JPH11183479A - Sensor for measuring solution and method for measuring solution component - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a solution measuring sensor and a method for measuring a solution component which show stable characteristics, superior safety and ease of handling, enable measurement with a small quantity of a solution to be measured in a continuous reaction process and can be used repeatedly. SOLUTION: Solenoid valves 8a and 8b and a liquid feed pump 9 are provided, which switch and feed a solution such as a phosphoric acid buffer solution 3, a liquid 4 to be measured, etc., to a flow through cell element 1a, and solenoid valves 8d and 8c are set, which discharge or collect the solution out of the element 1a. Since an antigen and an antibody bonded with each other can be dissociated efficiently by an acid solution 5, the element 1a can be used repeatedly. Moreover, a quantity of the solution required and a quantity of the solution consumed can be reduced to a minimum by switching the feed solution and discharge solution by the solenoid valves.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a sensor for measuring the concentration of a specific component contained in a solution and a measuring method.

[0002]

2. Description of the Related Art Conventionally, there is a method of measuring the concentration of a specific component in a solution by changing the oscillation frequency of a quartz oscillator. The crystal oscillator is a piezoelectric element obtained by cutting a crystal having a specific crystal orientation into thin pieces and forming electrodes such as gold and silver on both surfaces thereof by a vacuum deposition method or the like, and oscillates at a frequency unique to the oscillator. When a substance is attached to the surface of the electrode of the crystal unit by adsorption or the like and the weight of the electrode changes, the oscillation frequency changes with the change in the weight. Therefore, a weight change can be detected by measuring a change in the oscillation frequency. As such a microbalance sensor, in a gas phase system, an odor sensor or a gas sensor (for example, Asari Toshihiro et al., A quartz oscillator gas sensor that smells smell, an ultrasonic sensor)
TECHNO, March issue, 53-57, 1994). In the liquid phase system, there are immunosensors and DNA sensors (for example, Yoshio Okahata, DNA sensors using quartz oscillators, protein nucleic acid enzymes, Vo)
l.40, No. 2, 165-172, 1995).

[0003] The applicant of the present invention filed a patent application
In 8-247476, 2-methylisoborneol which is a musty odor substance in tap water using an antigen-antibody reaction (hereinafter, referred to as
A method for detecting 2-MIB that quantifies 2-MIB) is described. This is also an example of a crystal resonator application technique in a liquid phase system. In this detection method, 2-M
A camphor-ovalbumin complex in which camphor having a structure similar to that of IB is bound to ovalbumin is immobilized on the electrode surface of a quartz oscillator, and this is immersed in a solution to be measured mixed with a known concentration of antibody, and the antibody is immersed in the antibody. On the other hand, camphor and 2-MIB in the solution to be measured are made to react competitively.
Anti-2-M bound to camphor ovalbumin complex
Since the amount of the IB antibody is detected as a change in the oscillation frequency of the quartz oscillator, the concentration of 2-MIB contained in the solution can be measured by measuring the change in the oscillation frequency.

[0004] In addition to the change in weight, when the change in the physical property value of the solution changes the oscillation frequency of the crystal unit,
This technique can be applied. As an example, measurement of the viscosity of a solution (for example, Hiroshi Muramatsu et al., Quartz Crystal Chemical Measurement System, Ultrasonic TECHNO, Vol. 7, No. 2, 28-34, 1995) can be mentioned. In order to oscillate the crystal oscillator, it is necessary to apply a voltage to electrodes formed on both surfaces of the crystal oscillator. Therefore, when using a quartz oscillator in a liquid phase system having conductivity, it is necessary to electrically insulate one electrode. Generally, as shown in FIG. 8, while securing a space in which the quartz oscillator 11 can vibrate, one of the electrodes 11 is made of a hydrophobic insulator 12 such as a silicone resin.
1b, and the electrodes 111a and 111b
It is necessary to prevent the current from flowing during this.

However, when the measurement is performed with the configuration as shown in FIG. 8, there are the following problems and it is not practical. (1) The work of forming the hydrophobic insulator 12 on the electrode 111b of the crystal unit 11 using a hydrophobic insulating material is relatively time-consuming, and furthermore, it is difficult to secure sufficient insulation, and oscillation Touching the circuit 61, the frequency counter 62, the personal computer 63, or the beaker 2 containing the solution 4 to be measured generates noise.

