JPH11352064A - Eletrochemiluminescence analytical equipment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly measure a concentration change taking place in a relatively short time by a measurement method which does not include a sampling operation by observing as time passes from a moment when a voltage is impressed to a time, when an electric double layer is formed which a potential distribution formation in a solution is resolved with time and measured with the use of an emission reagent. SOLUTION: In using a three-electrode system, a working electrode 11, a counter electrode 12, a reference electrode 13 are set. An emission generated on the working electrode 11 passes a transparent part of a flow through cell 1 and enters a photodetecting camera 4. A minimum time interval, when an emission distribution change is detected by the photodetecting camera 4, is set not larger than 0.1 sec. A system holding a solution between two electrodes is electrostatically series system of a resistance R of the solution and a capacitor C between the electrodes and the solution, and a time before an electric double layer is formed equal to the charge time of the capacitor and nearly three times R×C. A sample the electrolyte concentration of which is to be measured is supplied from a feed port 14 to the cell 1 and discharged from a discharge port 15. The concentration is measured at an electrolyte concentration calculation part 53 of an image recognition part 5.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analyzer for measuring a dynamic change in potential distribution on an electrode surface using electrochemiluminescence to analyze an electrode reaction.

[0002]

2. Description of the Related Art In order to automatically control an industrial process such as a water supply system or a chemical plant, it is necessary to measure a chemical quantity in a chemical process, that is, a concentration of a substance, and to make sure that a chemical reaction is performed normally as planned. You need to monitor it. For this reason, an automatic analysis method called an instrumental analysis method has been developed. Among them, the instrumental analysis method based on the electrochemical principle called the electrochemical analysis method has simpler equipment and lower equipment costs than other instrumental analysis methods,
There are many advantages such as the advantage of selective separation in the analysis of specific components in a multi-component sample, and the suitability for quantitative analysis of dilute components. Typical electrochemical analysis methods include current measurement methods such as polarography, potential difference measurement methods such as pH electrodes, impedance measurement methods for measuring conductivity when AC is applied, and titration methods for measuring the amount of reaction with reagents. It is a target.

In recent years, an electrochemiluminescence method (Electro-chemilu
A luminescence phenomenon caused by an electrochemical reaction called minescence (ECL) has been applied to microanalysis of biological samples such as hormones, drugs in hair, and DNA. Electrochemical analysis methods such as amperometry and potentiometry have already been applied to trace analysis of biological samples, but the ECL method is known to have higher sensitivity than conventional electrochemical analysis methods. Have been.
A known example of the ECL method is described in, for example, Japanese Patent Publication No. 4-502964. In the ECL method, generally, both a luminescent reagent and a reducing agent are added to an ECL sample.

In the specification of the present application, the ECL method described in JP-T-4-502964, that is, Tr
An ECL method using both is (2,2′-bipyridyl) ruthenium (II) and tripropylamine (N (CH ₂ CH ₂ CH ₃ ) ₃ ) as a reducing agent is taken as an example. Thereafter, Tris (2, 2 '
-Bipyridyl) ruthenium and _{(II) Ru (bpy) 3} 2+, or abbreviated as Ru complex. Also, tripropylamine is TPA
Abbreviated.

[0005] Ru (bpy) ₃ ²⁺ at the working electrode a positive potential, R
u (bpy) is oxidized to ₃ ^3+. TPA is also oxidized to TPA. ^{+ On the} positive potential working electrode. After this Ru (bpy) ₃
³⁺ becomes Ru (bpy) ₃ ²⁺ * by reaction with TPA ·,
Emits light having a wavelength of about 600 nm when returning to a stable ^{_{Ru (bpy) 3 2+ (nm}} ). Jonathan L. Leland and Mi
chael J. Powell, Journal of Electrochemical Society (J. Electrochem. Soc.) 137,3
127-3131 (1990).

[0006] Ru (bpy) ₃ ²⁺ to be measured (such as hormones, tumor markers, DNA) those labeled in the molecule that specifically binds to a luminescent reagent. After sufficient coupled with luminescence reagent to a measurement target, measured in Ru (bpy) ₃ ²⁺ photomultiplier luminescence from or an optical detector such as a photodiode. As described above, in the electrochemiluminescence method, the measurement target substance is quantified based on the amount of luminescence.

