JP2007057289A - Microsensor for analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microsensor for analysis which forms a capturing means on only either of two electrodes only by allowing medical fluid to flow in a structure formed by bonding a piezoelectric vibrator onto a fluid, and performing highly accurate measurement even when a reference vibrator and a detection vibrator are arranged close to each other. <P>SOLUTION: A groove is provided on the piezoelectric substrate surface constituting one side face of the passage, and a counter electrode is provided on the counter surface. A detection electrode is provided on the upper surface of the groove, and a reference electrode is provided on the bottom surface, and the side wall surface of the groove is constituted so as to be hydrophobic. When water-soluble medical fluid is sent into the passage, bubbles remain in the groove, and only the detection electrode is dipped into the medical fluid. Hereby, an adsorption film for capturing a specific material onto the detection electrode surface can be formed only by a process for sending the medical fluid, and simultaneously a fluctuation caused by a temperature change is detected from a signal of the piezoelectric vibrator measured by switching between the reference electrode and the detection electrode, and thereby even if the ambient temperature is changed, the mass of the specific material adhering onto the detection electrode is measured highly accurately. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microsensor for analysis using QCM (Quartz Crystal Microbalance), and more specifically, capture means for capturing a specific substance only in one electrode with respect to two electrodes provided in the vicinity of the same channel. It is possible to compensate for fluctuations due to changes in ambient temperature, and to belong to an analytical microsensor that detects the mass of a specific substance contained in a liquid with high accuracy.

  In recent years, a small analysis system called Lab-on-a-Chip has been actively developed. This is a configuration in which element structures such as a flow path, a reaction vessel, a valve, and a sensor are integrated on a small substrate, and an analysis process is performed on a gas or a liquid flowing inside this. When such a small analysis system is used, analysis can be performed at a high speed with a small amount of sample, and the burden on the side of providing the sample can be reduced.

  Sensors using various principles have been proposed for the sensor unit which is one of the components of the small analysis system. In particular, the use of QCM (Quartz Crystal Microbalance), which can be integrated with a substrate mounted on the system and has a small configuration, is expected.

  QCM is a technique for measuring a minute mass attached to a vibrator using a resonance phenomenon of a piezoelectric vibrator (particularly a quartz vibrator). More specifically, when an AC voltage is applied to the electrodes formed on both sides of the piezoelectric vibrator, resonance occurs at a specific frequency determined from the material characteristics and shape of the piezoelectric vibrator. Therefore, when a substance adheres to the electrode of the piezoelectric vibrator, the resonance frequency of the whole vibrator changes according to the attached mass. This is a technique for measuring the mass of a substance attached to an electrode by detecting a change in the resonance frequency.

  However, since such a mass measuring means cannot detect a specific substance, a configuration in which a means for adsorbing or capturing only a specific substance is fixed on an electrode and only the specific substance is detected is used. As an example, a technique using an antigen-antibody reaction for protein detection is known (see Patent Document 1). When the QCM having such a configuration is used, a minute mass of a specific measurement target substance can be measured. Therefore, if QCM is used for the sensor part of a small analysis system, the mass of the substance to be measured can be measured with high accuracy, and as described above, the structure can be integrated with the analysis system, and the small size can be obtained. The configuration can be maintained.

  However, while the QCM can detect with high sensitivity, there is a problem that the resonance frequency fluctuates due to changes in the temperature of the fluid in contact with the piezoelectric vibrator. Therefore, a piezoelectric vibrator not provided with a means for capturing the substance to be detected is installed adjacent to the piezoelectric vibrator used for detection, and the attached mass using the resonance frequency of the piezoelectric vibrator without the attachment means. A compensation method has been used in which only the change in resonance frequency due to the above is taken out (see Non-Patent Document 1).

However, since a small analysis system such as Lab-on-a-Chip analyzes a very small amount of sample in a microchannel formed on a substrate, a chemical reaction used for analysis (for example, adsorption provided on an electrode). Or the coupling between the capture means and the substance to be measured) occurs rapidly and locally. This requires local measurement and real-time compensation. In order to realize this by the above QCM compensation method, it is necessary to place the reference vibrator and the detection vibrator close to each other and simultaneously vibrate the reference vibrator and the detection vibrator to perform measurement and compensation simultaneously. There is also a method of connecting a piezoelectric vibrator having both an electrode (detection electrode) having means for capturing a detection target substance and an electrode (reference electrode) not having a capture means to the flow path (see Patent Document 2). Proposed.
JP 2000-338022 A Japanese Patent Laid-Open No. 2003-307481 J. et al. Aug et al. , Sensors and Actuators B, 26-27 (1995) 181-186.