(2) The quartz resonator 11 covered with the hydrophobic insulator 12 has a large volume. When the whole of the quartz resonator 11 is immersed in the solution 4 to be measured, a large amount (at least 30 mL) of the solution is required. Therefore, the consumption of the solution to be measured and the reagent necessary for the measurement increases. (3) Since the measurement is of a batch type, the change in the oscillation frequency during the reaction process between the polymer membrane (not shown) and the component to be measured in the solution 4 to be measured, or the process of washing the electrode surface with a buffer solution, etc. It is difficult to measure it. It is also unsuitable for continuous online measurement.

As a component measuring sensor capable of solving the above-mentioned problems, a flow-through cell type component measuring sensor (Norio Miura et al., An immunoreaction type crystal vibration sensor for highly sensitive detection of methamphetamine, CHEMICAL S
ENSORS, Vol.7B, 53-56, 1991). The structure of the sensor is as shown in FIG. 9, and a pair of silicone rubbers 15 are interposed between a holding substrate 14 made of acrylic resin and a cover having a solution inflow passage and a discharge passage. A quartz oscillator 11 having electrodes (111 and the like) is held. The central portion of the silicone rubber on the cover side is removed, and a flow-through cell is formed by the cover, the crystal unit 11, and the silicone rubber. The solution supplied from the inflow passage flows over the electrode 111 of the crystal unit 11 and
Is discharged from the outflow side. Lead wires 13 are connected to the upper and lower gold electrodes (111 and the like) of the crystal unit 11, respectively. In addition, platinum black is formed on the surface of the electrode 111 on the side of the quartz oscillator 11 that contacts the solution.

When measuring the concentration of methamphetamine with a sensor having such a configuration, the following two methods are performed. In the first method, a monoclonal antibody solution is passed through a flow-through cell, and the monoclonal antibody (molecular weight: about 160,000) is adsorbed on platinum black on the surface of the electrode 111.
After washing with a phosphate buffer, a methamphetamine solution or a complex of methamphetamine and albumin (with a molecular weight of about
67,000) solution is allowed to flow, and methamphetamine or a complex of methamphetamine and albumin is bound by an antigen-antibody reaction, and the frequency change at that time is measured to measure the methamphetamine concentration.

In the second method, first, a complex solution of methamphetamine and albumin is allowed to flow and the electrode 111
After adsorbing the complex to the platinum black on the surface and washing with a phosphate buffer, a monoclonal antibody at a certain concentration is circulated and the monoclonal antibody is bound by an antigen-antibody reaction, and the frequency change at that time is measured and methamphetamine is measured. The concentration of is measured.

When these two methods are compared, it is said that the latter having a larger weight change when measuring a change in frequency has better reproducibility. It is also said that the once bound antigen and antibody can be dissociated by flowing a hydrochloric acid solution of glycine at pH 2.

[0011]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the conventional solution measuring sensor as shown in FIG. 8 and fully utilizes the features of the solution measuring sensor shown in FIG. A solution that can be used, has stable properties, can be measured with a small amount of the solution to be measured, can continuously measure the reaction process, can be used repeatedly, and is excellent in safety and ease of handling It is an object to provide a measurement sensor and a solution component measurement method.

[0012]

According to the present invention, there is provided a solution measuring sensor for measuring a specific component in a solution to be contacted with the solution to be measured, wherein a quartz oscillator having a polymer film formed thereon is used. In the flow-through cell type solution measurement sensor, the polymer film is a film containing an antigen or an antibody immobilized on the electrode surface of the quartz oscillator on the side that comes into contact with the solution,
There is provided a switching supply means for switching the solution to be measured or the solution to which the antibody is added, the phosphate buffer and the acidic solution and supplying the solution to the surface of the polymer membrane (the invention of claim 1).

By the switching supply means, the supply of various solutions to the polymer film of the crystal unit can be continuously switched, so that the reaction process can be continuously measured and the characteristics are stabilized. And handling becomes easy. In addition, since the replacement of the solution becomes very smooth by using the flow-through cell structure, the required amount of the solution such as the solution to be measured can be reduced. Furthermore, the supply of the acidic solution allows for repeated use.

