JP2003307481A - Multi-channel biosensor - Google Patents

Abstract

(57) [Problem] To provide a multi-channel biosensor which can easily reduce an error component due to a change in viscosity or density in a local sample space and can measure a change in a minute mass with high accuracy. I do. In a multi-channel biosensor in which a plurality of piezoelectric vibrators are arranged on a substrate, at least one specific channel is provided in a channel adjacent to an inspection channel on which a sensitive film for capturing only a specific analyte is immobilized. A correction channel that does not immobilize a sensitive membrane that captures only the analysis target is arranged, and an error in the inspection channel due to a change in viscosity / density of the space containing the sample due to a change in electrical characteristics due to the correction channel. It is configured to correct the components.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a multichannel biosensor for measuring changes in minute mass. In particular, QCM (Quartz Crystal Micro) for immobilizing biochemical substances and measuring chemical substances such as enzymes, antibodies, proteins and hormones that are specifically adsorbed thereto.
balance) type biosensor.

[0002]

2. Description of the Related Art FIG. 2 shows a schematic view of a crystal unit which constitutes a QCM sensor. Basically, electrodes 2a and 2b are formed on both surfaces of a quartz plate 1 that is thinly processed by etching and / or polishing. When this crystal oscillator is used as a biosensor, a sensitive film that captures only the analysis target is immobilized on one surface of the electrode. When an alternating electric field is applied to both electrodes 2a, 2b, vibration of a certain frequency is excited by the inverse piezoelectric effect, but when the analysis target is captured by the sensitive film, a mass increase Δm accompanies, and as a result, the oscillator The resonance frequency changes by Δf. This bioreaction includes a DNA hybridization reaction,
Various substances can be used, including in gas and in liquid, such as antigen-antibody reaction, protein binding, and enzyme reaction.

The relationship between the mass addition amount Δm and the vibration frequency change amount Δf is derived by Sauerbrey (G. Sauerbrey, Z. Phys. 1).
55, 1959, 206), and can be expressed by the following equation. Here, f ₀ is the main fundamental frequency of the crystal unit, A is the area of the electrode, μ _q is the shear elastic modulus of the crystal, and ρ _q is the density of the crystal. For example, in the case of a crystal oscillator cut out on an AT-cut surface, there is a relationship of the following expression between the thickness t of the crystal plate and the main fundamental frequency. Therefore, it can be seen that the higher the main fundamental frequency, that is, the thinner the crystal plate, the higher the sensitivity as the mass sensor.

As an example, an AT-cut crystal with a thickness of 335 μm
In the case of a plate, the main fundamental frequency is about 5MHz,
Elastic modulus μ_qIs 2.95 × 10¹¹dyn / cm²,crystal
Density ρ_qIs 2.65 g / cm³From about 18 ngHz
^-1cm^-2It becomes the sensitivity of. By the way, QCM type bar in liquid
When operating the Io sensor, the
The resonance frequency can be further increased by immersing the oscillator in the pull solution.
It causes a decrease in wave number. This relationship is Where μ_LOf the sample solution in which the crystal oscillator is immersed
Viscosity, ρ _LIs also the density of the sample solution. This
The higher the main fundamental frequency, the greater the fluctuation of the vibration frequency.
You can see that it will be affected.

In recent years, the development of bio-inspection chips called Lab-on-a-Chips or bio-micromachines has become popular. Lab-on-a
Since the Chip type biosensor is compact and inexpensive, it is said to be suitable for, for example, periodically inspecting human health conditions at home or analyzing special gas or odor on the spot. ing. In a biochip used for such a purpose, it is desirable that the sample be operated in a small amount.

The QCM type biosensor described in the present invention is
Lab-on-a that operates with a small amount of sample solution as described above, not only when the QCM sensor unit is immersed in a sample solution or gas that has been conventionally used for inspection.
-The description also includes an application example in which a QCM sensor is incorporated in the inspection portion of a Chip-type biochip and a multi-channel is formed so that multiple types of analysis targets can be detected simultaneously.