  However, when such a method is adopted, there has been a problem that the detection sensitivity is lowered due to contamination of the detection electrode and the reference electrode due to heating at the time of bonding or use of an adhesive. In addition, it is very difficult to selectively provide the capture means only on one of the two adjacent electrodes only by the step of flowing the chemical solution in the flow channel after joining the vibrator and the flow channel. Further, when the two vibrators are arranged close to each other, there is a problem that electro-mechanical coupling occurs between the reference vibrator and the detection vibrator, noise due to unintended vibration occurs, and measurement accuracy is lowered.

  In view of this, the present invention allows a chemical solution to flow through a structure in which a piezoelectric vibrator is joined to a flow path, and forms a capturing means only on one electrode, and at the same time, arranges a reference vibrator and a detection vibrator in close proximity. Another object of the present invention is to provide an analytical microsensor capable of measuring with high accuracy.

  The present invention has a capture means for capturing a specific substance contained in a sample flowing in a flow channel on a detection electrode, and in an analysis microsensor for measuring a mass captured by the capture means, A piezoelectric substrate having a first surface provided with a groove-shaped convex / concave portion having a hydrophobic side wall surface and having a counter electrode on a second surface opposite to the first surface, and the concave / convex A detection vibrator is constituted by the detection electrode provided at the top of the part, and the detection vibrator has a resonance characteristic by the piezoelectric substrate and a reference electrode provided at the bottom of the convex recess. A piezoelectric vibrator having a function of constituting a different reference vibrator, a switching section for switching connection to the detection vibrator and the reference vibrator, and the detection vibrator obtained by switching by the switching section. Resonance frequency and previous The above-mentioned problem has been solved by providing an analysis microsensor characterized by comprising: a measuring means for measuring the mass captured by the capturing means based on a difference value from a resonance frequency of a reference vibrator Is.

  According to the microsensor for analysis of the present invention, the capture means can be formed on one of the electrodes only by the process of flowing the chemical solution in the flow path, and the surrounding temperature change can be compensated for, even though it is a simple configuration. Measurement becomes possible. Furthermore, even if the reference vibrator and the detection vibrator are arranged close to each other, electro-mechanical coupling does not occur between the vibrators, and high-precision measurement is possible.

  Hereinafter, the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited by the following embodiment.

  FIG. 1 is a diagram illustrating the configuration of an analytical microsensor 1000 according to the present invention. FIG. 2 is a view for explaining the structure of the piezoelectric substrate 100 constituting the analysis microsensor 1000.

  The analysis microsensor 1000 is configured by laminating the piezoelectric substrate 100 and the flow path substrate 200 and integrally bonding or adhering them. The external dimensions of the analytical microsensor 1000 are about 20 to 100 [mm] in length, about 10 to 20 [mm] in width, and about several [mm] in thickness.

  First, the piezoelectric substrate 100 will be described. The piezoelectric substrate 100 is provided with a comb-shaped groove 101 having a plurality of grooves connected at one end. A reference electrode 602 is provided on the bottom surface of the groove portion 101, a detection electrode 601 is provided on the upper surface of the groove portion 101, and a hydrophobic surface is formed on the side wall surface of the groove portion 101. Further, a counter electrode 603 is provided on a surface facing the surface on which the groove portion 101 is provided. Further, an adsorption film 609 that adsorbs only a specific substance is provided on the surface of the detection electrode 601.

  The channel substrate 200 is provided with a liquid inlet 202 and a liquid outlet 203 which are grooves and through holes extending to the grooves. In addition, a part of the groove has a wide structure.

  The piezoelectric substrate 100 and the flow path substrate 200 are integrated to form an analysis microsensor 1000. Specifically, the surface of the piezoelectric substrate 100 provided with the groove 101 and the surface of the flow path substrate 200 provided with the groove are joined or bonded to be integrated. The flow path 400 is formed between the groove and the surface of the piezoelectric substrate 100, and the region where the width of the groove is further expanded becomes the reaction tank 500, and the detection electrode 601 and the reference electrode 602 are provided in the reaction tank 500. Become. Therefore, when a liquid flows in from the liquid introduction port 202, a flow that reaches the liquid discharge port 203 through the flow path 400 and the reaction tank 500 is generated.

  When attention is paid to the surface of the piezoelectric substrate 100 in the channel 400, the side wall surface in the groove 101 is hydrophobic. Therefore, when an aqueous solution (sample) containing a specific substance is flowed, bubbles remain in the groove 101 (FIG. 3). Therefore, only the detection electrode 601 among the three electrodes provided on the piezoelectric substrate 100 is immersed in the sample.