Further, the polymer membrane is a camphor ovalbumin complex as an immobilized antigen (the invention of claim 2). Since the solution measurement sensor in which the antigen contained in the polymer membrane is a camphor-ovalbumin complex measures the output change due to the binding of the antibody, it is particularly effective for detecting a low-molecular-weight substance to be measured, and is excellent. With high sensitivity.

Further, the acidic solution is a citric acid solution, a mixed solution of sodium hydrogen phosphate and citric acid, or a glycine hydrochloric acid solution (the invention of claim 3). These acidic solutions can effectively dissociate the bound antigen and antibody, are easily available, and are excellent in safety and ease of handling. The above three inventions are related to a sensor for measuring a solution, and the following inventions are provided as a method for measuring a solution component.

A method for measuring a solution component using the sensor for solution measurement according to claim 1, wherein a phosphate buffer solution is passed at least to the electrode surface of the crystal unit on which the polymer film is formed, and the reference oscillation frequency is adjusted. A reference frequency measuring step of measuring
The solution to be measured or the solution to which the antibody is added is circulated to the electrode surface of the crystal resonator on which the polymer film is formed, and the antigen and the antibody are immunoreacted, and then the phosphate buffer is circulated to cause an immunoreaction. An immune reaction measuring step of measuring the frequency change due to the immune reaction, and flowing the acidic solution to the electrode surface of the crystal resonator on which the polymer film is formed, and allowing the antigen and the antibody bound by the immune reaction to flow. A dissociation process for dissociation,
(Invention of claim 4).

By including the above three steps, the solution measuring sensor can be used continuously and repeatedly. In the solution component measuring method according to claim 4, when the solution is switched by the switching supply means,
During the time interval in which the solution recovered from the flow-through cell may be a mixed solution of the solution before switching and the solution after switching, the collected solution is discharged to the waste liquid side, and the time interval in which the mixed solution is not mixed During the period, the solution is returned to the original solution (the invention of claim 5).

By recovering the recoverable solution in this way, the solution such as the solution to be measured can be effectively reused.

[0019]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of a solution measuring sensor according to the present invention will be described with reference to examples. The same reference numerals are used for portions having the same functions as the conventional technology. FIG. 1 is a conceptual diagram showing the configuration of an embodiment of a liquid measurement sensor according to the present invention. FIG. 2 is a flow-through cell element 1a in this embodiment. FIG. 3 is an exploded perspective view showing the structure of a quartz oscillator 11 shown in FIG.
FIG. 2 is a perspective view showing a positional relationship between the flexible substrate 13 and the flexible substrate 13.

First, the flow-through cell element 1a will be described. Electrodes 111a and 111b of crystal unit 11 and terminals 131a and 131b of flexible substrate 13 as a wiring member
Are arranged and brought into contact as shown in FIG. That is, the right side of the crystal unit 11 is inserted into the lower side of the flexible substrate 13 so that the electrode 111b on the front side of the crystal unit 11 contacts the terminal 131b exposed on the right back surface of the flexible substrate 13. On the other hand, the electrode 111a on the back side of the crystal unit 11 is a terminal 131a exposed on the left side surface of the flexible substrate 13.
The left side of the crystal unit 11 is aligned with the upper side of the flexible substrate 13 so as to come into contact with the flexible substrate 13. Note that the crystal unit 11
As shown in FIG. 3, the portions where the electrodes 111a and 111b are in contact with the terminals 131a and 131b project rightward on the front side and project leftward on the rear side.

The structure of the flow-through cell element 1a is as follows. First, on the holding substrate 14 made of an acrylic resin plate having holes 142 through which fixing screws 17 are inserted at four corners, similar holes 15 are formed at the four corners.
2 and a polymer elastic sheet 15a made of silicone rubber having a hole 151a formed in the center corresponding to the electrode 111 of the crystal unit 11 is placed thereon. The aligned crystal unit 11 and flexible substrate 13 are overlapped. Holes 132 similar to the holding substrate 14 are also formed at the four corners of the flexible substrate 13. Further, a polymer elastic sheet 15b processed in the same shape as the polymer elastic sheet 15a is overlaid on the crystal unit 11 and the flexible substrate 13, and an acrylic resin flow-through cell lid 16 is placed on the top. Can be In this state, the fixing screw 17 is passed from the holding substrate 14 side and screwed into the screw holes 165 formed at the four corners of the flow-through cell lid 16, and the whole is tightened integrally. The inflow passage 163 and the outflow passage 164 provided in the flow-through cell lid 16 are formed by holes (liquid contact holes in FIG. 2) of the polymer elastic sheet 15b.
It is open at 151b.

