WO2016043402A1 - Interdigitated microelectrode biosensor - Google Patents

Interdigitated microelectrode biosensor Download PDF

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Publication number
WO2016043402A1
WO2016043402A1 PCT/KR2015/004697 KR2015004697W WO2016043402A1 WO 2016043402 A1 WO2016043402 A1 WO 2016043402A1 KR 2015004697 W KR2015004697 W KR 2015004697W WO 2016043402 A1 WO2016043402 A1 WO 2016043402A1
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Prior art keywords
cross
electrode
signal
impedance
electrodes
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PCT/KR2015/004697
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French (fr)
Korean (ko)
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황교선
조원우
김태송
박종배
유용경
고경옥
김혜진
이준옥
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한국과학기술연구원
주식회사 캔티스
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Publication of WO2016043402A1 publication Critical patent/WO2016043402A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • 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

Definitions

  • the present invention relates to a cross-electrode biosensor, and more particularly, to a cross-electrode biosensor which detects the presence and the concentration of a biomaterial such as a protein through an impedance measurement between electrodes.
  • biosensors have been developed to detect the presence and concentration of various biological materials such as genes and proteins by electrical methods.
  • One example is the use of interdigitated microelectrodes. It is evaluated that even if the concentration of the biomaterial is low, the measurement is properly performed because the region in which the receptor that specifically binds the biomaterial is fixed is substantially wide in a zigzag form.
  • the problem to be solved by the present invention is a cross-electrode capable of accurately detecting the presence and concentration of a small amount of biological material through the measurement of the impedance between the electrodes, even if the conductive particles are not used so that the current flows between the electrodes To provide a biosensor.
  • Comb-shaped two electrodes are installed on the substrate in the form of interlocking staggered spaced apart by a predetermined interval, the cross-electrode portion is installed between the two electrodes to capture the target material;
  • a driving signal applying unit applying a driving frequency between the two electrodes
  • An impedance measuring unit measuring an impedance between the two electrodes By being made, including
  • the target material is analyzed by measuring a change in impedance which appears depending on whether the target material is captured.
  • the driving frequency has a frequency range of 10 Hz to 100 Hz.
  • the gap between the two electrodes is preferably 3 ⁇ 7 ⁇ m.
  • a protective cap is installed on the cross-electrode portion so that a channel is formed on the cross-electrode portion, and an adsorption prevention layer is coated on a surface inside the channel where the receptor is not installed.
  • the adsorption prevention layer is preferably made of BSA (Bovine Serum Albumin).
  • Two electrodes having a comb shape are installed on the substrate in an interlocking manner while being spaced apart from each other by a predetermined interval, and the reference cross-electrode portion does not capture the target material;
  • a signal cross-electrode unit disposed on the substrate in a shape in which two comb-shaped electrodes are alternately engaged with each other at a predetermined interval, and the target material is captured by a receptor provided between the two electrodes;
  • a driving signal applying unit installed to apply a driving frequency to the reference crossing electrode unit and the signal crossing electrode unit, respectively;
  • a reference impedance measuring unit which measures an impedance between two electrodes of the reference cross-electrode unit as a reference impedance
  • a signal impedance measuring unit which measures an impedance between two electrodes in the signal crossing electrode unit as a signal impedance
  • a differential amplifier installed to differentially amplify the reference impedance and the signal impedance from the reference impedance measuring unit and the signal impedance measuring unit;
  • the target material may be analyzed by changing the signal impedance relative to the reference impedance.
  • the driving frequency applied by the driving signal applying unit preferably has a frequency range of 10 Hz to 100 Hz.
  • the gap between the two electrodes forming the reference cross-electrode portion is 3 to 7 ⁇ m, and the gap between the two electrodes forming the signal cross-electrode portion is 3 to 7 ⁇ m.
  • Protective caps are respectively provided on the reference cross-electrode portion and the signal cross-electrode portion so that channels are formed on the reference cross-electrode portion and the signal cross-electrode portion, respectively, and an adsorption preventing layer on the surface inside the channel where the receptor is not installed. It is preferred to be coated.
  • the adsorption prevention layer is preferably made of BSA (Bovine Serum Albumin).