[0007]

DISCLOSURE OF THE INVENTION The conventional QC described above
When the crystal plate is thinned for the purpose of further increasing the sensitivity of the M biosensor, as can be seen from the formula (3), the resonance frequency of the QCM is greatly influenced by the change in the viscosity and the density of the sample solution. Become. To solve the influence of the density and viscosity of the sample solution, JP-A 2000-
In Japanese Patent No. 258324, a correction electrode is provided adjacent to the QCM working electrode, and the viscosity and density of the solution are corrected from the obtained signal. However, when applying such a method of correcting the density and viscosity of the sample solution to a multi-channel type QCM sensor capable of simultaneously measuring many kinds of biochemical reactions, it is possible to use a method for dipping in a small amount of sample solution. In some cases, the accuracy of inspection may be reduced due to local changes in the solution.

Therefore, the present invention solves the above-mentioned problems, and can easily reduce the error component due to the local fluctuation of the viscosity and the density of the sample space, and it becomes possible to measure the change of the minute mass with high accuracy. It is intended to provide a multi-channel biosensor.

[0009]

In order to achieve the above object, the present invention is characterized in that a multichannel biosensor is constructed as follows. That is, the present invention provides a multi-channel QCM sensor having at least 2
It is characterized in that the sensitive membrane for capturing only the analysis target can be operated without being immobilized in the channels above the location. By using changes in electrical characteristics such as resonance frequency in channels where the sensitive film is not immobilized, it is used to compensate for changes in environment around the channel such as viscosity and density in channels around the channel where the sensitive film is not immobilized. Is characterized by. At least one of the plurality of channels adjacent to the channel on which the sensitive film is immobilized has the non-immobilized sensitive film. Further, the present invention is characterized in that one channel in which one sensitive film is not fixed is arranged adjacent to one channel in which the sensitive film is fixed. By using changes in electrical characteristics such as resonance frequency due to channels in which each sensitive film is not immobilized, it is possible to correct the effects of environmental changes around the channels, such as more localized viscosity and density of adjacent inspection channels. Characterize. Further, the present invention is characterized in that the thickness of the quartz plate of the channel in which the pair of sensitive films is fixed and the thickness of the channel in which the sensitive film is not fixed are changed to make the wiring common. It is characterized in that the channel in which the pair of sensitive membranes are immobilized and the channel in which the sensitive membrane is not immobilized are simultaneously oscillated to sequentially monitor the process of the chemical reaction.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION By applying the above configuration,
It is possible to easily reduce the error component due to the local fluctuation of the viscosity or density of the sample space, and to realize a multi-channel biosensor that enables highly accurate measurement of changes in minute mass.

[0011]

EXAMPLES Examples of the present invention will be described below. [Embodiment 1] FIG. 1A is a top view of Embodiment 1 of the present invention, and FIG. 1B is a sectional view taken along line AA. In this embodiment,
4 × 4 multi-channels are formed, and correction channels 4 are provided at 4 positions out of a total of 16 channels.
This does not apply to the arrangement method and number of channels.
In the drawings, the detection channel 3 is shown in gray, the correction channel 4 is shown in black, and the wiring 5 on the back surface is shown in dotted lines.

When the multi-channel QCM type sensor of this embodiment is applied as a biosensor to, for example, an antigen-antibody reaction, the detection channel 3 is provided with a membrane 6 containing an antigen that captures only a specific antibody 7 as a channel electrode. Immobilize on top (see Figure 5). On the other hand, in the correction channel 4, the film containing the above-mentioned antigen is not immobilized on the electrode. In addition to the antigen-antibody reaction, various biochemical reactions can be applied to this biosensor, but the membrane immobilized on this electrode is a substance that can be adsorbed only to the substance to be analyzed. To use.