By being tightened by the fixing screw 17, the polymer elastic sheets 15a and 15b
The flexible substrate 13 and the electrode 111
1 is electrically contacted sufficiently, and at the same time, the holes 151a of the polymer elastic sheet 15a and the liquid contact holes 15 of the polymer elastic sheet 15b are
The portion excluding 1b is in close contact with the crystal unit 11 and the flexible substrate 13 to realize an airtight state to the outside. Furthermore,
The polymer elastic sheet 15a and the holding substrate 14, and the polymer elastic sheet 15b and the flow-through cell lid 16 are also in close contact with each other to realize an airtight state to the outside.

The sensor for liquid measurement using such a flow-through cell element 1a is composed of a flow-through cell element 1a and a phosphate buffer solution 3 (containing a beaker 2a for obtaining a zero-point output and for cleaning). pH 7), a solution 4 to be measured in a beaker 2b, and an acidic solution 5 in a beaker 2c for dissociating the bound antigen and antibody.
Solenoid valves 8a and 8b for switching the supply of these solutions 3, 4 and 5, and a liquid sending pump 9 (FLUID METERING-IN) for sending the solution from the inlet 161 to the flow-through cell element 1a.
C.), a solenoid valve 8d for switching between collecting and discharging the solution sent out from the outlet 162 of the flow-through cell element 1a, and a solenoid valve 8c for switching the recovered solution.
It is composed of

The solution fed into the flow-through cell element 1a from the inlet 161 reaches the liquid contact hole 151b through the inflow passage 163 shown in FIG. 2, comes into contact with the electrode 111b of the crystal unit 11, and flows out. The gas is sent from the outlet 162 through the passage 164 to a pipe outside the flow-through cell element 1a. The flow rate was 0.2 mL / min in this example. This flow velocity is set to the minimum required value, since if it is too large, an error will occur in the measurement due to the effect of its kinetic energy.

As the acidic solution 5, a citric acid solution of pH 2.2, a mixed solution of sodium hydrogen phosphate and citric acid of pH 3.0, or a hydrochloric acid solution of glycine of pH 2.2 is suitable. According to the study results of the inventors of the present invention, a quartz oscillator
The antibody or antigen immobilized on the surface of the eleventh electrode 111b is
It has been found that even when contacted with the acidic solution 5, about 10% of the solution is desorbed at an initial stage, but hardly desorbed thereafter. Therefore, the measurement can be repeated by subjecting the antigen and antibody to dissociation treatment with an acidic solution. Note that an acidic solution is superior in safety and ease of handling as compared with a solution that may be used for the dissociation treatment, such as an alkali solution and a surfactant.

FIG. 4 shows a flow-through cell element 1a having a quartz oscillator 11 having a camphor-ovalbumin complex immobilized on the surface of an electrode 111b, and a 2-MIB concentration of 0 mg / L as a solution 4 to be measured. Anti-MIB antibody concentration is 0.
Using a solution adjusted to 01 mg / mL, an acidic solution 5 was used as a solution of 0.1 mg / mL.
FIG. 3 is a diagram in which changes in the oscillation frequency of a quartz oscillator 11 are recorded using a hydrochloric acid solution (pH 2.2) containing 1 mol / L glycine.

The operation procedure is as follows. 1) The solenoid valves 8a and 8b are set to the phosphate buffer 3 side, the solenoid valve 8d is set to the outlet side, and the phosphate buffer 3 is flow-through cell element.
The liquid is sent to 1a for 30 minutes to stabilize the oscillation frequency of the crystal unit 11, and the oscillation frequency at this time is used as a reference (frequency change is 0H
z). 2) Switch the solenoid valves 8a and 8b to the acidic solution 5 side,
After the acidic solution 5 is supplied to the flow-through cell element 1a for 7 minutes while leaving the outlet side 8d, the electromagnetic valves 8d and 8c are switched to the beaker 2c to collect the acidic solution 4. The total time for feeding the acidic solution 4 is 10 minutes.