[0007] By adding the high sensitivity, which is the advantage of the ECL method, to the simplicity and selective separation, which are the advantages of the electrochemical analysis method, it is possible to produce a simpler electrochemical analysis device with higher detection sensitivity. At present, there is no application of the ECL method to the automatic management of industrial processes such as water supply systems and chemical plants.

[0008]

SUMMARY OF THE INVENTION In electrochemical analysis,
In general, it is considered that the electrode section is not a simple electric resistance but forms an impedance in which a resistance and a capacitance are combined in a complicated manner. Therefore, even when it is desired to examine only the capacitance component, it is inherently difficult to accurately measure only the capacitance component. This is the same even when it is desired to check only the resistance component. In the conductivity analysis method, there is a method of applying a synchronous rectifier to exclude a current related to a capacitance component. However, even with this method, it is difficult to separate a resistance and a capacitance portion.

In the above-described electrochemical analysis, if the resistance component and the capacitance component are separated, more accurate measurement can be performed in principle. The present invention enables separation of only the capacitance component by visualizing the potential distribution on the electrode surface. In addition, the use of the ECL method can provide an electrochemical analysis device having higher detection sensitivity than the conventional current measurement method.

In addition, in a liquid sampling type electrochemical analysis method in which a part of a sample liquid is sampled and measured, it takes some time until the sample solution is transmitted from a sampling point to a detection unit of an analytical instrument. Therefore, there is a problem that the instruction is delayed.

Therefore, in the present invention, a change in density occurring in a relatively short time is quickly measured by a measurement method which does not include a sampling operation. In addition, the titration method enables accurate measurement, but requires a complicated apparatus configuration. For the purpose of simply monitoring the change in concentration, it is desirable that the apparatus configuration be simpler in order to reduce the manufacturing cost and maintenance cost.

In the present invention, this instruction is not delayed.
An electrochemical analysis device having a simple device configuration is provided.

[0013]

In the present invention, a time-resolved measurement of the formation of a potential distribution in a solution is performed using an electrochemiluminescent reagent. The electrolyte concentration is measured using the fact that the rate of formation of the potential distribution in the solution is proportional to the electrolyte concentration in the solution. An electric double layer is formed on the electrode with the lapse of time from the moment of voltage application. Until the electric double layer is formed, the volume component of the solution is reflected. Therefore, by observing until the electric double layer is formed, only the capacitance component can be separated. Some conventional electrolyte concentration measuring devices use an analysis reagent, but there is no example in which the concentration is measured using an electrochemiluminescence reagent.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows the configuration of an apparatus according to an embodiment of the present invention. Electrochemical reactions are performed by measuring dynamic changes in the potential distribution on the electrode surface using electrochemiluminescent molecules. 1 shows an overall configuration of an analyzer for performing the above. In this embodiment, the concentration of the electrolyte in the solution is measured using the present apparatus.

This apparatus comprises an electrolyte concentration measurement flow-through cell 1, a potentiostat 2, a function generator 3, a light detection camera 4, and an image recognition unit 5.

Electrodes necessary for causing electrochemiluminescence are provided on the inner surface of the flow-through cell 1. The electrodes can be either a two-electrode type or a three-electrode type.
When the electrode method is used, the working electrode 11, the counter electrode 12,
The reference electrode 13 is provided. The working electrode 11 serves as a light emitting surface. Light emitted on the working electrode 11 passes through the transparent part of the flow-through cell 1 and enters the light detection camera 4.

As a material constituting the flow-through cell 1 for mounting the present invention, a transparent material such as glass, acryl, polymethyl methacrylate or the like is used, and a plastic material is used for other opaque portions. , A metal such as Al or stainless steel, or a ceramic. Further, as a material for forming the working electrode 11 and the counter electrode 12, Pt having high corrosion resistance or a metal such as Al or Ag generally used as an electrode can be used.

The light detection camera 4 is, for example, a light detection CCD (Charge C) having a photon counting mode.
oupled Device) A camera can be used. The minimum time interval for detecting the change in the light emission distribution by the light detection camera 4 is preferably set to 0.1 second or less.

