WO2006082668A1

WO2006082668A1 - Biosensor and biosensor chip

Info

Publication number: WO2006082668A1
Application number: PCT/JP2005/013881
Authority: WO
Inventors: Sumio Hosaka; Hayato Sone; Haruki Okano
Original assignee: National University Corporation Gunma University; Tokyo Sokki Kenkyujo Co., Ltd.
Priority date: 2005-02-01
Filing date: 2005-07-28
Publication date: 2006-08-10
Also published as: JP2006214744A

Abstract

A biosensor comprising a cantilever, an actuator for oscillating the cantilever, a sensor provided on the cantilever to detect the oscillatory state of the cantilever, a means for controlling the actuator to resonate the cantilever basing on the oscillatory state detected by the sensor, and a means for detecting the amount of substance adhering to the cantilever basing on the variation in oscillatory state detected by the sensor under a state where the cantilever is controlled to resonate.

Description

Specification

Biosensor and biosensor chip

Technical field

[0001] The present invention relates to a biosensor and a biosensor chip that can measure antigen-antibody reaction and protein with high sensitivity, and in particular, is dissolved in a liquid using a cantilever. The present invention relates to a biosensor capable of measuring a substance with high sensitivity and a biosensor chip used in the nanosensor.

Background art

As a noise sensor, an easy and simple high-sensitivity sensor is required. One of these is the development of biosensors that use crystal resonators. The detection sensitivity of this biosensor is approximately 30 pgZHz, which is insufficient for use as a biosensor.

On the other hand, a cantilever used in an atomic force electron microscope has a resonance point, and the resonance point is shifted by the force received from the outside. Ton) is used as a sensor that can measure force. Furthermore, recently, this sensor has a component that the weight of the cantilever changes due to the substance adhering to the cantilever and the resonance point changes, and this sensor can be applied to a biosensor using this change in resonance point. Is being considered.

[0004] As a paper using cantilevers as biosensors, a sensor published by Lang et al. ("Artificial Nose" (Analytica Chamica Acta No. 39 3 卷 (1999) p. 59)) is known! / RU

As shown in FIG. 9, this sensor is a sensor that detects changes in the resonance frequency of a small cantilever 1 using an optical lever to detect physical quantities, chemical quantities, temperatures, stresses, or the like. In this sensor, the laser light emitted from the semiconductor laser 2 is condensed on the back surface of the cantilever 1 using a lens, and the laser light reflected by the cantilever is incident on a position detector 3 composed of a photodiode or the like. An optical system is required. In FIG. 9, 4 is an actuator that vibrates the cantilever, 5 is a detection circuit, and 6 is the drive of the actuator. The operation circuit 7 is an arithmetic circuit composed of a computer that calculates physical quantities and the like based on the output of the detection circuit.

However, since conventional biosensors require an optical system including a semiconductor laser, the optical axis of the optical system is adjusted in the air and then immersed in a liquid having a refractive index different from that of the air. However, when the optical path length is changed, it is necessary to adjust the optical axis again because the optical path length changes, and there is a problem that it cannot be used easily.

[0007] Conventionally, as column f, Special Table 2004-506872, JP-A-9-304409, Special Table 2002-54

3403, JP 2004-125706, US4, 549, 427, US5, 719, 324, US5, 631, 4

10 and JP-A-6-323845.

Patent Document 1: Special Table 2004- 506872

Patent Document 2: JP-A-9-304409

Patent Document 3: Japanese Translation of Special Publication 2002-543403

Patent Document 4: Japanese Patent Laid-Open No. 2004-125706

Patent Document 5: U.S. Pat.No. 4,549,427

Patent Document 6: U.S. Pat.No. 5,719,324

Patent Document 7: U.S. Pat.No. 5,631,410

Patent Document 8: JP-A-6-323845

Non-patent document 1: "Artificial nose" (Analytica Chamica Acta 393 (1999) p. 59)

Disclosure of the invention

Problems to be solved by the invention

[0008] The present invention has been made to solve the above-described problems, and a biosensor that can be used easily without readjustment when used, and a nanosensor chip that can be used for the biosensor The purpose is to provide.