  • the capture of the target material occurs only at the signal cross-electrode part or the signal cross-over part.
  • the receptor may be provided only in the electrode portion, and the target material may be provided to both the reference cross electrode portion and the signal cross electrode portion so that the capture of the target material occurs only at the signal cross electrode portion.
  • the presence or absence of a biological substance and its concentration can be accurately detected through impedance measurement without using conductive particles as in the prior art.
  • the precise detection can be made while using a low frequency of 10Hz ⁇ 100Hz so as not to denature or damage the target biomaterial.
  • the use of differential amplification has the advantage of enabling precise detection of small amounts of biomaterials.
  • FIG. 1 is a view for explaining the principle of the cross-electrode biosensor according to the present invention.
  • FIG. 2 is a graph for explaining the change in the impedance of the cross-electrode portion 20 by the reaction of the receptor 31 and the target biomaterial 32;
  • FIG. 3 is a view for explaining a cross-electrode biosensor according to the present invention to which a differential amplifier is applied;
  • FIG. 4 is a view for explaining the manufacturing process of the cross-electrode portion 20 of FIG. 1 and the specific binding of the target biomaterial 32;
  • 6 and 7 are diagrams for explaining the detection capability of the cross-electrode biosensor of FIG. 3, and look at the contribution to the specific binding of the target biomaterial 32;
  • FIG. 8 is a graph illustrating a beta amyloid detection test result at a concentration of 100 pg / mL for explaining the detection capability of the cross-electrode biosensor of FIG. 3, and looks at the contribution of the differential amplifier 300.
  • driving signal applying unit 10 substrate
  • reference cross-electrode unit 225 reference impedance measuring unit
  • FIG. 1 is a view for explaining the principle of the cross-electrode biosensor according to the present invention.
  • the cross-electrode part 20 is installed on the substrate 10.
  • the interdigitated microelectrode part (IME part) 20 is installed in a form in which two electrodes 21 and 22 having a comb shape are interlocked with each other at predetermined intervals.
  • the impedance between the two electrodes 21, 22 is arranged as follows.
  • Z is impedance
  • R is resistance
  • X reactance
  • C is capacitance
  • w angular frequency.
  • the reactance X is divided into the inductor component X L and the capacitor component X C. Since the two electrodes 21 and 22 are not electrically connected directly, the inductor component X L is ignored and only the capacitor component X C is present. It can be said that.
  • a receptor (mainly an antibody, aptamer, etc.) 31 that specifically reacts with the target biomaterial 32 is fixed in the space between the two electrodes 21, 22 and the target biomaterial 32 is the receptor 31.
  • the impedance change between the two electrodes (21, 22) is confirmed when reacting to the) it is possible to quantitatively analyze the target biomaterial (32).
  • FIG. 2 is a graph for explaining the change in the impedance of the cross-electrode portion 20 by the reaction of the receptor 31 and the target biomaterial 32.
  • the receptor 31 and the target biomaterial 32 are specifically bound, the water (or buffer solution, serum, blood, etc.) existing between the two electrodes 21 and 22 is pushed out and the target biomaterial 32 is positioned.
  • the resistance increases.
  • reactance is the dielectric constant of water (or buffer solutions, serum, blood, etc.) decreases the value of the capacitance (C) by the nature of the small target biological material (32) than the value X C is greater according -X value C Will decrease.
  • the frequency of the driving frequency is high, the current mainly flows through the space above the specifically coupled target biomaterial 32, that is, root A, so that detection of the target biomaterial 32 is not properly performed.
  • the frequency is high, the target biomaterial 32 may be damaged by high frequency and may not be detected properly.
  • the present invention is characterized by using a low driving frequency of 10 Hz to 100 Hz so that the current can flow through the root B. Since the frequency is low, the target biomaterial 32 is also prevented from being damaged. Of course, in this case, since the frequency is low, there is a disadvantage in that it is difficult to detect a change in the fine impedance at the root B, but this disadvantage can be overcome by using a differential amplifier as described below.
