JP2005331443A - Flow cell type qcm sensor and measuring method using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable highly precise stable measurement by increasing a reaction speed and avoiding the interference of a resonance frequency. <P>SOLUTION: In this flow cell type QCM sensor, a sample solution is allowed to flow along the surface of a electrode 12 of a quartz vibrator 11 and the component of a sample is detected and determinated from a change in the resonance frequency of the quartz vibrator, a measuring instrument 20 measures the resonance frequency of the quartz vibrator. A base line measuring instrument 30 obtains the detection/determination measuring output of the component of the sample from the change quantities of both of the measured value f<SB>1</SB>of the measuring instrument 20 immediately before the start of measurement and the measured value f<SB>2</SB>after the sufficient reaction of the sample solution. A flow channel is made lower than the height causing the interference of resonance frequency caused by the longitudinal wave component propagating through the sample solution to increase the reaction speed of the sample solution. Gas with a sufficient low density and viscosity as compared with the sample solution is injected in the flow channel by a three-way valve 15, The flow channel is made of a PDMS resin or an acrylic resin having high hydrophilicity with respect to the sample solution. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a QCM (Quarz Crystal Microbalance) sensor for detecting and quantifying a component of a sample from a change in a resonance frequency or a change in impedance of the crystal unit when an electrode surface of the crystal unit is exposed to a sample gas or a sample solution. In particular, the present invention relates to a flow cell type QCM sensor and a measurement method for increasing the response speed of component detection / quantification.

  In the fields of chemistry, biochemistry and electrochemistry, it is important to quantify the amount of reaction and the amount of product, but it was difficult to obtain sufficient detection sensitivity for very small amounts of reaction with conventional devices. .

  In recent years, chemical and biosensors that apply the microbalance principle using AT-cut quartz resonators have attracted attention. AT-cut quartz resonators exhibit a phenomenon in which the main resonance frequency is inversely proportional to the thickness of the resonator, and when a sample component is formed on the electrode surface or when adsorption of the material occurs, the unit of the material present on the surface A frequency shift corresponding to the weight per plane area occurs.

  The QCM sensor is an application of the frequency shift phenomenon described above. Since the AT-cut quartz resonator has a stable frequency over a wide temperature range, a stable detection sensitivity can be expected. Substance detection is possible in real time. Equation (1) shows the relationship between the amount of adsorbed material and the amount of frequency shift.

Here, ΔF: Resonance frequency change amount, F: Resonance frequency, Δt: Quartz plate thickness change amount, t: Quartz plate thickness, Δm: Adsorbed substance amount, ρ _q : Quartz density, μ: Quartz elastic modulus , A: electrode area.

  As can be seen from the above equation (1), among the substances adsorbed on the crystal resonator, only the electrodes of the crystal resonator can be detected and quantified, so the receptor corresponding to the component to be detected and quantified is an electrode. It will be formed on the surface.

  In actual measurement, when the sample to be detected and quantified is dispersed in the solution, the above-described receptor-mounted crystal resonator is installed in the cell configuration shown in FIG. 5 or FIG. However, these cell configurations have the following problems.

  1) In the stationary solution type cell shown in FIG. 5, since the binding between the receptor and the sample to be detected / quantified is limited by the diffusivity of the sample in the solution, the reaction rate is slow.

  2) The sample solution forced stirring type cell shown in FIG. 6 has an effect of suppressing the diffusion rate limiting of the sample solution by stirring, but the sample component in the solution does not always reach the crystal resonator electrode as the receptor. Absent. In particular, it is considered that the stirring effect decreases as the sample components in the solution become dilute.

As a method for increasing the reaction rate, a flow cell type has been proposed (see, for example, Patent Document 1 and Patent Document 2). The flow cell type measuring apparatus according to these patent documents has a structure in which a flow cell is provided that forms a flow path through which a solution flows into and out of the crystal resonator. Patent Document 1 proposes a flow cell structure in which the width, depth, and length of the flow path are defined in order to prevent the flow of the solution on the quartz resonator from being disturbed.
Japanese Patent No. 2905064 JP-A-11-183479

  When the flow cell structure is applied to a QCM sensor, the reaction rate can be increased as compared with a conventional solution forced stirring type cell or the like.

