JP2002122600A - Sensor chip for biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor chip for biosensor in which a substance in the humor of an organism can be detected repeatedly with high accuracy while reducing the detection cost regardless of the external environment in the detection process. SOLUTION: The sensor chip in a biosensor for capturing a substance in the humor of an organism has positive and negative electrodes arranged relatively at a specified interval. A chip material composed of a substance being detected is placed on the negative electrode while being insulated electrically and under a state where an organic or inorganic material gas is introduced into a reaction container containing the chip material and regulated to a specified gas pressure, a voltage is applied between the positive and negative electrodes to generate glow discharge. Organic or inorganic substance in the material gas, in the form of plasma, is deposited on the chip material to form a film and then the chip material is dissolved and removed to form a recess matching the substance being detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a biological component (cell,
The present invention relates to a sensor chip used for a biosensor for detecting a target substance to be detected from among proteins, DNAs, hormones and the like.

[0002]

For example, in various analytical tests, when detecting a substance to be detected from a biological component, a change in ligand can be detected with high sensitivity without using a labeling substance such as a fluorescent substance. A biosensor utilizing surface plasmon resonance (SPR) is used.

A sensor chip of the SPR biosensor has a structure in which a physiologically active substance (cell, protein, lipid, etc.) that binds only to a desired substance to be detected is supported on a gold thin film formed on a glass substrate. are doing.

[0004] A porous material made of synthetic fiber, natural fiber or the like is formed, and the porous material carries a physiologically active substance such as an enzyme or an antibody that binds only to a desired substance to be detected.

[0005] When a biological component liquid containing the substance to be detected flows on the gold film, the substance to be detected binds to the physiologically active substance, the mass increases, and the dielectric constant changes. The substance to be detected is detected by detecting the change in the dielectric constant based on the SPR angle.

In a quartz oscillator sensor used for the same purpose, a similar sensor chip is attached to a vibrating electrode plate, and a vibration of the quartz oscillator is caused by a mass change due to a combination of a physiologically active substance and a substance to be detected. The change in the number enables detection of the substance to be detected.

However, in any of the sensor chips, the physiologically active substance that contributes to the detection of the substance to be detected is often a protein, and greatly affected by the external environment (temperature, pH, buffer capacity, ionic strength, cofactor, etc.). It was easily affected and deactivated. For this reason, even if it can be fixed efficiently to the gold surface, it is deactivated due to environmental influences and changes in the three-dimensional structure due to the fixation, and at present the efficiency of capturing the test substance is significantly reduced. Was.

[0008] In addition, the physiologically active substance for capturing a test substance is usually very expensive in many cases, and the quantity available on the market is small.

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned conventional drawbacks, and an object thereof is to provide a biosensor capable of detecting a substance to be detected in a biological component liquid with high accuracy. It is to provide a sensor chip for use.

[0010] Another object of the present invention is to provide a sensor chip for a biosensor which can repeatedly detect and reduce the detection cost.

Another object of the present invention is to provide a sensor chip for a biosensor capable of detecting a substance to be detected with high accuracy without being affected by an external environment in a detection process.

[0012]

SUMMARY OF THE INVENTION The present invention relates to a biosensor for detecting a target substance in a biological component by capturing it on a sensor chip. The sensor chip comprises a positive electrode and a negative electrode arranged at a predetermined interval. In a state in which a raw material gas of an organic or inorganic compound is introduced into a reaction vessel in which a chip material made of a substance to be detected is placed in an electrically insulated state on the negative electrode and adjusted to a predetermined gas pressure. The glow discharge generated by the voltage applied between the positive electrode and the negative electrode converts the organic or inorganic substance in the raw material gas into plasma, attaches and deposits it on the chip material, forms the film, and then dissolves and removes the chip material to be detected. It has a concave portion corresponding to the substance.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below with reference to the drawings. Embodiment 1 The present embodiment is applied to an SPR biosensor. In FIGS. 1 to 4, gold (Au) or silver (Ag) is provided on the upper surface of a glass substrate 3 which constitutes a part of the SPR biosensor 1. Metal thin film 5 of a predetermined thickness (5 to 50 n
m).

On the lower surface of the glass substrate 3, a semi-cylindrical prism 7 having an optical refractive index substantially equal to that of the glass substrate 3 (1.4 to 2.0) is attached in close contact with a matching oil, a silicon rubber sheet or the like. ing. A light irradiating device 9 is symmetrically disposed on the outer peripheral side on the left side of the center vertical line of the illustrated semi-cylindrical prism 7, and a light receiving device 11 is symmetrically disposed on the outer peripheral side on the right side.