  • the gap between the two electrodes 21 and 22 is preferably 3 to 7 ⁇ m. If the gap is too small and less than 3 ⁇ m, the deviation of the detection signal is too large to perform a reliable test. If the gap is too large and larger than 7 ⁇ m, the sensitivity is insufficient to detect a small amount of the biomaterial 32. Because. Considering the deviation and sensitivity, the case of 5 ⁇ m is most preferable.
  • FIG. 3 is a view illustrating a cross-electrode biosensor according to the present invention to which a differential amplifier is applied.
  • the signal cross-electrode portion 120 and the reference cross-electrode portion 220 are installed on the substrate 1, and each of the signal cross-electrode portion 120 and the reference cross-electrode portion 220 is illustrated in FIG. 1.
  • Two electrodes 21 and 22 having a comb-like shape, such as the cross-electrode portion 20 of, are arranged in a staggered manner while being spaced apart by a predetermined interval.
  • the signal cross-electrode unit 120 captures the target biomaterial 32 by the receptor 31 disposed between the two electrodes 21 and 22, while the capture cross-electrode unit 220 does not capture the target biomaterial 32. .
  • the receiver 31 may be provided only in the unit 120, and the target biomaterial 32 may be provided to both the signal cross-electrode unit 120 and the reference cross-electrode unit 220.
  • the driving frequency is applied to the signal cross electrode unit 120 and the reference cross electrode unit 220 through the driving signal applying unit 1. Then, the impedance at the signal cross-electrode unit 120 is measured as the signal impedance in the signal impedance measuring unit 125, and the impedance at the reference cross-electrode unit 220 is measured as the reference impedance in the reference impedance measuring unit 225. .
  • the signal impedance will be the impedance between the two electrodes 21 and 22 for the case where the target biomaterial 32 is captured between the two electrodes 21 and 22, and the reference impedance will be between the two electrodes 21 and 22. Will be the impedance between the two electrodes 21, 22 for the case where the target biomaterial 32 is not captured.
  • the differential amplifier 300 receives the signal impedance and the reference impedance, respectively, and differentially amplifies the signal impedance and outputs a result signal.
  • FIG. 4 is a view for explaining the manufacturing process and the specific binding of the target biomaterial 32 of the cross-electrode portion 20 of FIG.
  • a 500 nm thick silicon oxide film SiO 2 , 11 is formed on the silicon substrate 10 by thermal oxidation, and then sputtering on the silicon oxide film 11 is performed.
  • a metal layer 20a is formed by sequentially stacking Ti 30 nm and Pt 150 nm by sputtering. The Ti layer is used as an adhesion layer for increasing the bonding force between the Pt layer and the silicon oxide film 11.
  • a photoresist film is coated on the metal layer 20a and the photoresist film is patterned by a photolithography process to form a photoresist pattern 40.
  • the metal layer 20 is etched using inductively coupled plasma reactive ion etcher (ICP-RIE) until the silicon oxide film 11 is exposed using the photoresist pattern 40 as an etch mask.
  • ICP-RIE inductively coupled plasma reactive ion etcher
  • Specific binding of the target biomaterial 32 is performed as follows.
  • a Calixcrown Self-Assembled Monolayer is formed as a connecting molecule layer 33 for selectively fixing the beta amyloid antibody on the surface of the silicon oxide film 11 between the two electrodes 21 and 22.
  • the beta amyloid antibody is fixed to the linking molecule layer 33 as the receptor 31.
  • the beta amyloid which is the target biomaterial 32, is selectively bound to the receptor 31.
  • FIG. 5 is a diagram for describing the channel 55.
  • the protective cap 50 is installed on the two electrodes 21 and 22 so that the two electrodes 21 and 22 are placed in the channel 55.
  • the channel 55 by the protective cap 50 also serves to help the sample enter the specific binding region.
  • the protective cap 50 is preferably made of a polydimethylsiloxane (PDMS) material.
  • the target biomaterial 32 When a sample containing various components is introduced into the channel 55, only the target biomaterial 32 that specifically reacts with the receptor 31 is bound to the receptor 31.
  • the target biomaterial 32 will be beta amyloid.
  • the adsorption preventing layer 51 is preferably made of BSA (Bovine Serum Albumin).
  • FIG. 6 and 7 illustrate the detection capability of the cross-electrode biosensor of FIG. 3 and illustrate the contribution to specific binding of the target biomaterial 32.