Means for solving the problem

[0009] In order to achieve the above object, according to the first aspect of the present invention, a biosensor is provided in the cantilever, an actuator that vibrates the cantilever, and a vibration state of the cantilever. The detected sensor and the sensor Control means for controlling the actuator so that the cantilever resonates based on the issued vibration state, and the vibration state detected by the sensor in a state where the cantilever is controlled to resonate. Detecting means for detecting the amount of the substance adhering to the cantilever based on the change.

[0010] According to this feature, when a substance adheres to the cantilever while the cantilever is controlled to resonate, the vibration state such as the resonance frequency of the cantilever changes. Since the change in the vibration state and the mass of the substance attached to the cantilever are correlated, the amount of the substance attached to the cantilever can be detected based on the change in the vibration state detected by the sensor.

[0011] Since the sensor of this feature is provided on the cantilever, it is self-detecting, and therefore it can be used conveniently without having to adjust it again when using the biosensor. The cantilever of the biosensor can be used in a liquid such as a reaction liquid.

[0012] In the second aspect of the present invention, the cantilever can be attached to an insulator thin film, a thin film to which a substance to be detected can adhere, or the insulator thin film and the substance to be detected coated thereon. It can be covered with a laminated film with a thin film. By covering the cantilever with an insulating film, current leakage when using the biosensor in liquid or the like can be prevented. Moreover, the mass of the target substance can be detected by coating a thin film to which the substance to be detected can adhere.

[0013] According to the third feature of the present invention, the actuator can be constituted by a piezoelectric element, a capacitance element, or an electromagnetic induction element.

[0014] In the fourth aspect of the present invention, the sensor includes a strain resistance element whose resistance changes according to vibration of the cantilever, a capacitance element whose capacitance changes according to vibration of the cantilever, or a cantilever of the cantilever. Include a piezoelectric element or electromagnetic induction element that generates a voltage in response to vibration.

[0015] Further, in the fifth feature of the present invention, the biosensor chip has a surface covered with an insulator thin film, a thin film to which a substance to be detected can adhere, or the insulator thin film and the insulator thin film. A cantilever made of a semiconductor coated with a laminated film with a thin film to which the substance to be detected can adhere, and the cantilever so as to detect a vibration state of the cantilever. It consists of a sensor built into the chiller.

[0016] This biosensor chip can be produced using a technique capable of manufacturing MEMS (Micro Electro Mechanical Systems), which is a system including a mechanical part that is movable as a part of a semiconductor element.

The invention's effect

In short, according to the present invention, since the self-detecting sensor for detecting the vibration of the cantilever is provided in the cantilever, it can be used easily without having to adjust again when using the biosensor. The effect of is obtained.

Brief Description of Drawings

FIG. 1 is a schematic diagram showing a nanosensor according to a first embodiment of the present invention.

FIG. 2 is a flowchart showing a processing routine for detecting a substance attached to the cantilever according to the first embodiment.

FIG. 3 is a schematic diagram showing a modification of the first embodiment.

FIG. 4 is a schematic view showing another modification of the first embodiment.

FIG. 5 is a schematic view showing a second embodiment.

FIG. 6 is a schematic view showing a third embodiment.

FIG. 7 is a schematic view showing a fourth embodiment.

FIG. 8 is a schematic view showing a fifth embodiment.

FIG. 9 is a schematic view of a biosensor using a conventional optical lever.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. As shown in FIG. 1, the biosensor chip according to the first embodiment includes a thin plate-like cantilever 10 formed so as to be continuous with a base 12. The shape of the cantilever 10 may be a single triangle or elongated shape, as shown in Fig. 9, in which the base end is separated into two parts and the tip is connected to form a V shape. May be.

[0020] The pedestal 12 is attached with an actuator 14 made of a piezoelectric element that vibrates the cantilever 10 by exciting the pedestal. The actuator 14 is attached so as to be integrated with the pedestal by being attached or mechanically joined to the pedestal. Akuchu The position where the eta is attached can be any position where the cantilever can be vibrated in the thickness direction of the cantilever. As shown in the figure, the side where the cantilever is not formed or the side where the cantilever is formed Attached to.