  FIG. 7 shows a configuration example of a flow cell type QCM sensor, and FIG. 8 shows an example of measurement of antigen-antibody reaction rate by this QCM sensor. As a comparison, FIG. 9 shows a measurement example using a solution forced stirring type cell.

  For this measurement, CRP (C-reactive protein) is used as a detection substance, the forced stirring frame type is set to 900 rpm, and each concentration shown in the figure is added to a 9 mL buffer solution. The minimum sample concentration was determined. In the flow cell type, a sample solution adjusted to a concentration of 1.185 ng / mL in advance in a flow path having a width of 4.4 mm and a height of 1.5 mm was reacted under the condition of a flow rate of 400 μL / min.

  As is clear from these measurement examples, the reaction time can be shortened and a low-concentration sample can be detected by using the flow cell structure.

  However, when QCM sensors are deployed in a flow cell structure, as reported by Zuxuan Lin (Anal. Chem. 1995, 67, 685-693) et al. There is a problem of interference of the resonance frequency due to reflection from the flow path wall.

  Resonance frequency interference results from the propagation speed and flow path height of the solution used. As the verification, the influence of the reflected wave was confirmed from the change in the liquid level using the stationary solution type QCM sensor of FIG. FIG. 10 shows the measurement result of the resonance frequency by evaporation of 100% ethanol, and FIG. 11 shows the measurement result of FIG. 10 plotted with the amount of change of the resonance frequency with respect to the liquid level.

  The measurement result in FIG. 10 shows the dependence of the interference frequency on the change in the solution height, and even if the solution height (equal channel height) is constant, the resonance frequency of the crystal resonator changes. This suggests that the interference state changes.

  In FIG. 11, it is suggested that the QCM surface and the flow cell wall need to be higher than a certain height so as not to be affected by the reflected wave propagating through the solution. In the case of FIG. 11, the liquid surface height not affected by the reflected wave propagating through the solution is 0.9 mm or more. However, raising the solution flow path increases the volume of the flow path, resulting in a new problem that the reaction rate becomes slow.

  10 and 11, the liquid level height is the case where 100% ethanol is used as the measurement solution, and the liquid level height at which the influence of the reflected wave can be ignored varies depending on the solution.

  From the above, in the QCM sensor adopting the flow cell structure, it is necessary to increase the flow path to a position where the influence of the reflected wave can be ignored for each sample solution. However, as the flow path becomes higher, the flow volume of the sample solution increases. Therefore, there is a problem that the reaction rate decreases.

  An object of the present invention is to provide a flow cell type QCM sensor capable of increasing the reaction speed and avoiding interference at a resonance frequency and performing highly accurate and stable measurement, and a measurement method using this QCM sensor.

  In order to solve the above-mentioned problems, the present invention lowers the flow path height of the sample solution to increase the reaction speed, and accordingly, an interference phenomenon occurs in the resonance frequency and impedance of the QCM sensor, resulting in a decrease in measurement accuracy. In order to avoid stabilization, the sample solution is detected and quantitatively measured by the baseline measurement immediately before the start of measurement and the baseline measurement after the sample solution has sufficiently reacted. Features the device.

(Invention of method)
(1) In a flow cell type QCM sensor that detects and quantifies a sample component from a change in the resonance frequency or impedance of the crystal resonator caused by adsorption of a component in the sample solution through a flow path formed on the crystal resonator. ,
Performs a baseline measurement of the resonance frequency or impedance of the crystal unit immediately before the start of measurement and a baseline measurement of the resonance frequency or impedance of the crystal unit after the sample solution has sufficiently reacted, and crystal oscillation by both baseline measurements. It is characterized by detecting and quantitatively measuring sample components from changes in the resonance frequency or impedance of the child.

  (2) The baseline measurement is performed by setting the flow path height to be lower than the height at which interference of the resonance frequency or impedance caused by the longitudinal wave component propagating through the sample solution occurs to increase the reaction speed of the sample solution. And

  (3) The baseline measurement is performed by injecting a gas having sufficiently lower density and viscosity than the sample solution into the flow path.