  • the test unit 100 and the control unit 200 are divided into the test unit 100 and the control unit 200, respectively, as shown in FIG. 3.
  • the electrode portions 220 were formed in pairs.
  • the beta amyloid antibody is fixed to the signal cross-electrode unit 120 of the experiment unit 100, and the PSA (prostate-specific antigen) for selectivity control (negative control) to the signal cross-electrode unit 120 of the control unit 200.
  • Antibodies were fixed.
  • a PDMS chip having two microchannels was attached to each of the experiment unit 100 and the control unit 200.
  • 0.1X PBS buffer solution was injected into both channels, and the stabilization was performed by observing the signal until the impedance signal output from each electrode part remained stable and stable.
  • the beta amyloid was injected into the channel to observe the change in the impedance signal to confirm the antigen-antibody reaction of the beta amyloid.
  • FIG. 7 is a graph showing impedance changes in the test unit 100 and the control unit 200 when 10, 100, and 1000 pg / mL of beta amyloid was sequentially injected.
  • the impedance change amount of the experiment unit 100 increases as the beta amyloid concentration increases (green), but in the case of the control unit 200 in which the non-specific antibody is immobilized, the beta amyloid is injected. It can be seen that the signal change is very insignificant (blue). This is a result reflecting that the specific binding of the beta amyloid that is the target biomaterial 32 is made in the experiment unit 100, while the control unit 200 does not perform such specific binding.
  • FIG. 8 is a graph illustrating a beta amyloid detection test result at a concentration of 100 pg / mL for explaining the detection capability of the cross-electrode biosensor of FIG. 3, and looks at the contribution of the differential amplifier 300.
  • FIG. 8A illustrates the impedance output from the signal cross electrode unit 120 and the reference cross electrode unit 220 without being differentially amplified. As can be seen in FIG. 8A, it is very difficult to quantitatively detect the amount of the target biomaterial 32 because impedance changes of the signal cross-electrode portion 120 and the reference cross-electrode portion 220 are not properly distinguished.
  • FIG. 8B differentially amplifies the signal impedance (red) output from the signal cross electrode unit 120 with respect to the reference impedance (blue) output from the reference cross electrode unit 220 and outputs the result.
  • Figure 8b it can be seen that even if a small amount of 100pg / mL can be properly measured because the distinction between the signal impedance and the reference impedance.
  • the presence or absence of a biological substance and its concentration can be accurately detected through impedance measurement without using conductive particles as in the prior art.
  • the precise detection can be made while using a low frequency of 10Hz ⁇ 100Hz so that the target biomaterial 32 is not denatured or damaged.
  • the use of differential amplification has the advantage of enabling precise detection of small amounts of biomaterials.

Abstract

Signal impedance is impedance between two electrodes (21, 22) when a target biomaterial (32) is captured between the two electrodes (21, 22), and reference impedance is impedance between two electrodes (21, 22) when a target biomaterial (32) is not captured between the two electrodes (21, 22). A differential amplifier (300) receives an input of each of the signal impedance and the reference impedance and outputs a resultant signal while differentially amplifying the signal impedance. If the signal impedance is differentially amplified on the basis of the reference impedance, the impedance difference therebetween is definitely shown and therefore the quantity of even a small amount of target biomaterial (32) can be quantitatively and precisely detected.

Description

교차전극 바이오센서Cross Electrode Biosensor
본 발명은 교차전극 바이오센서에 관한 것으로서, 특히 단백질 등과 같은 생체물질의 유무 및 그 농도를 전극 사이의 임피던스 측정을 통하여 검출하는 교차전극 바이오센서에 관한 것이다. The present invention relates to a cross-electrode biosensor, and more particularly, to a cross-electrode biosensor which detects the presence and the concentration of a biomaterial such as a protein through an impedance measurement between electrodes.
