeatedly applied to perform the same experiment. As a result, quantitative measurement of the luminescence amount could be performed with high accuracy.

[Brief description of drawings]

FIG. 1 is a perspective view showing a preferred embodiment of the electrode for detecting electrochemical optical characteristics of the present invention. FIG. 2 is a diagram showing a configuration example of an electrochemical optical characteristic measuring device using the electrode for detecting electrochemical optical characteristic of the present invention. FIG. 3 is a graph showing the voltage applied to the working electrode. FIG. 4 is a graph showing the relationship between the luminol concentration and the maximum luminescence amount. DESCRIPTION OF SYMBOLS 1 ... Electrode for detecting electrochemical optical characteristics, 2 ... Working electrode, 3 ... Counter electrode, 4 ... Optical fiber, 5 ... Lead wire, 6 ... Joint part

Claims (3)

[Claims] 1. An electric appliance comprising a light-transmissive working electrode, a counter electrode, and an optical fiber, wherein the working electrode is integrally formed on a light incident surface of the optical fiber. Electrode for chemical and optical property detection. 2. An electrode having a light-transmissive working electrode, a counter electrode, and an optical fiber, wherein the working electrode is integrally formed on a light incident surface of the optical fiber. Is immersed in a test solution, a voltage is applied between the working electrode and the counter electrode, and the electrochemical optical property is detected. 3. The electrochemical optical property detection method according to claim 2, wherein a pulsed voltage is applied between the working electrode and the counter electrode.