In a system in which a solution is sandwiched between two electrodes as in the present detector, when viewed electrostatically, the resistance R of the solution and the capacitor C between the electrode and the solution are connected in series.
Therefore, the time until the ions have completely moved, that is, the time until the formation of the electric double layer, is equal to the time required for charging the capacitor (charging time), and is approximately 3 × R × C.
It is twice. In the case of 0.1 mol / l sulfuric acid, the above-mentioned charging time is about 0.03 seconds when calculated using the actually measured value. See Watanabe and Nakamura, Electron Transfer Chemistry, Asakura Shoten (1996).
The above charging time varies depending on the solution concentration and the type of ions, but considering the measurement concentration range of the electrolyte concentration to be measured, the minimum time interval for detecting a change in the light emission distribution is 0.1 second or less. It is desirable.

A sample whose electrolyte concentration is to be measured flows into the flow-through cell 1 through an inlet 14 and is discharged through an outlet 15. The image processing unit 5 includes, for example, an image memory unit 51, a light emission distribution change recognition unit 52, and an electrolyte concentration calculation unit 53.

It is known that the value of the current flowing through the working electrode has a correlation with the amount of chemical reaction occurring on the working electrode. Inoue, Aizawa, Fujishima, Electrochemical Measurement, Gihodo Publishing (198
4) See. An ammeter 6 may be provided to measure the amount of chemical reaction occurring on the working electrode. However, the ammeter 6 is not always essential in the present invention.

FIG. 2 is an arrangement diagram of the working electrode 11 and the counter electrode 12. FIG. 2A is a top view, and FIG. 2B is a side view. The working electrode 11 and the counter electrode 12 are preferably displaced in the vertical direction so as to sandwich the flow path through which the solution to be measured flows from above and below. Further, since the surface of the working electrode 11 serves as a light emitting surface, the counter electrode 12 is preferably slightly displaced in the horizontal direction. For example, from the end of the working electrode 11 to the counter electrode 12
The distance (α) to the end is preferably set to 2 mm (mm) or less.

The working electrode 11 is a 5 mm × 5 mm platinum plate,
The counter electrode 12 is a 1 mm × 5 mm platinum plate, and α is 0,
In the case of 0.5, 1, 1.5, and 2 mm, α increases and the signal-to-noise ratio (S / N) of the measurement of the electrolyte concentration decreases. Na S / N
Was not obtained. When one of the two counter electrodes 12 shown in FIG. 2 was removed, the S / N ratio was lower than that in the case where the two surfaces were used as they were. As shown in FIG. 2, two working electrodes 11 are provided for one working electrode 11,
It is preferable to displace them on both sides as viewed from the working electrode.

FIG. 3 shows the working electrode 11 and the counter electrode 1 of FIG.
Electrode arrangement A in which the two arrangements are arranged as one unit and arranged one-dimensionally
((A1) Top view (A2) side view) and two-dimensionally arranged electrode arrangement B ((B1) top view (B2) side view). As described above, when the counter electrode 12 corresponding to one surface of the working electrode 11 is only one surface, the S / N is higher than when two counter electrodes are used.
Decreased. Therefore, as shown in FIG. 3, two or more counter electrodes 12 may correspond to one surface of the working electrode 11.

FIG. 4 is a side sectional view of the flow-through cell 1. In the flow-through cell 1, a light detection camera 4,
There are a working electrode 11, a counter electrode 12, a reference electrode 13, a sample inlet 14, and a sample outlet 15. In the present invention, to place the electrochemiluminescent reagent on the working electrode, for example, beads are used (FIG. 4A) or chemical bonds are used (FIG. 4).
A method such as (B)) can be used. When the beads of (A) are used, the electrochemiluminescent reagent 21 and the magnetic beads 22 are combined with a binding molecule 23 such as an avidin-biotin molecule.
To join.

The magnetic beads 22 can be captured on the working electrode 11 by arranging the magnet 24 outside the wall surface of the working electrode 11 serving as a light emitting surface. This magnet 24 may be an electromagnet or a permanent magnet. The magnetic beads 22 captured on the working electrode 11 of the flow-through cell 1 by the magnetic force of the magnet, after the analysis is completed, cut off the current to the electromagnet or mechanically move the permanent magnet away from the flow-through cell 1. The flow can be discharged from the flow-through cell 1 by weakening the magnetic force.