3) Again, the solenoid valves 8a and 8b are
The phosphate buffer 3 is sent to the flow-through cell element 1a for 10 minutes while the solenoid valve 8d is set to the discharge port side.
Is stabilized, and the oscillation frequency at this time is used as a reference for a frequency change with respect to the solution to be measured. The steps 1), 2) and 3) are intended to desorb the camphor-ovalbumin complex which is unstablely immobilized on the surface of the electrode 111b.

Therefore, these three steps are usually employed when starting to use a new quartz oscillator 11 with the antibody or antigen immobilized on the electrode surface 111b, and the quartz oscillator 11 is not replaced. When used repeatedly as it is, it is often omitted. 4) The electromagnetic valves 8a and 8b are switched to the solution 4 to be measured, the electromagnetic valve 8d is kept at the outlet side, and the solution 4 to be measured is sent to the flow-through cell element 1a for 7 minutes. And 8c
Is switched to the beaker 2b side to recover the solution 4 to be measured. The total time for sending the solution 4 to be measured is 30 minutes.

5) The solenoid valves 8a and 8b are set three times with phosphate buffer solution 3
And the solenoid valve 8d is set to the discharge port side, and the phosphate buffer 3 is fed to the flow-through cell element 1a for 15 minutes.
And stabilize the oscillation frequency, and measure the oscillation frequency at this time. The difference between this oscillation frequency and the oscillation frequency in step 3) (the amount of change in frequency) corresponds to the amount of anti-2-MIB antibody bound to the camphor-ovalbumin complex on the crystal unit 11, and the amount of change in frequency The 2-MIB concentration in the solution to be measured is calculated using a calibration curve as shown in FIG.

With the above, the measurement of the solution to be measured is completed.
In order to dissociate the anti-2-MIB antibody bound to the surface of the electrode 111b from the surface of the electrode 111b by the immune reaction and prepare for the next measurement, 6) again, the solenoid valves 8a and 8b are switched to the acidic solution 5 side,
After the acidic solution 5 was sent to the flow-through cell element 1a for 7 minutes while the electromagnetic valve 8d was kept at the outlet side, the electromagnetic valves 8d and
8c is switched to the beaker 2c side, and the acidic solution 4 is recovered. The total time for feeding the acidic solution 4 is 10 minutes.

7) Four times, the solenoid valves 8a and 8b are set in phosphate buffer 3
The phosphate buffer 3 is fed to the flow-through cell element 1a with the solenoid valve 8d facing the discharge port, and the oscillation frequency of the crystal unit 11 is stabilized. Such as when it is measured repeatedly,
When the steps 1), 2) and 3) are omitted, the oscillation frequency at this time becomes a reference for the frequency change with respect to the solution to be measured.

As is clear from the above description, the frequency change amount (ΔF) of the oscillation frequency in step 3) and step 5)
Corresponds to the amount of the anti-2-MIB antibody bound to the camphor-ovalbumin complex immobilized on the surface of the electrode 111b. In FIG. 4, since the oscillating frequency in step 3) and the oscillating frequency in step 7) are substantially at the same level, most of the anti-2-MIB antibody bound to the camphor-ovalbumin complex is in acidic solution 4. You can see that it has been dissociated by

FIG. 5 is a diagram showing the variation of the oscillation frequency (frequency variation ΔF) in the case where the above-described measuring step is repeated three times, using the type of the acidic solution as a parameter. 0.
Hydrochloric acid solution (pH 2.2) containing 1 mol / L glycine, 0.2m
With a mixed solution of ol / L sodium hydrogen phosphate and 0.1 mol / L citric acid (pH 3.0), ΔF was found to decrease, and the binding and dissociation with the anti-2-MIB antibody were repeated. As a result, it is presumed that the anti-MIB antibody that cannot be dissociated accumulates, and as a result, the binding amount of the anti-2-MIB antibody decreases. However, assuming that the limit of use as a sensor is a point in time when the amount of frequency change due to the binding of the anti-2-MIB antibody becomes 50% of the initial value, about eight times repeated use can be expected.