  Here, an example of an electrical configuration connected to the detection electrode 601, the reference electrode 602, and the counter electrode 603 provided on the piezoelectric substrate 100 is shown. The detection electrode 601 and the changeover switch 704 are connected, and one electrode of the changeover switch 704 and the variable frequency AC power supply 701 is connected. A counter electrode 603 is connected to the other electrode of the AC power source 701. A reference electrode 602 is connected to the other side of the changeover switch 704. Further, an ammeter 702 is connected in series between the AC electrode 701 and the counter electrode 603. Calculation means 703 is connected to each of AC power supply 701 and ammeter 702, and AC power supply 701, ammeter 702, and calculation means 703 constitute measurement means 710 (see FIG. 4A).

  Next, a method for measuring the mass of a substance attached to the detection electrode 601 from these electrical configurations will be described. When an AC voltage is applied from the AC power source 701 to the detection electrode 601 and the counter electrode 603 via the changeover switch 704, a detection vibrator is formed between the detection electrode 601 and the counter electrode 603. The current flowing through the ammeter 702 changes according to the frequency of the applied voltage. Information on the voltage and frequency of the applied voltage from the AC power source 701 and information on the current flowing from the ammeter 702 are input to the computing means 703. The calculation means 703 records the input information, and further calculates and records the impedance of the vibrator, the phase difference between voltage and current, and the like from the input information. The calculation means 703 calculates and records the voltage and frequency of the applied voltage, the amplitude value of the current, the phase difference between the voltage and current, the impedance of the vibrator, and the like. The relationship between the frequency of the applied voltage and the amplitude of the current is shown by the solid line in FIG. The current flowing in the current becomes maximum at the frequency f1 in the figure, and this is the resonance frequency. After obtaining the resonance frequency f1, the changeover switch 704 is switched, an AC voltage is applied to the reference electrode 602 and the counter electrode 603, a reference vibrator is configured, and the resonance frequency f2 at which the current becomes the maximum value is obtained in the same procedure. Obtained (see broken line in FIG. 4B).

  Here, as an example, an AT-cut quartz plate is used for the piezoelectric substrate 100. The AT-cut quartz plate has a property that thickness-shear vibration is generated when a potential difference is periodically provided in the thickness direction. The resonance frequency (fn) at this time is determined by the elastic constant and density of the vibrator and the distance (t) between the electrodes providing the potential difference. In the case of an AT cut crystal vibrator, fn = 1670 / t. Therefore, the resonance frequency f2 of the reference vibrator having a small distance between the electrodes is larger than the resonance frequency f1 of the detection vibrator. Therefore, even if they are arranged close to each other, the detection vibrator and the reference vibrator are not electro-mechanically coupled.

  At this time, when the measurement target substance adheres to the detection electrode 601, the resonance frequency f1 of the detection vibrator changes according to the attached mass. However, the resonance frequency changes similarly when the temperature of the vibrator itself changes. Therefore, it cannot be determined whether the change in the resonance frequency is caused only by the attached mass or the change caused by the temperature change is included. Therefore, the fluctuation of the resonance frequency f2 of the reference vibrator is used. Since the measurement target substance does not adhere to the reference electrode, the fluctuation of f2 is caused only by the temperature change. Therefore, if the temperature change is obtained from the fluctuation of f2, and the fluctuation of the resonance frequency f1 due to the temperature change is subtracted, the fluctuation of f1 due to the adhesion mass can be obtained, and the adhesion mass can be obtained accurately.

  As described above, by measuring fluctuations in the two resonance frequencies of the detection vibrator and the reference vibrator, the mass of the substance attached to the detection electrode 601 can be measured with high accuracy even when the ambient temperature changes. Is possible.

  Next, a method for manufacturing the analytical microsensor 1000 of the present invention will be described. First, the AT cut crystal wafer is etched with hydrogen fluoride to form the groove 101. By immersing the wafer in an HMDS (hexamethyldisilazane) atmosphere and then performing a heat treatment, the wafer surface is replaced with a hydrocarbon group and becomes hydrophobic. Thereafter, titanium and gold are vapor-deposited in this order on both surfaces of the wafer, and a hydrophobic surface is formed on the detection electrode 601, the reference electrode 602, the counter electrode 603, wiring to each electrode, and the side wall of the groove 101. In addition, since the side wall of the groove 101 exists, the detection electrode 601 and the reference electrode 602 are not short-circuited. Next, after etching one surface of the silicon wafer to form a groove, etching is performed from the other surface to form a hole penetrating to the groove. Thereafter, a UV curing adhesive is applied to the piezoelectric substrate, the piezoelectric substrate and the silicon wafer are overlapped, irradiated with ultraviolet rays, bonded, and the wafer is cut by dicing.

  Although an example of the manufacturing process has been described here, the flow path substrate material is not limited to silicon, and the piezoelectric substrate 100 is not limited to quartz. The channel substrate 200 can be made of an inorganic material such as glass or a resin material such as polyimide or polydimethylsiloxane (PDMA). The piezoelectric substrate 100 can also use a single crystal piezoelectric material such as lithium tantalate or langasite. Furthermore, the quartz wafer and the substrate on which the groove is formed can be integrated to be integrated. For example, an aluminum thin film may be formed at a portion to be bonded to a crystal wafer, and anodic bonding may be performed with a glass substrate having grooves.