In addition, a strain resistance element 16 that is a self-detecting sensor is formed in a predetermined region including a boundary portion between the cantilever 10 and the base 12. When the cantilever is vibrated in the thickness direction by the actuator, tensile and compressive stress is generated at the boundary between the cantilever base and the resistance value of the strain resistance element 16 changes. The state can be detected.

The cantilever 10 can be formed integrally with the pedestal by etching a semiconductor substrate such as silicon into a thin plate while leaving a portion corresponding to the pedestal. The strain resistance element 16 is formed by forming a pair of electrodes using semiconductor technology at the boundary with the cantilever pedestal and forming a strain resistance pattern by ion implantation of impurity atoms such as boron between the electrodes. can do. The resistance value of the strain resistance is preferably 2 kΩ or less. Note that the cantilever and the pedestal are preferably formed of a silicon substrate, but an electrode that is not ion-implanted may be formed and the strain resistance element may be attached.

A detection circuit 18 for detecting a change in the resistance value of the strain resistance element is connected to the electrode of the strain resistance element 16. The detection circuit 18 includes a bridge circuit that forms a Wheatstone bridge together with the strain resistance element 16, and a power source that applies a voltage to the bridge circuit. The detection circuit 18 detects a resistance change of the strain resistance element 16 as a voltage change, and detects the detected signal. Is output. The detection circuit 18 is connected to a positive feedback circuit 20 for driving the actuator 14 to resonate the cantilever 10 and an F—V conversion circuit (FM demodulation circuit) 22 for converting the frequency into a voltage. A personal computer 26 for data processing and display is connected to the F—V conversion circuit 22!

[0024] Next, the principle for detecting the mass of the substance attached to the cantilever of this embodiment will be described. The cantilever sags when an external force is applied, and the resonance frequency changes when the mass changes in a resonating state. Considering a cantilever as a harmonic oscillator, the operation of a force cantilever can be expressed by the following force equation (1).

[0025] [Equation 1]

[0026] where m is the effective mass of the cantilever, Z is the amount of strain of the cantilever, k is the spring constant of the cantilever, ξ is the viscosity of the liquid in which the cantilever is immersed, F is the excitation force of the actuator, ω is the frequency of the actuator, that is, the cantilever.

[0027] From the above equation (1), the resonance frequency ω of the cantilever 10 is the effective mass m of the cantilever 10

0

And the following equation (2) using the spring constant k.

[0028] [Equation 2]

[0029] Here, with the cantilever resonating at a frequency ω, the mass of the cantilever is Δ

0

When m is increased, the above equation (1) is expressed as the following equation (3).

[0030] [Equation 3]

[0031] When these equations are represented by a change in resonance frequency Δω, the following equation (4) is obtained.

[0032] drawings

△ _ω = Ichi ^^! _{Δ ιη}

2 m ... (4 ^

[0033] Therefore, from the above equation (4), by detecting the change in the frequency of the cantilever, the change in the mass of the force cantilever, that is, the mass of the substance attached to the cantilever can be detected. Since the change in frequency can be measured with an accuracy of 1 Hz or less, the above equation (4) means that the change in the mass of the cantilever can be measured with a picogram or femtogram. For example, the spring constant k is lNZm, the resonance frequency ω is 100 kHz,

0

If the effective mass m is lOng, the mass of the substance attached to the cantilever can be detected with a sensitivity of about lpgZHz. [0034] Further, since the above equation (4) can be described as the following equation (5), the detection sensitivity can be reduced by reducing the mass of the cantilever and / or increasing the resonance frequency. Can be made higher.

[0035] [Equation 5]

△ m—— „m

Δ ω ω ₀ …)

[0036] A micromachine process can be used to reduce the mass of the cantilever.

[0037] According to the present embodiment, the resonance frequency gradually decreases as the mass of the adhering material to the cantilever increases. Therefore, the resonance frequency changing force detected by the vibration detection sensor formed of the strain resistance element is used. It is possible to detect the mass and therefore the weight of the deposits attached to the cantilever.

