1. Field of the Invention
The present invention relates to a sensing method for recognizing and quantifying a substance to be sensed contained in a sample solution, based on a frequency of a piezoelectric resonator such as a quartz resonator.
2. Description of the Related Art
As a piezoelectric sensor sensing and measuring a trace substance contained in a sample solution, there has been known a quartz sensor using a quartz resonator. In this quartz sensor, an adsorption layer made of a biological substance film or the like recognizing and reacting with a specific substance to be sensed is formed on a front surface of a metal electrode (excitation electrode) provided on a quartz piece. When reacting with the substance to be sensed present in a sample solution to adsorb the substance to be sensed, the adsorption layer changes in mass, which causes a change in natural frequency of the quartz resonator. By utilizing this operation, the concentration of the substance to be sensed is measured.
As the biological substance, used is, for example, a film of an antibody reacting with a specific antigen, and the film of the antibody adsorbs the antigen. A method of fabricating such a quartz sensor is as follows. A buffer solution is supplied into the quartz sensor including therein the quartz resonator, and then a solution containing a predetermined amount of antibody is supplied into the quartz sensor, so that the antibody is adsorbed by the front surface of the metal electrode of the quartz resonator. Next, a solution containing a predetermined amount of a substance for blocking (blocker) made of, for example, protein is injected into the quartz sensor so that the front surface of the metal electrode adsorbs the blocker. A reason why the front surface of the metal electrode is made to adsorb the blocker is to prevent the antigen from being adsorbed by the front surface of the metal electrode, thereby forming an environment where the antigen is adsorbed only by the antibody, and to ensure high accuracy in a correspondence relation between an amount of the antigen captured by the antibody and the frequency.
However, since it is difficult to separate the antigen and the antibody once the antigen and the antibody react with and bind to each other, the quartz sensor using the antigen-antibody reaction is usable only once and then is discarded. This requires a troublesome work of replacing the quartz sensor every time the sample is changed and also is a waste of resources.
Patent document 1 describes a measuring device including eight quartz sensors which are attachably/detachably provided in a measuring device main body measuring a frequency change of a quartz resonator, thereby facilitating a work of measuring the concentration of a substance to be sensed and reducing the time for the measuring work. However, this art is not capable of solving the aforesaid problem since the quartz sensors are discarded after use.
[Patent Document 1] Japanese Patent Application Laid-open No. 2006-194868 (paragraph 0012 and FIG. 1)
The present invention was made under such circumstances and has an object to provide a sensing method capable of measuring a sample a plurality of times with one piezoelectric sensor.
The sensing method may be structured as follows.
A sensing method of the present invention is a method in which a piezoelectric resonator having an excitation electrode formed on a piezoelectric piece is oscillated by an oscillator circuit while being in contact with a liquid and an antigen in a sample solution is sensed based on a change in natural frequency of the piezoelectric resonator,
the piezoelectric resonator being a piezoelectric resonator in which a base layer made of protein adsorbing an immunoglobulin and desorbing the immunoglobulin under an atmosphere of an acid liquid is formed on the excitation electrode, the method including:
a step of supplying a liquid containing the immunoglobulin to the piezoelectric resonator to make the base layer adsorb the immunoglobulin;
a first measuring step of oscillating the piezoelectric resonator which has adsorbed the immunoglobulin and measuring a frequency corresponding to an oscillation frequency of the oscillator circuit;
a step, which follows the first measuring step, of supplying the piezoelectric resonator with the sample solution containing the antigen which is a substance to be sensed captured by the immunoglobulin;
a second measuring step, which follows the step of supplying the sample solution, of oscillating the piezoelectric resonator which has adsorbed the antigen and measuring a frequency corresponding to an oscillation frequency of the oscillator circuit;
a step of finding a difference between the frequencies obtained in the first measuring step and the second measuring step respectively; and
a step, which follows the step of finding the difference, of supplying the acid liquid to the piezoelectric resonator to make the base layer desorb the immunoglobulin.