In the operation of such a biosensor, for example, the sensor oscillator is immersed in the sample solution 8 or gas while simultaneously oscillating all channels at the resonance frequency.
Hereinafter, the analysis environment will be described only in the liquid. When the sample solution 8 contains a substance that can be captured by the membrane immobilized on the channel as described above, the substance is captured on the corresponding channel, and as a result, the mass change on the channel electrode is caused. As a result, the captured amount of the target can be identified through the change in the resonance frequency. The oscillating operation at the resonance frequency of each channel may not be performed simultaneously, but may be sequentially oscillated for each channel. In this case, it is not true real-time measurement, but interference between channels can be reduced.

In the case of such a biosensor, the density and viscosity of the solution in which the sensor is immersed change due to the progress of chemical reaction on the sensor, environmental temperature fluctuations, and the like. In particular, when a QCM-type multi-channel biosensor as shown in FIG. 5 is incorporated as one function of a biochip system such as Lab-on-a-Chip, the solution amount of the sample is very large in order to fully utilize its merit. There are few, and changes in viscosity and density are likely to occur. Further, the change may not be uniform on the biochip and may occur locally.

FIG. 6 shows an outline of an example in which the multi-channel QCM biosensor is incorporated into a multifunction small type system such as Lab-on-a-Chip. In this example, the solution or gas to be analyzed is put into the sensor system through the sample inlet 11, the sample is separated and extracted in the pretreatment element 10, and then introduced into the sensor unit. The pretreatment process may require various other functions such as mixing / reaction, filters, valves, etc.
I will not describe them in detail here. In the pretreatment process,
A special reagent such as a reaction may be required, and a route and an inlet for introducing the reagent may be required separately. On the other hand, for moving the sample, a mechanical one using a pump, an electric one using electroosmosis, etc. are widely used. The sensor section has QCs arranged for multi-channel.
There is an M sensor, and wiring is provided for each channel (the wiring on the back side is not shown in FIG. 6). After the detection, the sample solution or gas is discharged from the outlet 1.
It is discharged from 2.

Local changes in the viscosity and density of the sample solution 8 are detected by the channel in which the sensitive membrane capable of capturing only the substance to be analyzed prepared in advance is not fixed, that is, the correction channel 4. It is possible. Specifically, the correction channel 4 not covered with the capture molecule / membrane shows a decrease in the resonance frequency according to the equation (3) if the viscosity or density of the sample solution 8 changes.
If there is no change, the resonance frequency does not change. If the correction channel 4 and the inspection channel 3 have the same vibration characteristics (such as the thickness of the crystal plate of the channel portion and the electrode area), the difference between the vibration frequencies of the two channels is the sample solution 8. This is a net inspection amount that excludes errors due to changes in the viscosity and density. Oscillation of these two types of channels can be performed simultaneously or alternately without any problem. It is desirable that the correction channel 4 be adjacent to the inspection channel 3 in order to correct local changes in the viscosity and density of the sample solution 8. In the case of the 4 × 4 multi-array shown in this embodiment, at least four correction channels 4 are required.

[Embodiment 2] FIG. 3A is a top view of Embodiment 2 of the present invention, and FIG. 3B is a sectional view taken along line AA. In this embodiment, 4 × 4 multi-channels are formed, and the correction channels 4 are provided at 8 positions out of a total of 16 channels, but the arrangement method and number of channels are not limited to this. In the drawings, the detection channel 3 is shown in gray, the correction channel 4 is shown in black, and the wiring 5 on the back surface is shown in dotted lines.

When the multi-channel QCM type sensor of this embodiment is applied as a biosensor to, for example, an antigen-antibody reaction, the detection channel 3 includes a membrane 6 containing an antigen that captures only a specific antibody 7. It is immobilized on the electrode (see FIG. 5). On the other hand, in the correction channel 4, the film containing the above-mentioned antigen is not immobilized on the electrode. In addition to the antigen-antibody reaction, various biochemical reactions can be applied to this biosensor, but the membrane immobilized on this electrode is a substance that can be adsorbed only to the substance to be analyzed. To use.