On the other hand, a 0.1 mol / L citric acid solution (pH 2.2
In the case of ()), such a decrease in ΔF is not recognized, so that a large number of repeated use can be expected. FIG.
Using a flow-through cell element 1a having the same configuration as that of FIG.
Using a mixed solution (pH 3.0) of sodium hydrogen phosphate and 0.1 mol / L citric acid in a case where the repetitive measurement was performed seven times with the solution sending time of 10 minutes being set to 10 minutes.
FIG. 4 is a diagram in which changes in the oscillation frequency are recorded.

In this case, since the salt concentration of the acidic solution 5 is higher than the salt concentration of the phosphate buffer 3, the amount of frequency change when the acidic solution 5 is sent is equal to that when the phosphate buffer 3 is sent. It is measured larger than the frequency change. As for the frequency change (ΔF) in the repeated measurement, the final value when the acidic solution 5 is fed gradually increases, and accordingly, the ΔF due to the solution 4 to be measured decreases, and the seventh ΔF becomes the first time. ΔF
About 67% of the total. The limit of use as a sensor is that the amount of frequency change due to the binding of the
%, It can be seen that seven or more repetitive uses are possible.

FIG. 7 shows a flow-through cell element 1a for 2-MIB measurement in which a camphor-ovalbumin complex is immobilized on the surface of an electrode 111b on the side of the crystal unit 11 which comes into contact with the solution 4 to be measured. 4 and the like, the concentration of 2-MIB in the solution 4 to be measured was 0.0001 to 0.0001.
Frequency change (Δ) measured in the range of 0.1 mg / L
It is a diagram (calibration curve) showing F). The horizontal axis is the concentration of 2-MIB in the solution to be measured, and the vertical axis is ΔF. Also in this case, the concentration of the anti-2-MIB antibody in the solution to be measured is 0.01%.
It was fixed to mg / mL.

Anti-2-MI bound to a camphor-ovalbumin complex immobilized on the electrode 111b of the crystal unit 11
It can be seen that the frequency change (ΔF) due to the B antibody decreases as the concentration of 2-MIB in the solution to be measured increases, and converges to a constant value above a certain concentration. By using such a calibration curve, the variation ΔF of the oscillation frequency can be obtained.
Can determine the unknown concentration of 2-MIB in the solution.

The reason why the above-mentioned quantification of 2-MIB is determined by the competition method is that the molecular weight of 2-MIB is about 170, whereas the molecular weight of anti-2-MIB antibody is about 160,000, which is an order of magnitude higher. is there. That is, the electrode 111b of the crystal unit 11
When an anti-2-MIB antibody is immobilized thereon and 2-MIB in the solution to be measured is directly bound to the antibody for measurement,
Since the mass change is caused by the binding of 2-MIB, the mass change is small and the measurement sensitivity is low. In comparison, the crystal unit 11
Of the camphor-ovalbumin complex was immobilized on the electrode 111b, and the anti-camphor ovalbumin complex was immobilized on the electrode 111b.
This is because when the -MIB antibody is bound, the mass change is increased by about three orders of magnitude, so that the sensitivity of the measurement is remarkably increased and high-precision measurement becomes possible.

Unlike the case of the above embodiment, when the molecular weight of the substance (antigen) whose concentration is to be measured is large,
By using a solution measurement sensor in which an antibody corresponding to the substance is immobilized on the electrode 111b of the crystal unit 11,
The concentration of the substance can be measured directly from the solution to be measured. In this embodiment, the case of 2-MIB has been described. However, by changing the type of antibody or antigen immobilized as a polymer film on the electrode 111b of the crystal unit 11,
-It is also possible to quantify odor substances other than MIB, pesticides, oils and the like.

The numerical conditions such as the amount of material used, the processing time, the processing temperature and the like in the above embodiment are merely examples, and it is apparent that the present invention is not limited to these numerical values. Furthermore, in the above example, the solution to be measured is a solution in which an antibody solution of a known concentration is mixed with the solution to be measured, but the solution to be measured 4 is used. Electrode 11 of crystal unit 11
It will also be apparent that the sensor for solution measurement can be immobilized on 1b and the solution to be measured can be the solution 4 to be measured.