최근 들어 유전자, 단백질 등의 다양한 생체물질의 유무 및 그 농도를 전기적 방법으로 검출하는 바이오센서가 많이 개발되고 있다. 그 하나의 예가 교차전극(interdigitated microelectrode)을 이용하는 것이다. 이는 생체물질과 특이적 결합을 하는 수용체가 고정되는 영역이 지그재그 형태로서 실질적으로 매우 넓기 때문에 생체물질의 농도가 낮더라도 측정이 제대로 이루어진다는 평가를 받고 있다. Recently, many biosensors have been developed to detect the presence and concentration of various biological materials such as genes and proteins by electrical methods. One example is the use of interdigitated microelectrodes. It is evaluated that even if the concentration of the biomaterial is low, the measurement is properly performed because the region in which the receptor that specifically binds the biomaterial is fixed is substantially wide in a zigzag form.
대한민국 등록특허 제777973호(2007.11.29.공고)에 개시된 내용이 이와 같은 교차전극을 이용하는 것이다. 그러나 상기 등록특허의 경우에는 전극 사이에 흐르는 전류를 통하여 농도 측정이 이루어지기 때문에, 전극 사이에 전류가 통하도록 도전성입자를 별도로 이용해야 하는 번거로움이 있다. The contents disclosed in Republic of Korea Patent No. 777973 (announced November 29, 2007) is to use such a cross electrode. However, in the case of the registered patent, because the concentration measurement is made through the current flowing between the electrodes, there is a hassle to use the conductive particles separately so that the current flows between the electrodes.
[선행기술문헌][Preceding technical literature]
대한민국 등록특허공보 제777973호(2007.11.29.공고)Republic of Korea Patent No.777973 (announced November 29, 2007)
따라서 본 발명이 해결하고자 하는 과제는, 전극 사이에 전류가 통하도록 도전성 입자를 사용하지 않더라도, 전극 사이의 임피던스 측정을 통해서 소량의 생체물질의 존재 유무 및 그 농도를 정밀하게 검출할 수 있는 교차전극 바이오센서를 제공하는 데 있다. Therefore, the problem to be solved by the present invention is a cross-electrode capable of accurately detecting the presence and concentration of a small amount of biological material through the measurement of the impedance between the electrodes, even if the conductive particles are not used so that the current flows between the electrodes To provide a biosensor.
상기 과제를 달성하기 위한 본 발명의 일예에 따른 교차전극 바이오센서는, Cross-electrode biosensor according to an embodiment of the present invention for achieving the above object,
빗 모양을 하는 두 전극이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 기판 상에 설치되며, 표적물질의 포획을 위하여 상기 두 전극 사이에 수용체가 설치되는 교차전극부;Comb-shaped two electrodes are installed on the substrate in the form of interlocking staggered spaced apart by a predetermined interval, the cross-electrode portion is installed between the two electrodes to capture the target material;
상기 두 전극 사이에 구동주파수를 인가하는 구동신호 인가부; 및A driving signal applying unit applying a driving frequency between the two electrodes; And
상기 두 전극 사이의 임피던스를 측정하는 임피던스 측정부; 를 포함하여 이루어짐으로써, An impedance measuring unit measuring an impedance between the two electrodes; By being made, including
상기 표적물질의 포획 여부에 따라 나타나는 상기 임피던스의 변화를 측정하여 상기 표적물질을 분석하는 것을 특징으로 한다. The target material is analyzed by measuring a change in impedance which appears depending on whether the target material is captured.
상기 구동주파수는 10Hz ~100Hz 의 주파수 범위를 갖는 것이 바람직하다. Preferably, the driving frequency has a frequency range of 10 Hz to 100 Hz.
상기 두 전극 사이의 간극은 3~7㎛ 인 것이 바람직하다. The gap between the two electrodes is preferably 3 ~ 7㎛.
상기 교차전극부 상에 채널이 형성되도록 상기 교차전극부 상에 보호캡이 설치되고, 상기 수용체가 설치되지 않는 상기 채널 내부의 면에 흡착방지층이 코팅되는 것이 바람직하다. It is preferable that a protective cap is installed on the cross-electrode portion so that a channel is formed on the cross-electrode portion, and an adsorption prevention layer is coated on a surface inside the channel where the receptor is not installed.
상기 흡착방지층은 BSA(Bovine Serum Albumin)로 이루어지는 것이 바람직하다. The adsorption prevention layer is preferably made of BSA (Bovine Serum Albumin).