In the operation of such a biosensor, for example, the sensor oscillator is immersed in the sample solution 8 or gas while simultaneously oscillating at the resonance frequency in all channels.
Hereinafter, the analysis environment will be described only in the liquid. When the sample solution 8 contains a substance that can be captured by the membrane immobilized on the channel as described above, the substance is captured on the corresponding channel, and as a result, the mass change on the channel electrode is caused. Through the change in resonance frequency,
It is possible to identify the capture amount of the target. The oscillating operation at the resonance frequency of each channel may not be performed simultaneously, but may be sequentially oscillated for each channel. In this case, it is not true real-time measurement, but interference between channels can be reduced. In the case of such a biosensor, the density and viscosity of the solution in which the sensor is immersed change due to the progress of chemical reaction on the sensor, environmental temperature fluctuations, and the like. In particular, when a QCM type multi-channel biosensor as shown in FIG. 5 is incorporated as one function of a biochip system such as Lab-on-a-Chip,
In order to make full use of the merit, the amount of the sample solution is very small, and the viscosity and density are likely to change. Further, the change may not be uniform on the biochip and may occur locally.

FIG. 6 shows an outline of an example in which the multi-channel QCM biosensor is incorporated in a small multifunctional type system such as a Lab-on-a-Chip. In this example, the solution or gas to be analyzed is put into the sensor system through the sample inlet 11, the sample is separated and extracted in the pretreatment element 10, and then introduced into the sensor unit. The pretreatment process may require various other functions such as mixing / reaction, filters, valves, etc.
I will not describe them in detail here. In the pretreatment process,
A special reagent such as a reaction may be required, and a route and an inlet for introducing the reagent may be required separately. On the other hand, for moving the sample, a mechanical one using a pump, an electric one using electroosmosis, etc. are widely used. The sensor section has QCs arranged for multi-channel.
There is an M sensor, and wiring is provided for each channel (the wiring on the back side is not shown in FIG. 6). After the detection, the sample solution or gas is discharged from the outlet 1.
It is discharged from 2.

The correction channel 4 of the multi-QCM sensor shown in this embodiment is arranged alternately with the inspection channel 3 for the purpose of more strictly correcting such locally-generated changes in viscosity and density of the sample solution 8. Has been done. In FIG. 3, the wiring 5 is individually provided on both surfaces of each channel, but one surface may be covered with electrodes. Local changes in the viscosity and density of the sample solution 8 can be detected by the channel in which the sensitive membrane capable of capturing only the substance to be analyzed prepared in advance is not immobilized, that is, the correction channel 4. Is. Specifically, if the viscosity or density of the sample solution 8 changes, the correction channel 4 not covered with the capture molecule / membrane shows a decrease in the resonance frequency according to the equation (3), and if there is no change. No change in resonance frequency occurs. If the correction channel 4 and the inspection channel 3 have the same vibration characteristics (such as the thickness of the crystal plate of the channel portion and the electrode area), the difference between the vibration frequencies of the two channels is the sample solution 8. This is a net inspection amount that excludes errors due to changes in the viscosity and density. Oscillation of these two types of channels can be performed simultaneously or alternately without any problem.

In the case of this embodiment, the inspection channel 3 is always arranged adjacent to the plurality of correction channels 4, so that it is desirable that the inspection channels 3 and the correction channels 4 are arranged alternately. By averaging the error amount due to the viscosity and temperature of the sample solution 8 obtained from the adjacent correction channels, it is possible to strictly reduce the local error cause. For this reason, 4 shown in the present embodiment
In the case of a x4 multi-array, eight correction channels 4 are required.