In any case, the dissociation of the immune reaction by the acidic solution 5 and the phosphate buffer 3 by the switching means are used.
By switching between the solution 4 to be measured and the acidic solution 5 and switching between disposal and recovery, the characteristics are stable, measurement can be performed with a small amount of the solution to be measured, and the reaction process can be continuously measured. It is possible to provide a solution measurement sensor and a solution component measurement method that can be repeatedly used and are excellent in safety and ease of handling.

[0043]

According to the present invention, there is provided a solution measuring sensor for measuring a specific component in a solution to be measured by coming into contact with the solution to be measured, the flow-through using a quartz oscillator having a polymer film formed thereon. In the cell-type solution measurement sensor, the polymer film is a film containing an antigen or an antibody immobilized on the electrode surface of the crystal unit on the side in contact with the solution, and the polymer film to be measured to which the solution or the antibody to be measured is added. Since the switching supply means for switching between the solution, the phosphate buffer and the acidic solution and supplying the solution to the surface of the polymer film is provided, the supply of various solutions to the polymer film of the crystal unit can be continuously switched. . As a result, it is possible to continuously measure the reaction process, and it is easy to stabilize the characteristics, and the handling becomes easy. In addition, since the replacement of the solution becomes extremely smooth by adopting the flow-through cell structure, the required amount of the solution such as the solution to be measured can be reduced. Furthermore, the supply of the acidic solution allows for repeated use.

Therefore, the characteristics are stable, the measurement can be performed with a small amount of the solution to be measured, the reaction process can be continuously measured, the repetitive use is possible, and the safety and the ease of handling are excellent. A solution measurement sensor can be provided (the invention of claim 1). Further, the polymer membrane is a camphor-ovalbumin complex as an immobilized antigen. Since the solution measurement sensor in which the antigen contained in the polymer membrane is a camphor-ovalbumin complex measures the output change due to the binding of the antibody, it is particularly effective for detecting a low-molecular-weight substance to be measured, and is excellent. With high sensitivity. Therefore, it has excellent selectivity and sensitivity to 2-MIB, has stable properties, can be measured with a small amount of the solution to be measured, can continuously measure the reaction process, and can be repeatedly used. Thus, it is possible to provide a solution measurement sensor that is excellent in safety and ease of handling (the invention of claim 2).

Further, since the acidic solution is a citric acid solution, a mixed solution of sodium hydrogen phosphate and citric acid, or a glycine hydrochloric acid solution, the bound antigen and the antibody can be effectively dissociated, In addition, it is easily available, and is particularly excellent in safety and ease of handling. Therefore, the characteristics are stable and can be measured with a small amount of the solution to be measured.
It is possible to provide a solution measurement sensor that can continuously measure a reaction process, can be repeatedly used, and is particularly excellent in safety and ease of handling (claim 3).

The above invention relates to a sensor for measuring a solution, and the following invention is provided as a method for measuring a solution component. 2. A solution component measuring method using the solution measuring sensor according to claim 1, wherein a reference oscillation frequency is measured by circulating a phosphate buffer solution at least over an electrode surface of the crystal unit on which the polymer film is formed. After the reference frequency measurement step and the solution to be measured or the solution to which the antibody is added is allowed to flow to the electrode surface of the crystal resonator on which the polymer film is formed, and the antigen and the antibody are immunoreacted, a phosphate buffer solution The immune reaction by measuring the frequency change caused by the immune reaction by stabilizing the immune reaction, and by flowing the acidic solution to the electrode surface of the crystal unit on which the polymer film is formed, and binding by the immune reaction. And a dissociation step of dissociating the antigen and the antibody thus performed, so that the solution measurement sensor can be used continuously and repeatedly.