A light irradiation device 9 outputs a laser light source 13 for outputting a laser light of a predetermined wavelength, and a laser light from the laser light source 13 passes through the semi-cylindrical prism 7 to a predetermined position at a boundary between the glass substrate 3 and the metal thin film 5. And an optical lens 15 that converges to the area width.

The laser beam may be converged to a predetermined beam diameter by the optical lens 15 with respect to the boundary. In this case, the semi-cylindrical prism 7
The light irradiating device 9 and the light receiving device 11 are rotated in a direction opposite to each other by a predetermined angle width with respect to the outer peripheral surface of the device.

The light receiving device 11 includes a glass substrate 3 and a metal thin film 5.
An optical lens 17 for forming laser light reflected from the boundary into parallel light, and a light receiving member 19 such as a photodiode array or a CCD in which a large number of light receiving elements 19a are arranged at intervals according to the SPR angle detection resolution. Is done. Thereby, each light receiving element 19a receives the laser beam according to the reflection angle and outputs an electric signal according to the light intensity.

A sensor chip 21 described later is fixed on the upper surface of the metal thin film 5. When the sensor chip 21 is made of hydrocarbon, a chemical bond using a C—H group is suitable as a method for fixing the sensor chip 21.

The cell block 23 is provided on the upper surface of the metal thin film 5.
Are in close contact with each other via a matching oil or a silicone rubber sheet. A flow cell 25 is formed on a relative surface of the metal thin film 5 in the cell block 23, and a supply flow path member 27 is provided in the cell block 23 corresponding to one end of the flow cell 25, and a flow channel member 27 is provided in accordance with the other end of the flow cell 25. A discharge channel member 29 is provided in each of the cell blocks 23, and the biological component liquid 22 containing the substance to be detected flows in the flow cell 25.

In FIG. 4, a positive electrode 35 and a negative electrode 37 are disposed at a predetermined interval in a reaction vessel 33 of a plasma film forming apparatus 31 as a sensor chip manufacturing apparatus. A high-voltage DC power supply 39 is connected to 37. A DC voltage of 0.5 to 3 KV is applied between the electrodes 35 and 37.

Normally, the negative electrode 37 is connected to the ground, and a DC voltage of (+) potential is applied between the positive electrode 35 and the negative electrode 37. However, the positive electrode 35 is connected to the ground and the negative electrode 37 is connected to the (-) potential. And a DC voltage of the (−) potential may be applied between them.

A vacuum exhaust device 41 is connected to the reaction vessel 33.
The inside of the reaction vessel 33 is 1.3 × 10 ^-FourHigh true below Pa
Form in the sky. The raw material gas is contained in a part of the reaction vessel 33.
An introduction section 43 is provided, and the gas is introduced through the source gas introduction section 43.
Osmium gas, hydrocar
Raw material consisting of organic or inorganic compounds such as Bongas
Supply gas. And the reaction vessel into which the raw material gas was introduced
The gas pressure in 33 is based on the amount of exhaust by the vacuum
The feed rate of the feed gas to about 7.98 to 13.3 Pa
Is set.

As the raw material gas, in addition to the above-mentioned gases, as the metal of the metal compound, for example, Group I to VIII of the periodic table are used.
Group metals are available and are used as simple metals or organic or inorganic metal compounds.

As a method of supplying the raw material gas into the reaction vessel 33, for example, in the case of methane carbon gas or ethylene gas, a method of supplying these gases directly from a gas cylinder filled with compression or a method such as osmium tetroxide is used. Any sublimation material may be introduced into the reaction vessel 33 to be sublimated and supplied.

A chip material 45 formed on the sensor chip 21 is placed on the upper surface of the negative electrode 37 via an electric insulator 47 such as a glass plate, a silicon plate, or a ceramic plate. The electrical insulator 47 has a height of the chip material 45.
Is set in the negative glow layer (1 to 5 mm from the upper surface of the negative electrode 37) of the glow discharge generated between the positive electrode 35 and the negative electrode 37. The chip material 45 maintains a predetermined shape by freeze-drying or heat-drying a biological substance such as a protein or DNA as a substance to be detected.

The chip material 45 is manufactured on the sensor chip 21 as follows. First, the chip material 45 is set on the negative electrode 37 via the electric insulator 47, and the vacuum exhaust device 41 is driven in a state where the chip material 45 is located in the negative glow layer, so that the inside of the reaction vessel 33 is, for example, 1.3 ×. A high vacuum of 10 ⁻⁴ Pa or less is applied to remove impurities from inside the reaction vessel 33.

Next, a source gas is introduced from the source gas introduction unit 43 and filled. At this time, the source gas pressure in the reaction vessel 33 is about 1.33 to 13.3 by introducing the source gas in a state where the exhaust amount of the vacuum exhaust device 41 is reduced.
Adjust to Pa.