The diameter of the magnetic beads is, for example, several micrometers or less. Alternatively, if it is magnetic fine particles extracted from magnetic bacteria, fine particles of several tens to several hundreds of nanometers or less can be used. These magnetic fine particles can be incorporated into, for example, a biological cell, and the electrolyte concentration in the cell can be measured even when the cell is alive.

When the chemical bond (B) is used, the surface of the working electrode is modified, and the electrochemiluminescent reagent 21 is arranged on the working electrode by the immobilized molecule 25 using, for example, an SH bond. be able to. The distance between the electrochemiluminescent reagent 21 and the working electrode 11 can be controlled by changing the length of the immobilized molecule 25, for example, the number of carbon atoms constituting the carbon chain.

To [0029] of 1, ruthenium Toribi pyridyl complexes electrochemiluminescence reagent ^{_{21, Ru (bpy) 3 2+}} , tripropylamine the reducing agent, electrochemiluminescence using TPA (Electro-c
2 shows a chemical reaction formula relating to hemiluminescence (ECL). Jonathan L. Leland and Michael J. Powell, J. Elec
trochem. Soc. 137, 3127-3131 (1990).

[0030]

Embedded image

In order for ECL to emit light, first, Ru (bp
y) ₃ ²⁺ is, is oxidized on the electrode, it is necessary to Ru (bpy) ₃ ³⁺ are produced (equation 1). The TPA molecule is also oxidized on the electrode (Equation 2). It is believed that the oxidized TPA molecules release protons and become radicalized (Equation 3). The radicalized TPA reacts with Ru (bpy) ₃ ³⁺ to produce an excited state Ru (bpy) ₃ ²⁺ * (Equation 4). The excited state Ru (bpy) ₃ ²⁺ * is also produced by the reaction of Formulas 5 and 6. This expression occurs at 4 and Equation 6 Ru (bpy) ₃ ²⁺ * results in emission wavelength of about 600nm when returning to the stable state Ru (bpy) ₃ ²⁺ (Equation 7). The emission of this wavelength of about 600 nm
It is called electrochemiluminescence or ECL and is detected by an optical sensor.

[0032] In the embodiment shown in FIGS. 5-7 below, by electrochemiluminescence using Ru (bpy) ₃ ²⁺ and TPA expressed in Chemical formula 1, an example of measurement of the electrolyte concentration.

In this example, as shown in FIG. 4A, the electrolyte concentration was measured using magnetic beads. ECL
At the time of measurement, a constant voltage of +1.4 V was applied between the working electrode 11 and the reference electrode 13 for one second. Working electrode 11 is 5
A platinum plate having a size of 5 mm × 5 mm and a thickness of 0.2 mm is separated from the working electrode 11 by about 1 mm in the vertical direction, and two wire-shaped platinum plates (1 mm in width) are used as the counter electrode 12.
1 was set in parallel. The two counter electrodes 12 were parallel to each other. The reference electrode 13 is a silver / silver chloride electrode (Ag /
AgCl).

The measurement sample used in this example is a sample in which a Ru complex is bonded to the surface of a magnetic bead and TPA is added. Phosphate buffer 300 mmol · dm ^-3 , TP
The A180mmol · dm ^-3, and biotin -Ru (bpy) ₃ ²⁺ in 0.133mg · dm ^-3, and mixing magnetic beads coated with streptavidin 0.72mg · dm ^-3 (φ2.8μm) Incubate for about 10 minutes at 28 ° C.
(bpy) ₃ ²⁺ and the magnetic beads 22 Avidin - used was biotin-conjugated as a sample.

FIG. 5A shows a state immediately after a voltage is applied to the working electrode of the flow-through cell 1 in a stepwise manner. At this time, the electrochemiluminescent reagent 21 did not emit light. However, a few milliseconds later, light emission started as shown in FIG. 5B, and light was emitted in order from the luminescent reagent 21 near the counter electrode 12. After about 100 milliseconds, as shown in FIG. 5C, all the electrochemiluminescent reagents 21 on the working electrode 11 emit light.