A sensing method according to another aspect of the present invention is a method in which a piezoelectric resonator having an excitation electrode formed on a piezoelectric piece is oscillated by an oscillator circuit while being in contact with a liquid and an immunoglobulin in a sample solution is sensed based on a change in natural frequency of the piezoelectric resonator,
the piezoelectric resonator being a piezoelectric resonator in which a base layer made of protein adsorbing the immunoglobulin and desorbing the immunoglobulin under an atmosphere of an acid liquid is formed on the excitation electrode, the method including:
a step of supplying the piezoelectric resonator with the sample solution containing the immunoglobulin which is a substance to be sensed to make the base layer adsorb the immunoglobulin;
a first measuring step of oscillating the piezoelectric resonator which has adsorbed the immunoglobulin and measuring a frequency corresponding to an oscillation frequency of the oscillator circuit;
a step, which follows the first measuring step, of supplying the piezoelectric resonator with a liquid containing an antigen captured by the immunoglobulin;
a second measuring step of oscillating the piezoelectric resonator which has adsorbed the antigen and measuring a frequency corresponding to an oscillation frequency of the oscillator circuit;
a step of finding a difference between the frequencies obtained in the first measuring step and the second measuring step respectively; and
a step, which follows the step of finding the difference, of supplying the acid liquid to the piezoelectric resonator to make the base layer desorb the immunoglobulin.
The sensing method may further include a step of supplying a liquid containing the immunoglobulin to the piezoelectric resonator which has adsorbed the antigen to make the antigen adsorb the immunoglobulin, so as to sandwich the antigen between the immunoglobulin and the immunoglobulin which has already adsorbed the antigen, wherein
the second measuring step may be a step of measuring the frequency while the antigen is sandwiched by the immunoglobulins.
A self-assembled monolayer may be formed between the excitation electrode and the base layer. Further, a blocking substance for preventing the adsorption of the antigen may be adsorbed by a portion, on the excitation electrode, which has not adsorbed the immunoglobulin.
In the present invention, protein which has a property of reacting specifically with an immunoglobulin and desorbing the immunoglobulin when it comes into contact with the acid liquid is attached as the base film on the electrode of the piezoelectric resonator, and the immunoglobulin is made to capture the antigen by an antigen-antibody reaction or the antigen is further made to capture the immunoglobulin. Then, one of the immunoglobulin and the antigen is sensed as a substance to be sensed based on a change in the frequency. Then, the base film is made to desorb the immunoglobulin by the supply of the acid liquid after the measurement of the frequency. Therefore, it is possible to reproduce a new adsorption layer made of the immunoglobulin by repeating a series of the processes, which makes it possible to conduct the measurement a plurality of times with one piezoelectric sensor. As a result, in a work of preparing a calibration curve or in a work of measuring a substance to be sensed by using a calibration curve, it is possible to reduce running cost and to save the trouble of changing piezoelectric sensors every time a sample is measured.
a) to
An embodiment of a sensing instrument according to the present invention will be described by using the drawings.
As shown in
On the excitation electrode 22 on the front surface side (side in contact with a sample solution) provided on the quartz piece 21, an adsorption layer 10 adsorbing a substance to be sensed is formed. A method of forming the adsorption layer 10 will be described by using
Next, for example, an ethanolamine solution is supplied to the self-assembled monolayer 11, so that a blocker 13 as ethanolamine binds to the self-assembled monolayer 11. Further, an immunoglobulin 14 such as IgG which is an antibody is adsorbed by the base layer 12 made of the protein, whereby the adsorption layer 10 made of the immunoglobulin is formed. The immunoglobulin 14 is capable of capturing a substance to be sensed (antigen) 15 in a sample solution. Further, the immunoglobulin 14 has a property of reacting specifically with, for example, the protein A and separating from the protein when brought into contact with an acid liquid.