상기 과제를 달성하기 위한 본 발명의 다른 예에 따른 교차전극 바이오센서는, Cross-electrode biosensor according to another embodiment of the present invention for achieving the above object,
빗 모양을 하는 두 전극이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 기판 상에 설치되며, 표적물질의 포획이 이루어지지 않는 기준교차전극부;Two electrodes having a comb shape are installed on the substrate in an interlocking manner while being spaced apart from each other by a predetermined interval, and the reference cross-electrode portion does not capture the target material;
빗 모양을 하는 두 전극이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 상기 기판 상에 설치되며, 두 전극 사이에 설치되는 수용체에 의하여 표적물질의 포획이 이루어지는 신호교차전극부;A signal cross-electrode unit disposed on the substrate in a shape in which two comb-shaped electrodes are alternately engaged with each other at a predetermined interval, and the target material is captured by a receptor provided between the two electrodes;
상기 기준교차전극부와 신호교차전극부에 구동주파수를 각각 인가하도록 설치되는 구동신호 인가부;A driving signal applying unit installed to apply a driving frequency to the reference crossing electrode unit and the signal crossing electrode unit, respectively;
상기 기준교차전극부에 있는 두 전극 사이의 임피던스를 기준임피던스로서 측정하는 기준임피던스 측정부;A reference impedance measuring unit which measures an impedance between two electrodes of the reference cross-electrode unit as a reference impedance;
상기 신호교차전극부에 있는 두 전극 사이의 임피던스를 신호임피던스로서 측정하는 신호임피던스 측정부; 및A signal impedance measuring unit which measures an impedance between two electrodes in the signal crossing electrode unit as a signal impedance; And
상기 기준임피던스 측정부 및 신호임피던스 측정부로부터 상기 기준임피던스와 신호임피던스를 각각 입력받아 차동 증폭하도록 설치되는 차동증폭기; 를 포함하여 이루어짐으로써, A differential amplifier installed to differentially amplify the reference impedance and the signal impedance from the reference impedance measuring unit and the signal impedance measuring unit; By being made, including
상기 기준임피던스에 대한 상기 신호임피던스의 상대적 변화를 통하여 상기 표적물질을 분석하는 것을 특징으로 한다. The target material may be analyzed by changing the signal impedance relative to the reference impedance.
상기 구동신호 인가부에서 인가되는 구동주파수는 10Hz ~100Hz의 주파수 범위를 갖는 것이 바람직하다. The driving frequency applied by the driving signal applying unit preferably has a frequency range of 10 Hz to 100 Hz.
상기 기준교차전극부를 이루는 두 전극 사이의 간극은 3~7㎛ 이고, 상기 신호교차전극부를 이루는 두 전극 사이의 간극은 이 3~7㎛ 인 것이 바람직하다. The gap between the two electrodes forming the reference cross-electrode portion is 3 to 7 μm, and the gap between the two electrodes forming the signal cross-electrode portion is 3 to 7 μm.
상기 기준교차전극부와 신호교차전극부 상에 채널이 각각 형성되도록 상기 기준교차전극부와 신호교차전극부 상에 보호캡이 각각 설치되고, 상기 수용체가 설치되지 않은 상기 채널 내부의 면에 흡착방지층이 코팅되는 것이 바람직하다. Protective caps are respectively provided on the reference cross-electrode portion and the signal cross-electrode portion so that channels are formed on the reference cross-electrode portion and the signal cross-electrode portion, respectively, and an adsorption preventing layer on the surface inside the channel where the receptor is not installed. It is preferred to be coated.
상기 흡착방지층은 BSA(Bovine Serum Albumin)로 이루어지는 것이 바람직하다. The adsorption prevention layer is preferably made of BSA (Bovine Serum Albumin).