FIG. 6 shows a temporal change of the light emission distribution observed by the light detection camera 4 as shown in FIG. (A1) to (D1) show observation images of the time change of the light emission distribution on the working electrode surface observed in the photon counting mode.
(A2) to (D2) correspond to (A1) to (D
It is a state in which the emission intensity distribution of 1) is digitized. (A1)
(A2) is immediately after voltage application, (B1) and (B2) are 1/30 second after voltage application, and (C1) and (C2) are 1/15 of voltage application.
(D1) and (D2) are the light emission distributions after 1/10 second from the application of the voltage. By the numerical processing from (A2) to (D2), the speed (mm / sec) at which the end portion of the light emitting surface moves can be obtained. This moving speed changed depending on the concentration of the electrolyte such as sodium chloride.

FIG. 7 shows the concentration of sodium chloride (mol / l) and the moving speed (mm / mm) of the light emitting surface end obtained from three experiments.
Second). For comparison, the relationship between the concentration of sodium chloride (mol / l) and the peak value of the current value measured by the ammeter 6 is shown. The value of the current flowing through the working electrode 11 has a maximum value (peak value) at the moment of voltage application and decreases with time. Both the movement speed of the light emitting surface end and the peak current value increased with the increase of the sodium chloride concentration.

The moving speed of the end of the light emitting surface ranges from 17.03 mm / sec when the concentration of sodium chloride is zero to 49.45 mm / sec when the concentration of sodium chloride is 0.1 mol / l.
A change of 0.1 mol / l is about 2.9 times (49.45 / 1
7.03 = 2.904). On the other hand, the current value was 4.79 mA when the sodium chloride concentration was zero,
6.01 m when the concentration of sodium chloride is 0.1 mol / l
Up to A, about 1.3 times (6.1.
01 / 4.79 = 1.255).

The phenomenon of this embodiment will be considered with reference to FIG. FIG.
Is a potential distribution curve, which represents the potential between the positive electrode and the negative electrode. FIG.
(A) As shown on the left, the moment of voltage application to the electrolyte solution,
A linear potential difference occurs from the positive electrode to the negative electrode. Due to this potential difference, ions in the solution move, and an ion distribution on the right in FIG. 8A is formed. The distribution on the right side of FIG. 8A is called an electric double layer. In the present specification, the time from the moment of voltage application to the formation of the electric double layer is referred to as relaxation time (τ). ECL emission does not occur unless there is a local potential difference equal to or greater than the threshold value near the molecule of the ECL reagent.

The slope of the potential distribution curve in FIG. 8 indicates the potential difference. As shown in FIG. 8B, the area where the potential difference is large (the portion where the slope of the potential distribution curve is large) increases with time after the application of the voltage. You can see that At the time when the electric double layer is formed, the potential difference on the entire surface of the electrode increases, and the entire surface emits light. Therefore, the time from the moment of voltage application to the time when the entire electrode emits light corresponds to the relaxation time (τ).

As described above, according to the present invention, not only the electrolyte concentration can be measured, but also the formation of the electric double layer can be visualized. There is no known method for directly observing the formation of the electric double layer. The present invention provides the observation method for the first time. Although the electric double layer on the electrode seems to be disturbed by the presence of the magnetic beads, the influence of the beads can be reduced by reducing the diameter of the beads.

When the electroluminescent reagent 21 is bonded to the electrode by a chemical bond as shown in FIG. 4B, the length of the chemical bond is controlled to form an electric double layer within several molecules from the electrode. Can be observed.

In an existing enzyme sensor, an enzyme is immobilized on a polymer film, and a substance to be measured is selectively measured from an electrochemical oxidation-reduction reaction of the enzyme. In order to know the measurement result, the existing enzyme sensor measures the time of the current I (t), t, and obtains its first derivative (dI (t) / dt) or its second derivative (dI).
² (t) / dt ² ). In the present invention, the measurement is performed using the moving speed of the light emitting surface end instead of the current value. As shown in the specification of the present application, the sensitivity is higher when measurement is performed at the moving speed of the light emitting surface end than when the current value is used. Therefore, by applying the present invention to an existing enzyme sensor, selective measurement can be performed with higher sensitivity.