Next, the sensor unit 7 will be described with reference to
The quartz pressing member 4 is formed in a shape corresponding to the wiring board 3 by using an elastic material, for example, silicon rubber. As shown in
As shown in
Further, as shown in
The support 71 has the recessed portion 72 housing and holding the wiring board 3, and in the recessed portion 72, engagement projections 73 extend in a vertical direction to be engaged with engagement holes 37a, 37b of the wiring board 3 and engagement holes 46a, 46b of the quartz pressing member 4, thereby fixing the positions of the wiring board 3 and the quartz pressing member 4.
In the above, the quartz resonator 2, the wiring board 3, and the sealing member 3A correspond to a piezoelectric sensor of the present invention. The rear surface side of the quartz resonator 2 is exposed to the airtight atmosphere, and therefore, the piezoelectric sensor forms a Languban-typed quartz sensor.
Next, the whole structure of the sensing instrument according to the embodiment of the present invention will be described. The sensing instrument includes the sensor unit 7, an oscillator circuit 50, a measuring circuit part 51, a display device 52, a buffer solution supply part 53, an immunoglobulin-containing liquid supply part 54, a sample solution supply part 55, an acid liquid supply part 58, a supply liquid switching part 63, a waste liquid storage part 56, a pump 62, and a control unit 100.
The oscillator circuit 50 is electrically connected to the electrodes 34, formed in the wiring board 3. The measuring circuit part 51 analog/digital-converts (A/D-converts) a frequency signal sent from the oscillator circuit 50 and applies predetermined signal processing to the resultant signal to measure a frequency of the frequency signal. The oscillator circuit 50 and the measuring circuit part 51 are provided in different casings respectively, and these casings are connected by a cable.
The buffer solution supply part 53, the immunoglobulin-containing liquid supply part 54, the sample solution supply part 55, and the acid liquid supply part 58 are connected to the supply liquid switching part 63 via supply channels 57a, 57b, 57c, 57d respectively. The supply liquid switching part 63 is connected to the liquid supply pipe 88a and has a function of switching a supply channel to be connected to the liquid supply pipe 88a, among the supply channels 57a to 57b. Further, the pump 62 is used to discharge a liquid in the sensor unit 7 to the waste liquid storage part 56 via the liquid discharge pipe 88b and a discharge channel 57e. The supply liquid switching part 63 and the pump 62 are controlled by the control part 100, and the control part 100 outputs a control signal so that a one-cycle operation to be described later is performed based on a computer program.
Next, the operation of the sensing instrument as structured above will be described with reference to
Subsequently, while the buffer solution is supplied to the sensor unit 7, the immunoglobulin-containing liquid supply part 54 supplies an immunoglobulin-containing liquid to the liquid storage part 45 of the sensor unit 7. At this time, the immunoglobulin-containing liquid passes through the liquid storage part 45 and is discharged through the liquid discharge pipe 88b. The immunoglobulin 14 contained in the immunoglobulin-containing liquid is adsorbed by the protein forming the base layer 12 on the excitation electrode 22 of the quartz resonator 2 (
Next, the sample solution is supplied to the liquid storage part 45 of the sensor unit 7. A method of supplying the sample solution is as follows. The supply liquid switching part denoted by reference 63 in
The quartz resonator 2 provided in the sensor unit 7 is oscillated by the oscillator circuit 50 and its oscillation output (frequency signal) is sent to the measuring circuit part 51. The measuring circuit part 51 analog/digital-converts (A/D-converts) the obtained frequency signal and applies the predetermined signal processing to the resultant signal to measure the frequency and also output a measured value of the frequency to the display device 52. As is seen in
Here, in order to find a variation of the natural frequency of the quartz resonator 2 corresponding to the concentration of the antigen 15, it is necessary to find a difference between an oscillation frequency of the quartz resonator 2 when it is put in the buffer solution and an oscillation frequency of the quartz resonator 2 when it is put in the sample solution, but the method of finding the difference between the oscillation frequencies of the quartz resonator 2 is not limited to the measurement of the oscillation frequencies themselves, but the oscillation frequency difference may be found in such a manner that, for example, a difference frequency between an output frequency of the quartz resonator 2 and a predetermined clock frequency is found, this frequency is evaluated as the frequency of the quartz resonator 2, and a difference between the difference frequencies in the both environments is found.
