WO2006082668A1 - Biocapteur et sa puce - Google Patents

Biocapteur et sa puce Download PDF

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Publication number
WO2006082668A1
WO2006082668A1 PCT/JP2005/013881 JP2005013881W WO2006082668A1 WO 2006082668 A1 WO2006082668 A1 WO 2006082668A1 JP 2005013881 W JP2005013881 W JP 2005013881W WO 2006082668 A1 WO2006082668 A1 WO 2006082668A1
Authority
WO
WIPO (PCT)
Prior art keywords
cantilever
biosensor
thin film
substance
sensor
Prior art date
Application number
PCT/JP2005/013881
Other languages
English (en)
Japanese (ja)
Inventor
Sumio Hosaka
Hayato Sone
Haruki Okano
Original Assignee
National University Corporation Gunma University
Tokyo Sokki Kenkyujo Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National University Corporation Gunma University, Tokyo Sokki Kenkyujo Co., Ltd. filed Critical National University Corporation Gunma University
Publication of WO2006082668A1 publication Critical patent/WO2006082668A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
    • G01N5/02—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by absorbing or adsorbing components of a material and determining change of weight of the adsorbent, e.g. determining moisture content
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/025—Change of phase or condition
    • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
    • G01N2291/0257—Adsorption, desorption, surface mass change, e.g. on biosensors with a layer containing at least one organic compound

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 4 is an actuator that vibrates the cantilever
  • 5 is a detection circuit
  • 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.
  • 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)
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the actuator can be constituted by a piezoelectric element, a capacitance element, or an electromagnetic induction element.
  • the senor 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.
  • 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
  • a cantilever of the cantilever include a piezoelectric element or electromagnetic induction element that generates a voltage in response to vibration.
  • 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.
  • 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.
  • MEMS Micro Electro Mechanical Systems
  • the self-detecting sensor for detecting the vibration of the cantilever 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 k ⁇ or less.
  • 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!
  • the cantilever sags when an external force is applied, and the resonance frequency changes when the mass changes in a resonating state.
  • the operation of a force cantilever can be expressed by the following force equation (1).
  • 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
  • ⁇ is the viscosity of the liquid in which the cantilever is immersed
  • F is the excitation force of the actuator
  • ⁇ is the frequency of the actuator, that is, the cantilever.
  • the resonance frequency ⁇ of the cantilever 10 is the effective mass m of the cantilever 10
  • the mass of the cantilever is ⁇
  • 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.
  • the spring constant k is lNZm
  • the resonance frequency ⁇ is 100 kHz
  • the mass of the substance attached to the cantilever can be detected with a sensitivity of about lpgZHz.
  • the detection sensitivity can be reduced by reducing the mass of the cantilever and / or increasing the resonance frequency. Can be made higher.
  • a micromachine process can be used to reduce the mass of the cantilever.
  • 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.
  • the pedestal 12 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.
  • the cantilever 10 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.
  • 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.
  • the voltage change detected by the detection circuit is an analog signal (output Force V) and input to computer 26.
  • 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.
  • step 104 the output acquired last time is compared with the output acquired this time, and the output change ⁇ is calculated.
  • 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.
  • 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.
  • the computer can process and calculate noise removal, reaction speed, and the like.
  • 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.
  • the constitutional ability has factors such as allergies.
  • allergens are produced in the human body.
  • 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.
  • the cantilever and the base are covered with an insulating film 28. May be.
  • the actuator may be covered with an insulating film as shown in FIG. 3, or may not be covered.
  • 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.
  • the bridge circuit of the detection circuit can detect the vibration of the cantilever and output the vibration signal.
  • 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.
  • the counter electrode of the second embodiment is used as an actuator.
  • 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.
  • 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.
  • an electromagnetic induction type actuator is used in place of the electrostatic induction actuator of the third 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.
  • a strain resistance element similar to that in the first embodiment is used.
  • 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.
  • 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.
  • 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.
  • 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.
  • electromagnetic induction element 66 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.
  • the example in which the cantilever is coated with an insulating film has been described, it may be covered with a natural acid film.
  • a PLL circuit, a quadrature demodulation circuit or the like may be used for detection of the frequency shift amount of the signal output from the detection circuit.
  • 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.
  • each of the above embodiments may be configured to use a plurality of chips at the same time.
  • the biosensor and the biosensor chip according to the present invention do not need to be readjusted when used, and can be used easily.
  • the antigen-antibody reaction and proteins are highly sensitive. Suitable for nanosensors and biosensor chips that can be measured with

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Acoustics & Sound (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Micromachines (AREA)

Abstract

L’invention concerne un biocapteur composé d’un cantilever, d’un actionneur permettant de faire osciller le cantilever, d’un capteur installé sur le cantilever afin de détecter l’état d’oscillation du cantilever, de moyens pour commander l’actionneur de manière à faire résonner le cantilever en fonction de l’état d’oscillation détecté par le capteur et d'un moyen permettant de détecter la quantité de substance adhérant au cantilever en fonction de la variation dans l’état d’oscillation détecté par le capteur dans un état où le cantilever est commandé pour résonner.
PCT/JP2005/013881 2005-02-01 2005-07-28 Biocapteur et sa puce WO2006082668A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005025021A JP2006214744A (ja) 2005-02-01 2005-02-01 バイオセンサ及びバイオセンサチップ
JP2005-025021 2005-02-01

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US20090263065A1 (en) 2006-08-07 2009-10-22 Nsk Ltd. Bearing unit raceway ring member, bearing unit, and method and apparatus for manufacturing bearing unit raceway ring member
JP4845649B2 (ja) * 2006-09-07 2011-12-28 キヤノン株式会社 微小物体の表面評価装置及び微小物体の表面評価方法
WO2008042199A2 (fr) 2006-09-29 2008-04-10 Cyberoptics Semiconductor, Inc. Capteur de particules en forme de substrat
US20100095774A1 (en) * 2006-12-05 2010-04-22 National University Corporation Gunma University Mass measuring device and cantilever
JP2008241619A (ja) * 2007-03-28 2008-10-09 Gunma Univ カンチレバー、バイオセンサ、及びプローブ顕微鏡
JP2009085777A (ja) * 2007-09-28 2009-04-23 National Institute Of Advanced Industrial & Technology 振動子チップ、検出センサ
JP2009133772A (ja) * 2007-11-30 2009-06-18 National Institute Of Advanced Industrial & Technology 検出センサ、振動子
JP2014190771A (ja) * 2013-03-26 2014-10-06 National Institute Of Advanced Industrial & Technology 質量測定方法及び質量測定装置
GB2599858B (en) * 2019-07-10 2024-01-03 Nec Corp Membrane-type surface stress sensor and analysis method using same

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JP2004028956A (ja) * 2002-06-28 2004-01-29 National Institute Of Advanced Industrial & Technology 吸脱着量測定方法及び装置
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JP2004028956A (ja) * 2002-06-28 2004-01-29 National Institute Of Advanced Industrial & Technology 吸脱着量測定方法及び装置
JP2004125706A (ja) * 2002-10-04 2004-04-22 Sony Corp 相互反応作用検出方法及びバイオアッセイ装置、並びにバイオアッセイ用基板

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