[0038] Hereinafter, a measurement method using the biosensor of the present embodiment will be described.

[0039] When an excitation signal is input from the positive feedback circuit 20 to the actuator 14, the pedestal 12 is vibrated, and thereby the cantilever 10 is vibrated in the thickness direction of the cantilever. When the cantilever 10 is immersed in the reaction solution in the container 24, the reaction solution adheres to the cantilever and the frequency of the cantilever decreases slightly due to the influence of the viscosity of the reaction solution. In addition, since various vibration modes are generated at this time, the cantilever resonates at a frequency different from the original resonance frequency.

[0040] Since the movement of the cantilever and the pedestal at this time is not integral, tensile and compressive stress is generated in the strain resistance element, and the resistance of the strain resistance element changes. For this reason, when a constant voltage is applied to the strain resistance element, the current changes according to the vibration of the cantilever. By detecting this current change as a voltage change by the bridge circuit of the detection circuit, the frequency of the cantilever can be detected, and thereby the vibration state of the cantilever can be detected.

[0041] The voltage change detected by the detection circuit 18 is amplified by the positive feedback circuit 20, the phases are aligned, and the voltage change is input to the actuator. As a result, the cantilever is vibrated at the resonance frequency. In addition, the voltage change detected by the detection circuit is an analog signal (output Force V) and input to computer 26.

Next, arithmetic processing by the computer will be described with reference to FIG. In step 100, the analog signal output from the F—V conversion is converted into a digital signal and taken in, and in step 102, the output V is stored in the memory of the computer.

[0043] In the next step 104, the output acquired last time is compared with the output acquired this time, and the output change Δν is calculated. In step 106, it is determined whether or not the output V has changed. If it is determined, the mass of the substance attached to the cantilever is calculated in step 108 based on the above equation (4). Thereby, the time change of the mass of the substance attached to the cantilever can be detected. In addition, the total amount of the substance attached to the cantilever within the predetermined time can be detected by accumulating the time change of the mass over the predetermined time.

[0044] The mass of the substance adhering to the cantilever detected in this way is displayed on a display device such as an LCD connected to the computer. In addition, the computer can process and calculate noise removal, reaction speed, and the like.

[0045] In order to use the biosensor of this embodiment for detection of an antigen-antibody reaction, the antibody is first attached to the surface of the cantilever and the cantilever is immersed in the reaction solution, and then the measurement sample having the antigen is reacted. Put into the reaction solution in container 24. As a result, it is clarified whether or not the constitutional ability has factors such as allergies. Contrary to this case, when an antibody is first placed in a reaction container and then an antigen is introduced, allergens are produced in the human body.

In the present embodiment, an example in which a piezoelectric element is used as an actuator has been described. In this embodiment, as shown in FIG. 3, each electrode portion of the piezoelectric element 14 is covered with an insulating film 28, and It may be electrically insulated. Also in this case, in the same manner as described above, one of the insulating films of the actuator is bonded or mechanically bonded to the pedestal so that the actuator is integrated with the pedestal. Since the actuator electrode is covered with the insulating film 28, measurement can be started immediately by immersing the cantilever in the reaction solution.

[0047] Further, as shown in FIG. 4, the cantilever and the base are covered with an insulating film 28. May be. In this case, the actuator may be covered with an insulating film as shown in FIG. 3, or may not be covered. As a result, when the cantilever is immersed in the reaction solution, leakage current can be prevented from flowing on the surface of the cantilever, and the current can be accurately measured by the detection circuit.

[0048] Next, a second embodiment of the present invention will be described. In the second embodiment, instead of the strain resistance element of the first embodiment, a capacitance element whose capacitance changes according to the vibration of the cantilever is used as a sensor for detecting a change in resonance frequency. It is a thing.

In the present embodiment, as shown in FIG. 5, the counter electrode 30 is fixed to the base 12 so as to face and parallel to the cantilever 10, and the capacitance between the cantilever 10 and the counter electrode 30 is fixed. An element is configured. The cantilever 10 and the counter electrode 30 are connected to a detection circuit 18 having a bridge circuit that forms a Wheatstone bridge together with the electrostatic capacitance element in the same manner as described above.