(Invention of the device)
(4) A flow cell type QCM sensor that allows a sample solution to flow through a channel formed on a crystal resonator, and detects and quantifies the components of the sample from changes in the resonance frequency or impedance of the crystal resonator due to adsorption of components in the sample solution. In
A measuring instrument for measuring the resonance frequency or impedance of the crystal unit;
A baseline measuring device that obtains sample component detection / quantitative measurement output from the amount of change between the measured value of the measuring device immediately before the start of measurement and the measured value of the measuring device after the sample solution has sufficiently reacted It is characterized by that.

  (5) The flow path is structured to have a height that increases the reaction speed of the sample solution by lowering the resonance frequency or impedance interference caused by the longitudinal wave component propagating through the sample solution. And

  (6) A gas injection means for injecting a gas having a density and viscosity sufficiently lower than that of the sample solution into the channel is provided.

  (7) The flow path is made of PDMS resin or acrylic resin having high hydrophilicity with respect to the sample solution.

  As described above, according to the present invention, since the sample solution is detected and quantitatively measured by the baseline measurement immediately before the start of measurement and the baseline measurement after the sample solution has sufficiently reacted, the resonance frequency and impedance of the QCM sensor are adjusted. It is possible to prevent the measurement accuracy from being lowered or unstable due to an interference phenomenon.

  In addition, the flow rate of the sample solution can be lowered to increase the reaction rate.

  Furthermore, by performing baseline measurements before and after the reaction in the gas phase, frequency interference due to the longitudinal wave component propagating in the solution can be ignored, so the flow cell channel height can be further reduced, and the reaction The speed can be further improved and the measurement time can be shortened. Further, since the reaction amount is measured in the gas phase, the Q value of the crystal resonator can be made high, and highly sensitive measurement is possible.

  Further, since the flow path is made of a hydrophilic material, a smooth flow of the sample solution can be obtained, and interference can be further reduced.

FIG. 1 is an apparatus configuration diagram of a QCM sensor showing an embodiment of the present invention. The QCM sensor main body 10 has a flow cell type structure as in FIG. In this flow cell type structure, the electrode 12 and its lead wire 13 are formed on one surface of the quartz substrate 11, the electrode 12 'and its lead wire 13' are formed on the other surface, and the electrode 12 side is inside the flow cell container 14. Exposed to. The sample solution injected from the three-way valve 15 through the injection tube 16 flows into the flow cell container 14 through the surface of the electrode 12 and is discharged from the discharge tube 17. The three-way valve 15 switches between the sample solution and the nitrogen gas (N ₂ ) so that it can be injected into the flow cell container 14.

  Here, the flow path height of the sample solution (the height of the inner wall of the flow cell container 14) is sufficiently low in order to increase the reaction rate between the sample solution and the receptor at the electrode 12. In this case, as described above, the problem of the interference of the resonance frequency due to the longitudinal wave component that is the vibration component of the crystal resonator propagates in the solution and is reflected from the flow path wall remains, but the measurement accuracy decreases due to this interference. The problem of instability is solved by the following measurement method and QCM sensor configuration.

(1) Baseline measurement The resonance frequency measuring device 20 measures the resonance frequency of the crystal resonators (electrodes 12 and 12 ′ and the crystal substrate 11) of the QCM sensor body 10. The configuration and measuring method of the measuring instrument 20 are the same as those in the prior art, and detailed description thereof is omitted.

The baseline measuring device 30 receives the resonance frequency signal measured by the resonance frequency measuring device 20 as an input. As shown in the measurement flow in FIG. 2, the baseline measuring device 30 is immediately before injecting the sample solution into the QCM sensor main body 10 (before the reaction between the receptor on the electrode 12 and the sample solution), that is, nitrogen in the flow cell container 14. A resonance frequency signal in an atmosphere in which gas (N ₂ ) is injected is obtained and its value f ₁ is stored. Thereafter, the sample solution is injected into the flow cell container 14 and the receptor and the sample solution react sufficiently. After a certain period of time required, solution injection is stopped and nitrogen gas (N ₂ ) is reinjected to obtain a resonance frequency signal in an atmosphere in which the inside of the flow cell container 14 is replaced with nitrogen gas, and the value f ₂ is stored. Based on the difference Δf between the resonance frequencies f ₁ and f ₂ , a measurement output of the adsorbed substance amount Δm is obtained from the above equation (1).