Subsequently, the acid liquid supply part 58 supplies the sensor unit 7 with an acid liquid, for example, glycine. The acid liquid is supplied to the liquid storage part 45 in the sensor unit 7 via the supply channel 57d, the supply liquid switching part 63, and the liquid supply pipe 88a, and is further discharged from the liquid storage part 45 to the waste liquid storage part 56 via the liquid discharge pipe 88b, the pump 62, and the discharge channel 57e. Then, the acid liquid comes into contact with the immunoglobulin 14 which forms the adsorption layer 10 of the quartz resonator 2 and has captured the substance to be sensed, so that the immunoglobulin 14 is separated from the protein 12 (
Thereafter, the base layer 12 made of protein is again made to adsorb the immunoglobulin 14 in the above-described manner, whereby the adsorption layer 10 is formed, and then the frequency is measured while the sample solution is supplied to the piezoelectric sensor in the same manner. In this example, the process of supplying only the buffer solution at the time of the switching of the liquids is provided in a series of the processes, so that the previous liquid is pushed out from the inside of the piezoelectric sensor and in this state, the next liquid is supplied into the piezoelectric sensor. A cycle from the supply of the immunoglobulin 14 to the supply of the sample solution, and then to the supply of the acid liquid is automatically performed by, for example, the control unit 100, but the sample solution is changed by a worker.
Further, in the one example, the sample solution supply part 55 is changed every cycle, that is, a container for use is changed among a plurality of containers containing sample solutions different in the concentration of the antigen 15, but another possible structure may be to provide a flow rate regulating part and a flowmeter in each of the supply channels and adjust the concentration of the sample solution supplied into the piezoelectric sensor by adjusting a flow rate ratio of the buffer solution and the sample solution. By thus changing the concentration of the antigen 15 in the sample solution from cycle to cycle and repeating this cycle, it is possible to find the correlation between the concentration of the antigen 15 as the substance to be sensed in the sample solution and the aforesaid frequency difference (difference between the frequency of the quartz resonator 2 corresponding to the buffer solution and the frequency of the quartz resonator 2 corresponding to the sample solution), that is, it is possible to prepare the calibration curve. Such a calibration curve can be used when a user measures the concentration of the antigen 15 in the sample solution.
In the above-described embodiment, by utilizing the property that the immunoglobulin 14 separates from specific protein when coming into contact with the acid liquid, the immunoglobulin 14 is adsorbed by the base layer 12 made of protein which is formed in advance on the excitation electrode, and after the immunoglobulin 14 captures the antigen 15 as the substance to be sensed, the immunoglobulin 14 is brought into contact with the acid liquid to be separated from the base layer 12. Therefore, by repeating a series of these processes, the old adsorption layer 10 which has reacted with the antigen 15 is separated and a new adsorption layer 10 is reproduced.
As a result, it is possible to successively measure samples by using one piezoelectric sensor, which makes it possible not only to save the trouble of changing the piezoelectric sensor every time the measurement is conducted but also to reduce running cost.
The above-described example is an example where the present invention is applied to the preparation of the calibration curve showing the correspondence between the concentration of the antigen 15 and a variation of the frequency of the quartz resonator 2, but another possible example is to find a variation of the frequency of the quartz resonator 2 and detect the concentration of the antigen 15 in the sample solution based on the variation and the calibration curve prepared in advance.