[0050] Thereby, when the cantilever vibrates, the electrostatic capacitance of the capacitive element periodically changes. Therefore, the bridge circuit of the detection circuit can detect the vibration of the cantilever and output the vibration signal.

[0051] According to the present embodiment, the change in the resonance frequency is detected from the vibration signal output from the detection circuit, and the change force of this resonance frequency is changed over time in the mass of the substance attached to the cantilever as described above. Can be detected.

Next, a third embodiment of the present invention will be described with reference to FIG. In the present embodiment, the counter electrode of the second embodiment is used as an actuator. As a sensor for detecting the vibration of the cantilever, a strain resistance element similar to that in the first embodiment is used.

The strain resistance element 16 is connected to the bridge circuit of the detection circuit 18 as in the first embodiment. Further, the base end side of the cantilever 10 is grounded, and the counter electrode 30 constituting the capacitance element is connected to the positive feedback circuit 20.

According to the present embodiment, the voltage change of the strain resistance element 16 is detected by the bridge circuit of the detection circuit 18, the detected signal is input to the positive feedback circuit 20, and the excitation signal is transmitted to the counter electrode 30. Therefore, the cantilever 10 is controlled to resonate and vibrate. The signal detected by the bridge circuit of the detection circuit 18 is input to the computer 26 via the FV conversion circuit 22, and the resonance frequency changing force in the computer 26 is the mass of the substance adhering to the cantilever as described above. A change in time is detected.

[0055] Next, a fourth embodiment of the present invention will be described with reference to FIG. In the present embodiment, an electromagnetic induction type actuator is used in place of the electrostatic induction actuator of the third embodiment. In the present embodiment, the electromagnetic induction coil 32 is fixed to the pedestal 12 so as to face and substantially parallel to the cantilever 10, and the surface of the cantilever 10 is coated with a magnetic thin film 34 made of a magnetic material. The As a sensor for detecting the vibration of the cantilever, a strain resistance element similar to that in the first embodiment is used.

According to the present embodiment, similarly to the above, the signal from the strain resistance element 16 is detected by the bridge circuit of the detection circuit 18, and the detected signal is input to the positive feedback circuit 20 and the electromagnetic induction coil 32. Therefore, the cantilever 10 is resonantly vibrated. In addition, the signal detected by the bridge circuit of the detection circuit 18 is input to the computer 26 through the FV conversion circuit 22 in the same manner as described above, and the resonance frequency changing force in the computer 26 is also a substance attached to the cantilever. Changes in the mass of the time are detected. In Fig. 7, only one side is coated, but it may be coated on both sides, or on the opposite side of Fig. 7.

[0057] FIG. 8 shows a fifth embodiment of the present invention, in which a special chemically reactive group is attached to the insulating film of the cantilever shown in FIG. 3 in order to attach a substance to be detected. The thin film 36 made of gold or the like is coated. The type of thin film is appropriately selected according to the substance to be deposited. As a result, it is possible to detect a change in the mass of a substance attached to a cantilever such as a protein, DNA, antibody, or antigen that has been artificially selected via a thiol group.

In the above-described embodiment, the example using the strain resistance element (the first embodiment, etc.) or the capacitance element (the second embodiment) as the self-sensing element has been described. A piezoelectric element, an electromagnetic induction element, a temperature detection element, or the like may be used (piezoelectric element 5 in FIG. 1). 6. Refer to electromagnetic induction element 66). Further, as the actuator, a temperature-driven actuator or an optically-driven actuator may be used instead of the piezoelectric element and the electrostatic-driven capacitive element. Furthermore, although the example in which the cantilever is coated with an insulating film has been described, it may be covered with a natural acid film. For detection of the frequency shift amount of the signal output from the detection circuit, a PLL circuit, a quadrature demodulation circuit or the like may be used.

[0059] In addition, in the above, a plurality of cantilevers may be provided on the force base described in the example using one cantilever, and the substance attached to each cantilever may be measured.

That is, each of the above embodiments may be configured to use a plurality of chips at the same time.