  Next, a method for forming the adsorption film 609 installed on the detection electrode 601 will be described. Here, as an example, a method for producing an adsorption film 609 in which an antibody is immobilized on a self-assembled film (hereinafter referred to as SAM) is shown. First, pure water is flowed to clean the inside of the flow path 400, and then a SAM reagent (carboxyl terminal disulfide type) is flowed to form SAM only on the detection electrode 601. After washing with a phosphate buffer, hydroxysuccinimide is run to activate SAM. After washing with a phosphate buffer again, the antibody to be immobilized on the phosphate buffer is mixed and flowed to immobilize the antibody on the SAM. Even if any reagent is allowed to flow, bubbles remain in the groove portion 101 of the piezoelectric substrate, so that the adsorption film 609 is not formed on the reference electrode 602 but only on the detection electrode 601.

  A process of detecting only a specific substance from a sample flowing in the analysis microsensor 1000 will be described. Here, detection of biopolymers, particularly proteins, will be described as an example.

  A sample flows into the liquid inlet 202 of the analysis microsensor 1000. The sample reaches the reaction tank 500 from the liquid inlet 202 through the flow path 400. When the reaction tank 500 is filled with the sample, bubbles remain in the groove 101, and the adsorption film 609 provided on the surface of the detection electrode 601 is immersed in the sample. At this time, the antibody of the adsorption film 609 captures and fixes a specific antigen contained in the sample, and the mass attached to the detection electrode 601 increases, so that the resonance frequency f1 of the detection vibrator changes. At this time, after the measuring unit 710 measures the fluctuation of the resonance frequency f1, the temperature change is measured from the fluctuation of the resonance frequency f2 of the reference vibrator, and only the fluctuation of the attached mass is calculated and stored. The measurement unit 710 repeatedly stores the measurement and calculation processing at high speed.

  As described above, although it is a simple configuration, the capturing means can be formed on one of the electrodes and the surrounding temperature change can be compensated only by the step of flowing the chemical solution in the flow path, so that highly accurate measurement is possible. Furthermore, even if the reference vibrator and the detection vibrator are arranged close to each other, electro-mechanical coupling does not occur between the vibrators, and high-precision measurement is possible.

1 is a structural diagram schematically showing the structure of an analytical microsensor of the present invention. It is the elements on larger scale for demonstrating the structure of the microsensor for analysis of this invention. It is explanatory drawing for demonstrating the flow of the sample which touches a piezoelectric substrate. It is a block diagram for demonstrating the electrical structure of the micro sensor for analysis of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Piezoelectric substrate 101 Groove part 200 Flow path board 202 Liquid introduction port 203 Liquid discharge port 400 Flow path 500 Reaction tank 601 Detection electrode 602 Reference electrode 603 Counter electrode 609 Adsorption film 701 AC power supply 702 Ammeter 703 Calculation means 704 Changeover switch 710 Measurement means 1000 Microsensor for analysis

Claims (6)

In the analysis microsensor for measuring the mass captured by the capture means having a capture means for capturing the specific substance contained in the sample flowing in the flow channel on the detection electrode,
The first surface is provided in the flow path, and the side wall surface is formed with a groove-shaped convex / concave portion having hydrophobicity, and has a counter electrode on a second surface opposite to the first surface. A detection vibrator is constituted by the piezoelectric substrate and the detection electrode provided on the top of the convex recess, and the detection vibrator is constituted by the piezoelectric substrate and a reference electrode provided on the bottom of the convex recess. A piezoelectric vibrator having a function of constituting a reference vibrator having different resonance characteristics;
A switching unit that switches connection between the detection vibrator and the reference vibrator;
A measuring unit that measures the mass captured by the capturing unit based on a difference value between a resonance frequency of the detection vibrator and a resonance frequency of the reference vibrator obtained by switching by the switching unit;
An analytical microsensor characterized by comprising:   The analysis microsensor according to claim 1, wherein the first surface has the plurality of groove-shaped convex concave portions provided in parallel.   The analysis microsensor according to claim 1, wherein the detection electrode is gold.   The analysis microsensor according to any one of claims 1 to 3, wherein a longitudinal direction of the groove-shaped concave portion forming the convex concave portion is arranged perpendicular to a direction in which the sample flows.   5. The analytical microsensor according to claim 1, wherein the capturing means is a self-assembled film and an antibody fixed to the self-assembled film.   6. The analytical microsensor according to claim 1, wherein the piezoelectric substrate is made of a single crystal of quartz, lithium tantalate, or ligasite.

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