Therefore, the characteristics are stable, the measurement can be performed with a small amount of the solution to be measured, the reaction process can be continuously measured, the repetitive use is possible, and the safety and the ease of handling are excellent. A solution component measuring method can be provided (the invention of claim 4). In the solution component measuring method according to claim 4, when the solution is switched by the switching supply unit, the solution recovered from the flow-through cell may be a mixed solution of the solution before the switching and the solution after the switching. During a certain time interval, the recovered solution is drained to the waste liquid side,
Since the original solution is returned during the time interval other than the mixed solution, the solution such as the solution to be measured can be effectively reused. Therefore, the required amount of solution can be further reduced,
At the same time, the amount of waste liquid can be reduced (the invention of claim 5).

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing a configuration of an embodiment of a liquid measurement sensor according to the present invention.

FIG. 2 is a perspective view showing a configuration of a flow-through cell element in an embodiment.

FIG. 3 is a perspective view showing a positional relationship between the crystal unit and a flexible substrate in FIG. 2;

FIG. 4 is a diagram showing an example of a measurement result by the measurement system of FIG. 1;

FIG. 5 is a diagram showing the relationship between the type of acidic solution and the results of repeated measurements.

FIG. 6 is a diagram showing repeated measurement results when a mixed solution of sodium hydrogen phosphate and citric acid is used as an acidic solution;

FIG. 7 is a diagram showing a calibration curve obtained by using 2-MIB having a known concentration.

FIG. 8 is a conceptual diagram showing a configuration of a measurement system using an example of a liquid measurement sensor according to the related art.

FIG. 9 is a cross-sectional view illustrating the configuration of another example of a liquid measurement sensor according to the related art.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Solution sensor 1a Flow-through cell element 11 Crystal oscillator 111, 111a, 111b Electrode 12 Hydrophobic insulator 13 Lead wire 13a Flexible substrate 131, 131a, 131b Terminal 132 hole 14 Holding substrate 142 hole 15 Silicone rubber 15a, 15b Polymer elastic sheet 151a 151b Liquid contact hole 152 hole 16 Flow-through cell lid 161 Inlet 162 Outlet 163 Inflow passage 164 Outflow passage 165 Screw hole 17 Fixing screw 18 Connector 2, 2a, 2b, 2c Beaker 3 Phosphate buffer 4 Solution to be measured 5 Acid solution 61 Oscillation circuit 62 Frequency counter 63 Personal computer 8a, 8b, 8c, 8d Solenoid valve 9 Liquid pump

Claims (5)

[Claims] 1. A solution measurement sensor for measuring a specific component in a solution in contact with a solution to be measured, wherein the sensor is a flow-through cell type solution measurement using a quartz oscillator having a polymer film formed thereon. In the sensor, the polymer film is a film containing an antigen or an antibody immobilized on the electrode surface of the crystal unit on the side that comes into contact with the solution, and comprises a solution to be measured or a solution to which the antibody is added and a phosphate buffer. A solution measurement sensor, comprising: a switching supply unit that switches between a liquid and an acidic solution and supplies the solution to the surface of the polymer film. 2. The solution measurement sensor according to claim 1, wherein the polymer membrane is a camphor-ovalbumin complex as an immobilized antigen. 3. The sensor according to claim 1, wherein the acidic solution is a citric acid solution, a mixed solution of sodium hydrogen phosphate and citric acid, or a glycine hydrochloric acid solution. 4. A method for measuring a solution component using the sensor for measuring a solution according to claim 1, wherein at least a phosphate buffer solution is passed through at least the electrode surface of the crystal resonator on which the polymer film is formed. A reference frequency measurement step for measuring the oscillation frequency; and a solution to be measured or a solution to which the antibody was added was passed through the electrode surface of the quartz crystal resonator on which the polymer film was formed to cause an immunoreaction between the antigen and the antibody. After that, an immune reaction measuring step of circulating a phosphate buffer solution to stabilize the immune reaction and measure a frequency change due to the immune reaction, and circulating an acidic solution to the electrode surface of the crystal resonator having the polymer film formed thereon A dissociation step of dissociating the antigen and the antibody bound by an immune reaction by an immunoreaction. 5. A method according to claim 1, wherein when the solution is switched by the switching supply means, the solution recovered from the flow-through cell may be a mixed solution of the solution before switching and the solution after switching during a time interval. 5. The method according to claim 4, wherein the recovered solution is discharged to a waste liquid side and returned to an original solution during a time interval when the liquid mixture is not a mixed liquid.