Industrial applicability

[0061] As described above, the biosensor and the biosensor chip according to the present invention do not need to be readjusted when used, and can be used easily. In particular, the antigen-antibody reaction and proteins are highly sensitive. Suitable for nanosensors and biosensor chips that can be measured with

Claims

The scope of the claims

[1] A biosensor,

Cantilevers,

An actuator for vibrating the cantilever;

A sensor provided on the cantilever to detect a vibration state of the cantilever;

Control means for controlling the actuator so that the cantilever resonates based on a vibration state detected by the sensor;

Detection means for detecting the amount of substance attached to the cantilever based on a change in the vibration state detected by the sensor in a state where the cantilever is controlled to resonate;

A noise sensor comprising:

2. The biosensor according to claim 1, wherein the cantilever is immersed in a liquid.

[3] The cantilever is covered with an insulator thin film, a thin film to which a substance to be detected can adhere, or a laminated film of the insulator thin film and a thin film to which the substance to be detected can be attached. The biosensor according to claim 1.

4. The biosensor according to claim 1, wherein the actuator is configured by a piezoelectric element, a capacitance element, or an electromagnetic induction element.

[5] The sensor is connected to a strain resistance element whose resistance changes according to the vibration of the cantilever, a capacitance element whose electrostatic capacity changes according to the vibration of the cantilever, or a voltage according to the vibration of the cantilever. The biosensor according to claim 1, wherein the biosensor is configured to include a generated piezoelectric element or electromagnetic induction element.

[6] The biosensor according to claim 4, wherein the electrode portions of the piezoelectric element as the actuator are each covered with an insulating film.

7. The biosensor according to claim 1, wherein the detection means detects an adhesion amount of a substance attached to the cantilever based on a change in a resonance frequency of the cantilever.

8. The biosensor according to claim 7, wherein the detection means detects an adhesion amount (A m) of a substance attached to the cantilever based on the following formula. Δ πι = — 2 · Δ ω / ω, m

ο

In the above formula, m = effective mass of the cantilever

ω = cantilever resonance frequency

0

Δ ω = change in resonance frequency

Δ πι = cantilever effective mass change, that is, the amount of substances attached to the cantilever

[9] A neurosensor,

Cantilevers,

An actuator for vibrating the cantilever;

The detection result force of the sensor in a state where the cantilever is controlled to resonate. Detection means for calculating the change in the resonance frequency of the cantilever and detecting the amount of the substance attached to the cantilever based on the change When,

A noise sensor comprising:

10. The biosensor according to claim 9, wherein the detection means detects an adhesion amount (A m) of a substance attached to the cantilever based on the following formula.

Δ πι = — 2 ·. Ω / ω · πι

ο

In the above formula, m = effective mass of the cantilever

ω = cantilever resonance frequency

0

Δ ω = change in resonance frequency

11. The biosensor according to claim 9, wherein the cantilever is immersed in a liquid.

[12] The cantilever is formed by stacking an insulating thin film, a thin film to which a substance to be detected can be attached, or a thin film to which the above-described insulating thin film and the substance to be detected coated thereon can be attached. The biosensor according to claim 9, which is coated with a film.

13. The biosensor according to claim 9, wherein the actuator is configured by a piezoelectric element, a capacitance element, or an electromagnetic induction element.

[14] The biosensor according to claim 13, wherein the electrode portions of the piezoelectric element as the actuator are each coated with an insulating film.

[15] The sensor may be a strain resistance element whose resistance changes according to vibration of the cantilever, a capacitance element whose capacitance changes according to vibration of the cantilever, or a voltage according to vibration of the cantilever. The biosensor according to claim 9, wherein the biosensor is configured to include a generated piezoelectric element or electromagnetic induction element.

[16] A biosensor chip,

Cantilevers,

A sensor built in the cantilever to detect a vibration state of the cantilever,

The cantilever is made of a semiconductor, and the surface of the semiconductor can be attached to an insulator thin film, a thin film to which a substance to be detected can adhere, or the insulator thin film and the substance to be detected coated thereon. A biosensor chip characterized by being coated with a laminated film with a thin film.