Further, a method to be described next may be adopted as another embodiment. After the base layer 12 adsorbs the immunoglobulin 14, the frequency is measured in the state where the environment in which the quartz resonator 2 is put is replaced by the buffer solution. Thereafter, the sample solution containing the antigen 15 is supplied to the quartz resonator 2 so that the antigen 15 is captured by the immunoglobulin 14 adsorbed by the base layer 12. Next, the liquid containing the immunoglobulin 14 is supplied to the quartz resonator 2 so that the captured antigen 15 adsorbs the immunoglobulin 14 (
Alternatively, the substance to be sensed may be the immunoglobulin 14 instead of the antigen 15. Specifically, if an adsorption amount of the immunoglobulin 14 adsorbed by the base layer 12 is constant, a variation of the oscillation frequency of the quartz resonator 2 depends on the concentration of the antigen 15 in the sample solution, but if conversely the concentration of the antigen 15 in a liquid containing the antigen 15 (corresponding to the sample solution in the above-described example) is constant, a variation of the oscillation frequency of the quartz resonator 2 depends on an adsorption amount of the immunoglobulin 14 adsorbed by the base layer 12. That is, the larger the adsorption amount of the immunoglobulin 14, the larger a captured amount of the antigen 15, and the smaller the adsorption amount of the immunoglobulin 14, the smaller the captured amount of the antigen 15. The adsorption amount of the immunoglobulin 14 corresponds to the concentration of the immunoglobulin 14 in the immunoglobulin-containing liquid, and the specific antigen 15 is captured by the specific immunoglobulin 14. Consequently, it is possible to find the concentration of the specific immunoglobulin 14 in the liquid containing the immunoglobulin 14, by finding a frequency change of the quartz resonator corresponding to the amount of the antigen 15 captured by the immunoglobulin 14.
Therefore, the sample solution in this case is the immunoglobulin-containing liquid. In the case where the immunoglobulin 14 is the substance to be sensed, by using various sample solutions different in concentration of the immunoglobulin 14 and finding a difference between the oscillation frequency of the quartz resonator 2 when it is put in the buffer solution and the oscillation frequency of the quartz resonator 2 when it is put in the liquid containing the antigen 15, it is also possible to prepare a calibration curve showing the correspondence between the difference and the concentration of the immunoglobulin 14. Alternatively, by finding the aforesaid frequency difference using a sample solution whose concentration of the immunoglobulin 14 is not known, it is possible to know the concentration of the specific immunoglobulin 14 capturing the antigen 15 based on the frequency difference and the calibration curve prepared in advance.
The above-described instrument is also usable in the case where the immunoglobulin 14 is thus the substance to be sensed. For example, when the calibration curve is prepared, the concentration of the antigen 15 is set constant instead of changing the concentration of the antigen 15 at each cycle in a series of the above-described processes, and the concentration of the immunoglobulin 14 in the immunoglobulin-containing liquid is changed.
Further, the structure described below may be adopted besides the above-described embodiments. The liquid containing the immunoglobulin 14 is used as the sample solution, and after the base layer 12 adsorbs the immunoglobulin 14, the frequency corresponding to the oscillation frequency of the oscillator circuit is obtained in the state where the environment in which the quartz resonator 2 is put is replaced by the buffer solution. Thereafter, a liquid containing the antigen 15 with constant concentration is supplied to the quartz resonator 2 so that the antigen 15 is captured by the immunoglobulin 14. Next, the sample solution containing the immunoglobulin 14 is supplied so that the immunoglobulin 14 is adsorbed by the captured antigen 15, and thereafter the frequency is obtained. By finding a difference between the frequency obtained in the buffer solution environment and this obtained frequency, it is possible to prepare the calibration curve showing the correlation between the concentration of the immunoglobulin 14 in the sample solution and the frequency difference. When the captured antigen 15 further adsorbs the immunoglobulin 14, a variation of the frequency of the quartz resonator 2 corresponding to the adsorption amount of the antigen 15 becomes large, so that an accurate calibration curve can be prepared. In this case, the liquid which is added in order to cause the antigen 15 captured by the immunoglobulin to further adsorb the immunoglobulin may be a liquid whose concentration of the immunoglobulin is known in advance, instead of the sample solution.
As described above, in the present invention, it is possible to find the concentration of the antigen 15 and the concentration of the immunoglobulin 14, and therefore, the present invention is an art effective for a blood test or the like, for instance (the sample solution is blood).
Number | Date | Country | Kind |
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2008-131262 | May 2008 | JP | national |