In this state, when a DC voltage is applied between the positive electrode 35 and the negative electrode 37 to generate a glow discharge between them, the raw material gas between the positive electrode 35 and the negative electrode 37 as shown in FIG. Osmium atoms or carbon atoms are ionized (+) to form plasma. As a result, the (+) ionized osmium atoms or carbon atoms are attracted to the negative electrode 37 having the (-) potential, adhere to and deposit on the surface of the chip material 45, and are formed on the osmium thin film or the carbon thin film.

At this time, since the chip material 45 is located in the negative glow layer, osmium atoms or carbon atoms effectively wrap around and adhere to the periphery of the chip material 45, particularly, the undercut surface. Deposits to form a thin film of uniform thickness (about 10-500 °) over the entire surface.

The thickness of the thin film formed on the surface of the chip material 45 depends on parameters such as the concentration of the source gas to be introduced (source gas pressure), the applied voltage between the positive electrode 35 and the negative electrode 37, the applied current, and the time. It is usually determined that the film thickness is 1 to
These parameters are appropriately set so as to be 50 nm.

Next, the chip material 45 having the osmium thin film or the carbon thin film formed on the surface is immersed in a dissolving solution to be dissolved and removed, and the chip material 45, that is, the concave portion 21a having a shape coinciding with the biological substance to be detected. Is formed on the sensor chip 21 made of an osmium thin film or a carbon thin film having

Next, the detection operation of the substance to be detected by the SPR biosensor 1 will be described. First, the supply channel member 27
To supply the buffer solution to fill the inside of the flow cell 25 with the buffer solution. In this state, a laser beam is irradiated from the light irradiation device 9 to the entire area of the flow cell 25, and the light receiving device 11 receives reflected laser light from the boundary between the glass substrate 3 and the metal thin film 5.

Then, the SPR angle at which the intensity of the reflected light at each incident angle of the laser beam measured in a state where the buffer solution is caused to flow in the flow cell 25 is attenuated is stored in a storage member (not shown) as a reference SPR angle.

Next, as shown in FIG.
Then, instead of the buffer solution, a biological component liquid containing the substance to be detected is supplied from the supply channel member 27 to flow through the flow cell 25. As a result, of the biological substances contained in the biological component liquid, the substance to be detected (the substance to be detected is indicated by the symbol △ for the sake of convenience of description) passes through the upper surface of the sensor chip 21 in the recess 21 a. And the other non-detection substances (for the sake of explanation, non-detection substances are indicated by symbols ○ and □) flow directly without being fitted into the concave portions 21 a and flow out of the discharge flow path member 29. Collected.

At this time, if the substance to be detected fits into the recess 21a and is fixed, the SPR angle fluctuates due to a change in the dielectric constant near the metal film surface due to the fixed substance to be detected. A desired substance to be detected in the biological component liquid is detected based on the change in the SPR angle. As another detection example, the concentration of the target substance in the biological component liquid can be measured based on the amount of change in the SPR angle per unit time.

Embodiment 2 The biosensor of this embodiment is a quartz oscillator sensor 71.
Is characterized in that the sensor chip 73 is used.

7 and 8, the quartz oscillator 75 of the quartz oscillator sensor 71 has a characteristic of vibrating at the natural frequency f0. In addition, each surface of the crystal unit 75 has a first
And second electrode plates 77 and 79 are formed respectively. The crystal unit 75 is fixed on the insulating substrate 81 through a matching oil, a silicon rubber sheet, or the like.

On the insulating substrate 81 to which the crystal unit 75 is fixed, a cell block 83 is similarly tightly fixed via a matching oil, a silicon rubber sheet or the like. The cell block 83 is formed with a flow cell 85 having at least a capacity for accommodating the crystal resonator 75, and a supply block member 87 for supplying a biological component liquid is provided in the cell block 83 on one end side of the flow cell 85, and the other of the flow cell 85. The end-side cell block 83 is provided with a discharge channel member 89 for collecting a biological component liquid overflowing from the flow cell 85.

The sensor chip 73 is fixed on the second electrode plate 79 of the crystal unit 75 located in the flow cell 85. The sensor chip 73 is manufactured by the plasma film forming principle similarly to the sensor chip 21 of the first embodiment, and a detailed description thereof will be omitted.

Next, the operation of detecting the substance to be detected by the crystal oscillator sensor 71 will be described. Although the natural frequency f0 of the crystal unit 75 itself is determined in advance, the flow cell 85
The frequency at which the biological component liquid flows into the inside differs from the natural frequency f0 due to the mass of the biological component liquid. Therefore, the buffer is supplied from the supply flow path member 87 into the flow cell 85 prior to the detection, and the frequency f1 in a state where the buffer is filled in the flow cell 85 is measured.