The timer 40 measures a certain time (time T in FIG. 2) required for the receptor and the sample solution to sufficiently react, instructs a frequency measurement start to the resonance frequency measuring device 20, injects the solution into the three-way valve 15, and nitrogen gas. An injection switching command and timings for acquiring the frequencies f ₁ and f ₂ for the baseline measuring device 30 are generated.

  Therefore, the resonance frequency measurement in the QCM sensor main body 10 includes a baseline measurement immediately before the reaction between the receptor and the sample solution starts and a baseline measurement performed immediately after the reaction between the receptor and the sample solution is sufficiently stabilized. In the line measurement, the phenomenon in which the longitudinal wave component propagates in the solution and is reflected from the channel wall is almost cured, and resonance frequency interference due to this reflection does not occur, and stable resonance frequency measurement and adsorbed substance measurement Output can be obtained.

  In the case of a QCM sensor that measures the amount of adsorbed substance from the change in impedance of the crystal resonator instead of the frequency measurement by the resonance frequency measuring device 20, the baseline measuring device 30 absorbs from the impedance change immediately before the start of measurement and after a certain time. The measurement output of the substance amount can be obtained.

(2) Baseline measurement by gas injection In the QCM sensor having the above configuration, a gas having a density and viscosity sufficiently lower than the sample solution around the flow path (humidity controlled air, dry air, nitrogen atmosphere) Resonance frequency measurement and baseline measurement are performed in a state in which is injected. In the case of FIG. 1, the flow cell container 14 has an airtight structure, and nitrogen gas (N ₂ ) is injected into the flow cell container 14 by a three-way valve 15.

  According to this measurement method, when the sample solution flows on the surface of the electrode 12 in a gas phase having a low density and viscosity, the generation of reflected waves and the interference thereof can be reduced to perform more stable measurement. Further, by exposing the electrode 12 to a dried gas phase, the inside of the flow cell container 14 after the reaction of the sample solution is dried, and moisture contained in the atmosphere is prevented from being adsorbed on the electrode surface. In addition, the Q value (indicating the sharpness of resonance and reflected in the S / N ratio of the resonance circuit) of the crystal resonator can be increased, and high sensitivity measurement is possible.

As an experiment in the QCM sensor injected with N ₂ gas, when baseline measurement is performed in a nitrogen atmosphere (1 atm, 25 ° C.), the flow path height is lowered to about 30 μm, and the measurement is stable at a good reaction rate. And high sensitivity could be obtained.

(3) Configuration of the QCM sensor main body Since the flow cell type QCM sensor allows the sample solution to flow through a minute flow path, the flow path type QCM sensor has a flow path configuration that is highly hydrophilic to the sample solution, thereby obtaining a smooth flow of the sample solution. Is important. In addition, since inexpensiveness is an important factor, the following two types of QCM sensor configurations and manufacturing methods are proposed.

  (Example 1) Configuration and manufacturing method of a flow cell type QCM sensor using a PDMS resin channel.

  FIG. 3 shows a manufacturing procedure of the QCM sensor. In the manufacture of the crystal resonator, the electrode 12 and the lead wire 13 are formed on the surface of the crystal resonator (S1), and the main resonance frequency is adjusted by adjusting the shape and thickness of the electrode 12 (S2).

  In the flow path production, PDMS resin is injection molded into a urethane matrix to obtain a flow path molded body (S3), and the PDMS resin surface (flow path portion) is hydrophilized by oxygen plasma treatment (S4).

  Finally, using the self-adhesive property of the PDMS resin, the PDMS resin flow path is bonded to the crystal resonator (S5).

  The method according to this embodiment is characterized by a very simple manufacturing method while increasing the hydrophilicity of the QCM sensor main body.