[Embodiment 3] FIG. 4A shows a top view of Embodiment 3 of the present invention, and FIG. 4B shows a sectional view taken along line AA. In this embodiment, 4 × 4 multi-channels are formed, and the correction channels 4 are provided at 8 positions out of a total of 16 channels, but the arrangement method and number of channels are not limited to this. In the drawings, the detection channel 3 is shown in gray, the correction channel 4 is shown in black, and the wiring 5 on the back surface is shown in dotted lines.

When the multi-channel QCM type sensor of this embodiment is applied as a biosensor to, for example, an antigen-antibody reaction, the detection channel 3 includes a membrane 6 containing an antigen that captures only a specific antibody 7. It is immobilized on the electrode (see FIG. 5). On the other hand, in the correction channel 4, the film containing the above-mentioned antigen is not immobilized on the electrode. In addition to the antigen-antibody reaction, various biochemical reactions can be applied to this biosensor, but the membrane immobilized on this electrode is a substance that can be adsorbed only to the substance to be analyzed. To use.

In the operation of such a biosensor, for example, the sensor oscillator is immersed in the sample solution 8 or gas while simultaneously oscillating at all channels at the resonance frequency.
Hereinafter, the analysis environment will be described only in the liquid. When the sample solution 8 contains a substance that can be captured by the membrane immobilized on the channel as described above, the substance is captured on the corresponding channel, and as a result, the mass change on the channel electrode is caused. Through the change in resonance frequency,
It is possible to identify the capture amount of the target. The oscillating operation at the resonance frequency of each channel may not be performed simultaneously, but may be sequentially oscillated for each channel. In this case, it is not true real-time measurement, but interference between channels can be reduced.

In the case of such a biosensor, the density and viscosity of the solution in which the sensor is immersed change due to the progress of chemical reaction on the sensor, environmental temperature fluctuations, and the like. In particular, when a QCM-type multi-channel biosensor as shown in FIG. 5 is incorporated as one function of a biochip system such as Lab-on-a-Chip, the solution amount of the sample is very large in order to fully utilize its merit. There are few, and changes in viscosity and density are likely to occur. Further, the change may not be uniform on the biochip and may occur locally.

FIG. 6 shows an outline of an example in which the multi-channel QCM biosensor is incorporated into a multifunction small system such as a Lab-on-a-Chip. In this example, the solution or gas to be analyzed is put into the sensor system through the sample inlet 11, the sample is separated and extracted in the pretreatment element 10, and then introduced into the sensor unit. The pretreatment process may require various other functions such as mixing / reaction, filters, valves, etc.
I will not describe them in detail here. In the pretreatment process,
A special reagent such as a reaction may be required, and a route and an inlet for introducing the reagent may be required separately. On the other hand, for moving the sample, a mechanical one using a pump, an electric one using electroosmosis, etc. are widely used. The sensor section has QCs arranged for multi-channel.
There is an M sensor, and wiring is provided for each channel (the wiring on the back side is not shown in FIG. 6). After the detection, the sample solution or gas is discharged from the outlet 1.
It is discharged from 2.

The correction channel 4 of the multi-QCM sensor shown in this embodiment is arranged alternately with the inspection channel 3 for the purpose of more strictly correcting such a change in the viscosity and density of the sample solution 8 which occurs locally. Has been done. However, in order to reduce the influence of the wiring 5 on each channel, one resonance channel 4 and one inspection channel 3 are made into a set, and the resonance frequency is changed slightly by changing the thickness of the transducers slightly. It is possible to shift and oscillate at the same time with a set of wirings 5. This alleviates the problem of complicated wiring 5 and space. Local changes in the viscosity and density of the sample solution 8 can be detected by the channel in which the sensitive membrane capable of capturing only the substance to be analyzed prepared in advance is not immobilized, that is, the correction channel 4. Is. Specifically, if the viscosity or density of the sample solution 8 changes, the correction channel 4 not covered with the capture molecule / membrane shows a decrease in the resonance frequency according to the equation (3), and if there is no change. No change in resonance frequency occurs. In the case of the present embodiment, the vibration and other characteristics of the oscillators of the correction channel 4 and the inspection channel 3 do not match because the thickness of the crystal plate has changed, so the difference between the vibration frequencies of both channels. Does not indicate the net inspection amount excluding errors due to changes in the viscosity and density of the sample solution 8 as it is, and must be converted and corrected for each channel. Oscillation of these two types of channels can be performed simultaneously or alternately without any problem.