Next, after the buffer solution is discharged from the flow cell 85, the biological component liquid containing the substance to be detected is supplied from the supply flow path member 87, and the biological component liquid overflowing from the flow cell 85 is discharged from the flow cell member 89. And flow through the flow cell 85.

When the biological component solution flows in the flow cell 85, the target substance to be detected in the biological component solution is fitted and fixed in the concave portion 73 a of the sensor chip 73, and is fixed to the second electrode plate 79. The mass of the fixed sensor chip 73 increases, and the frequency of the crystal unit 75 changes from f1 to f2. Frequency displacement Δf of crystal resonator 75 = f2-f1
The substance to be detected in the biological component is detected based on As another measurement mode, the concentration of the substance to be detected in the biological component liquid is measured based on the frequency fluctuation per unit time.

[0043]

According to the present invention, a substance to be detected in a biological component liquid can be detected with high accuracy. In addition, the detection can be performed repeatedly, and the detection cost can be reduced. Further, the target substance can be detected with high accuracy without being affected by the external environment in the detection process.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an SPR biosensor.

FIG. 2 is an explanatory diagram showing a portion A of FIG. 1 in an enlarged manner.

FIG. 3 is an enlarged partial perspective view of a sensor chip.

FIG. 4 is an explanatory view showing an apparatus for manufacturing a sensor chip.

FIG. 5 is an explanatory diagram showing a film formation state.

FIG. 6 is an explanatory diagram showing a detection state.

FIG. 7 is an explanatory diagram of a crystal oscillator sensor.

8 is an explanatory diagram showing a portion B of FIG. 7 in an enlarged manner.

[Explanation of symbols]

1-SPR biosensor, 21-sensor chip, 2
1a-recess, 33-reaction vessel, 35-positive electrode, 37-negative electrode, 45-tip material

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Takato Sato 20-9, Sanbonmatsucho, Atsuta-ku, Nagoya F-term in Japan Laser Electronics Co., Ltd. (reference) 2G057 AA02 AB04 AB07 AC01 BA05 DB05 DC07 2G059 AA05 BB12 CC16 DD12 DD13 EE02 FF06 GG01 JJ11 JJ12 KK04

Claims (5)

[Claims] 1. A biosensor for detecting a target substance to be detected in a biological component by capturing the target substance on a sensor chip, wherein the sensor chip has a positive electrode and a negative electrode which are disposed relative to each other at a predetermined interval. A raw material gas of an organic or inorganic compound is introduced into a reaction vessel in which a chip material composed of a substance to be detected is placed in an electrically insulated state, and is applied between a positive electrode and a negative electrode while being adjusted to a predetermined gas pressure. After the organic or inorganic substance in the raw material gas is turned into plasma by the glow discharge generated by the applied voltage and adhered and deposited on the chip material to form a film, the chip material is dissolved and removed to have a concave portion corresponding to the substance to be detected. Sensor chip for biosensor. 2. The biosensor according to claim 1, wherein the biosensor comprises a substrate on which a metal thin film to which a sensor chip is fixed is formed;
A prism adhered to the substrate on the opposite side of the metal thin film, a light irradiation device for irradiating light to a boundary between the substrate and the metal thin film, and receiving reflected light from the boundary and outputting an electric signal according to light intensity It comprises a light receiving device and a cell block having a flow cell of a size corresponding to the sensor chip, being closely attached to the substrate, supplying and flowing a sample liquid containing a substance to be detected in the flow cell, and flowing through the flow cell. A sensor chip for a biosensor, wherein a substance to be detected can be detected by a surface plasmon resonance angle based on a change in dielectric constant caused by a substance to be detected in a sample liquid being captured by a concave portion of the sensor chip. 3. The crystal sensor according to claim 1, wherein the biosensor oscillates at a natural frequency, a first electrode plate closely attached to one side surface of the crystal oscillator, and a first electrode plate adhered to the other side surface of the crystal oscillator. And a second electrode plate to which the sensor chip is fixed. The detection target is detected based on a frequency displacement due to a mass change when the substance to be detected is captured in the concave portion of the sensor chip with the flow of the sample liquid to the sensor chip. A sensor chip for biosensors that can detect substances. 4. A sensor chip for a biosensor according to claim 1, wherein the raw material gas is a sublimation gas of osmium tetroxide. 5. A sensor chip for a biosensor according to claim 1, wherein the raw material gas is a mixed gas of methane and ethylene.