  (Example 2) Structure and manufacturing method of a flow cell type QCM sensor using an acrylic resin flow path.

  FIG. 4 shows a manufacturing procedure of the QCM sensor. Only the steps different from those in FIG. 3 will be described. In the flow path production, acrylic resin is injection-molded into a mold to obtain a flow path molded body (S6), and an adhesive is coated on the surface of the acrylic resin flow path molded body to which the crystal resonator is attached (S7). ). Here, a PMMA photoresist (ODUR series, manufactured by Tokyo Ohka) is used for spin coating.

  Thereafter, the acrylic channel coated with the adhesive and the crystal unit are bonded together (S5), and the adhesive is dried in a forced circulation dryer (S8).

  The method according to the present embodiment is characterized by a very simple manufacturing method while enhancing the hydrophilicity of the QCM sensor main body as in the first embodiment.

The apparatus block diagram of the flow cell type QCM sensor which shows embodiment of this invention. The measurement flow in embodiment. The manufacturing procedure of the flow cell type QCM sensor main body using the PDMS resin channel in the embodiment. The manufacturing procedure of the flow cell type QCM sensor main body using the acrylic resin flow path in the embodiment. Conventional stationary solution type cell structure. Conventional forced stirring solution type cell structure. Conventional flow cell type QCM sensor body structure. An example of measurement of antigen-antibody reaction using a flow cell type QCM sensor. The measurement example of the antigen antibody reaction using a solution forced stirring type cell. The example of the resonant frequency change of the crystal oscillator by evaporation of 100% ethanol. The example of the reflection liquid dependence of the resonant frequency with respect to the liquid level height (solution amount) of 100% ethanol.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 QCM sensor main body 20 Resonance frequency measuring device 30 Baseline measuring device 40 Timer 11 Crystal oscillator 12, 12 'Electrode 14 Flow cell container 15 Three-way valve

Claims (7)

In a flow cell type QCM sensor that flows a sample solution through a channel formed on a crystal unit and detects and quantifies the sample component from changes in the resonance frequency or impedance of the crystal unit due to adsorption of components in the sample solution.
Performs a baseline measurement of the resonance frequency or impedance of the crystal unit immediately before the start of measurement, and a baseline measurement of the resonance frequency or impedance of the crystal unit after the sample solution has sufficiently reacted. A measurement method using a flow cell type QCM sensor, characterized in that a sample component is detected and quantitatively measured from a resonance frequency change or impedance change of a child.   The baseline measurement is performed by setting the flow path height to be higher than a height at which interference of a resonance frequency or impedance caused by a longitudinal wave component propagated by the sample solution occurs to increase a reaction speed of the sample solution. Item 5. A measurement method using the flow cell type QCM sensor according to Item 1.   3. The measurement method using a flow cell type QCM sensor according to claim 1, wherein the baseline measurement is performed by injecting a gas having a density and viscosity sufficiently lower than the sample solution into the flow path. In a flow cell type QCM sensor that detects and quantifies a sample component from a change in the resonance frequency or impedance of a crystal resonator caused by adsorption of a component in the sample solution through a flow path formed on the crystal resonator.
A measuring instrument for measuring the resonance frequency or impedance of the crystal unit;
A baseline measuring device that obtains sample component detection / quantitative measurement output from the amount of change between the measured value of the measuring device immediately before the start of measurement and the measured value of the measuring device after the sample solution has sufficiently reacted. A flow cell type QCM sensor.   The flow path has a structure that has a height lower than a height at which interference of a resonance frequency or impedance caused by a longitudinal wave component propagating through the sample solution occurs to increase a reaction rate of the sample solution. Item 5. The flow cell type QCM sensor according to Item 4.   6. The flow cell type QCM sensor according to claim 4, further comprising gas injection means for injecting a gas having a density and viscosity sufficiently lower than that of the sample solution into the flow path.   The flow cell type QCM sensor according to any one of claims 4 to 6, wherein the channel is made of PDMS resin or acrylic resin having high hydrophilicity with respect to the sample solution.

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