In the case of this embodiment, the inspection channel 3 is always arranged adjacent to the plurality of correction channels 4, so that the inspection channels 3 and the correction channels 4 are preferably arranged alternately. By averaging the error amounts due to the viscosity, temperature, etc. of the sample solution 8 obtained from a plurality of adjacent correction channels, it is possible to strictly reduce the cause of the error locally. In the case of the 4 × 4 multi-array shown in this embodiment, eight correction channels 4 are required.

[0030]

According to the present invention, it is possible to easily reduce the error component due to the local fluctuation of the viscosity or density of the sample space,
A highly accurate multi-channel biosensor can be provided.

[Brief description of drawings]

FIG. 1 is a diagram showing a multi-channel type QCM sensor having a plurality of correction channels according to a first embodiment of the present invention, (a) is a top view thereof, and (b) is a sectional view taken along line AA.

FIG. 2 shows a basic configuration of a crystal unit.

3A and 3B are views showing a multi-channel type QCM sensor having the same number of correction channels / inspection channels in Embodiment 2 of the present invention, FIG. 3A is a top view thereof, and FIG. 3B is a sectional view taken along line AA.

4A and 4B are diagrams showing a multi-channel type QCM sensor in which wirings for a correction channel and an inspection channel are shared in Example 3 of the present invention, where FIG. 4A is a top view thereof, and FIG.
FIG.

FIG. 5 shows an outline of a chemical reaction on a multi-channel biochip.

FIG. 6: Lab-o on multi-channel biochip
An application example to the na-Chip is shown.

[Explanation of symbols]

1: Crystal plate 2a, 2b: electrodes 3: Detection channel 4: Correction channel 5: Wiring 6: Capture film 7: Target substance 8: Sample solution 9: flow path 10: Preprocessing element 11: Sample introduction port 12: Waste exit

Claims (7)

[Claims] 1. In a multi-channel biosensor in which a plurality of piezoelectric vibrators are arranged on a substrate, at least one is provided in a channel adjacent to an inspection channel in which a sensitive film capturing only a specific analysis target is immobilized. A correction channel in which a sensitive film that captures only a specific analysis target is not immobilized is arranged, and the change of the electrical characteristics due to the correction channel causes a change in the viscosity and density of the space containing the sample A multi-channel biosensor characterized by correcting an error component. 2. The multi-channel biosensor according to claim 1, wherein the piezoelectric vibrator is a crystal vibrator. 3. The multi-channel biosensor according to claim 1, wherein the substrate is connected to an element having a function other than detection for separating / extracting a sample through a sample flow path. . 4. In a multi-channel biosensor in which a plurality of piezoelectric vibrators are arranged on a substrate, an inspection channel in which a sensitive film capturing only a specific analysis target is fixed and a correction channel in which the sensitive film is not fixed. The channels have approximately the same number and are arranged so as to alternate with each other, and the error component of the inspection channel due to the change in the viscosity / density of the space containing the sample due to the change in the electrical characteristics due to the correction channel is A multi-channel biosensor, wherein correction is performed from an average of detection values obtained by a plurality of matching correction channels. 5. The wiring is shared by the pair of the inspection channel and the correction channel, the thicknesses of the crystal resonators of the two are slightly different from each other, and they are simultaneously operated. Multi-channel biosensor. 6. The multi-channel biosensor according to claim 4, wherein the piezoelectric vibrator is a crystal vibrator. 7. The multi-channel biosensor according to claim 4, wherein the substrate is connected to an element having a function other than detection for separating / extracting a sample through a sample flow path. .