[0044]

According to the present invention, the dynamic change of the potential distribution on the electrode surface is measured using electrochemiluminescence to analyze the electrode reaction. As an example of this, an example is shown in which a resistance component and a capacitance component related to electrochemical analysis are separated, and more accurate measurement is performed in principle. Separation of only the capacitance component is possible by visualizing the potential distribution on the electrode surface as in the present invention.
In addition, by using the ECL method, an electrochemical analyzer having higher detection sensitivity than the conventional amperometric method is provided.

[Brief description of the drawings]

FIG. 1 is a block diagram showing the overall configuration of an electrochemiluminescence analyzer according to one embodiment of the present invention.

FIG. 2 is a top view and a side view showing a basic arrangement of a working electrode and a counter electrode according to one embodiment of the present invention.

FIGS. 3A and 3B are a top view and a side view showing another arrangement example of a working electrode and a counter electrode. FIGS.

FIG. 4 is a schematic side sectional view of a measurement flow-through cell.

FIG. 5 is an explanatory diagram of a phenomenon when an electrolyte concentration is measured in a measurement flow-through cell.

FIG. 6 is a luminescence image and a luminescence intensity distribution diagram when an electrolyte concentration is measured in a measurement flow-through cell.

FIG. 7 is a measurement diagram showing a relationship between a NaCl concentration, a moving speed of an end portion of a light emitting surface, and a current value on a working electrode.

FIG. 8 is a diagram illustrating the reason why the light emitting surface changes.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Electrolyte concentration measurement flow-through cell, 2 ... potentiostat, 3 ... function generator, 4 ... light detection camera, 5 ... image recognition part, 6 ... ammeter, 11 ... working electrode, 12 ... counter electrode, 13 ... see Electrode, 14: sample inlet, 15: sample outlet, 21: electrochemiluminescent reagent, 22
... magnetic beads, 23 ... binding molecules, 24 ... magnets, 25 ... immobilized molecules, 51 ... image memory section, 52 ... emission distribution change recognition section, 54 ... electrolyte concentration calculation section.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Ken Ninomiya 1-280 Higashi Koigakubo, Kokubunji-shi, Tokyo Inside Central Research Laboratory, Hitachi, Ltd.

Claims (9)

[Claims] 1. A sample flow path through which a sample flows, a working electrode provided in the sample flow path, a counter electrode facing the working electrode,
In a flow-through cell including voltage application control means for controlling a voltage applied to the working electrode, a time change of a two-dimensional intensity distribution of light emission from the sample flow path is detected using a light detection camera. Electrochemiluminescence analyzer. 2. The method according to claim 1, wherein a change in the emission intensity distribution is detected.
The electrochemiluminescence analyzer according to claim 1, wherein the minimum time interval is set to 0.1 second or less. 3. The electrochemiluminescence analyzer according to claim 1, wherein a stepwise (step-like) voltage is applied using the voltage applying means. 4. A CCD (Charge Coupled Device) having a photon counting mode as the light detection camera.
e) The electrochemiluminescence analyzer according to claim 1, wherein a camera is used. 5. The electrochemiluminescence analyzer according to claim 1, wherein an electrochemiluminescence reagent is chemically bonded to the working electrode. 6. The electrochemiluminescence analyzer according to claim 1, wherein microparticles having an electrochemiluminescence reagent bound thereto are arranged on the working electrode. 7. A luminous intensity distribution image for each time is stored,
The electrochemiluminescence analyzer according to claim 1, wherein the light emitting end is extracted by image processing. 8. A working electrode is disposed on a first inner surface of a flow cell, and a counter electrode is disposed on a second inner surface of the flow cell, and light emitted from the working electrode serving as a light emitting surface is incident on a light receiving surface of a light detection camera. The electrochemiluminescence analyzer according to claim 1, wherein the arrangement is such that the arrangement is not prevented. 9. An electrochemiluminescence analyzer according to claim 1, having the electrode arrangement according to claim 8, wherein two or more counter electrodes correspond to one surface of the working electrode.