상기 기준교차전극부와 상기 신호교차전극부 모두에 상기 수용체를 설치한 후에 상기 신호교차전극부에만 상기 표적물질을 제공함으로써 상기 표적물질의 포획이 상기 신호교차전극부에서만 일어나도록 하거나, 상기 신호교차전극부에만 상기 수용체를 설치하고 상기 기준교차전극부와 신호교차전극부 모두에 상기 표적물질을 제공함으로써 상기 표적물질의 포획이 상기 신호교차전극부에서만 일어나도록 할 수 있다. By providing the target material only to the signal cross-electrode part after the acceptor is installed in both the reference cross-electrode part and the signal cross-electrode part, the capture of the target material occurs only at the signal cross-electrode part or the signal cross-over part. The receptor may be provided only in the electrode portion, and the target material may be provided to both the reference cross electrode portion and the signal cross electrode portion so that the capture of the target material occurs only at the signal cross electrode portion.
본 발명에 의하면, 종래와 같이 도전성 입자를 사용하지 않고서도 임피던스 측정을 통해서 생체 물질의 존재 유무 및 그 농도를 정밀하게 검출할 수 있다. 또한 표적 생체물질이 변성 또는 손상되지 않도록 10Hz ~100Hz의 낮은 주파수를 사용하면서도 정밀 검출이 이루어질 수 있다는 장점이 있다. 그리고 차동증폭을 이용함으로써 소량의 생체물질에 대해서도 정밀 검출이 가능하다는 장점이 있다. According to the present invention, the presence or absence of a biological substance and its concentration can be accurately detected through impedance measurement without using conductive particles as in the prior art. In addition, there is an advantage that the precise detection can be made while using a low frequency of 10Hz ~ 100Hz so as not to denature or damage the target biomaterial. In addition, the use of differential amplification has the advantage of enabling precise detection of small amounts of biomaterials.
도 1은 본 발명에 따른 교차전극 바이오센서의 원리를 설명하기 위한 도면;1 is a view for explaining the principle of the cross-electrode biosensor according to the present invention;
도 2는 수용체(31)와 표적 생체물질(32)의 반응에 의한 교차전극부(20)의 임피던스 변화를 설명하기 위한 그래프;2 is a graph for explaining the change in the impedance of the cross-electrode portion 20 by the reaction of the receptor 31 and the target biomaterial 32;
도 3은 차동증폭기가 적용된 본 발명에 따른 교차전극 바이오센서를 설명하기 위한 도면;3 is a view for explaining a cross-electrode biosensor according to the present invention to which a differential amplifier is applied;
도 4는 도 1의 교차전극부(20)의 제조과정 및 표적 생체물질(32)의 특이적 결합을 설명하기 위한 도면;4 is a view for explaining the manufacturing process of the cross-electrode portion 20 of FIG. 1 and the specific binding of the target biomaterial 32;
도 5는 채널(50)을 설명하기 위한 도면;5 is a diagram for explaining the channel 50;
도 6 및 도 7은 도 3의 교차전극 바이오센서의 검출능력을 설명하기 위한 도면들로서, 표적 생체물질(32)의 특이결합에 대한 기여를 살펴본 것;6 and 7 are diagrams for explaining the detection capability of the cross-electrode biosensor of FIG. 3, and look at the contribution to the specific binding of the target biomaterial 32;
도 8은 도 3의 교차전극 바이오센서의 검출능력을 설명하기 위한 100pg/mL 농도의 베타아밀로리이드 검출실험 결과 그래프로서, 차동증폭기(300)의 기여를 살펴본 것이다. FIG. 8 is a graph illustrating a beta amyloid detection test result at a concentration of 100 pg / mL for explaining the detection capability of the cross-electrode biosensor of FIG. 3, and looks at the contribution of the differential amplifier 300.
<부호의 설명><Description of the code>
1: 구동신호 인가부 10: 기판1: driving signal applying unit 10: substrate
11: 실리콘 산화막 20: 교차전극부11: silicon oxide film 20: cross-electrode part
20a: 금속층 21, 22: 전극20a: metal layer 21, 22: electrode
31: 수용체 32: 표적 생체물질31: Receptor 32: Target Biomaterial
33: 연결분자층 40: 감광막 패턴33: connecting molecule layer 40: photosensitive film pattern
50: 보호캡 55: 채널50: protective cap 55: channel
100: 실험부 120: 신호교차전극부100: experimental unit 120: signal cross-electrode unit
125: 신호임피던스 측정부 200: 대조부125: signal impedance measurement unit 200: control unit
220: 기준교차전극부 225: 기준임피던스 측정부220: reference cross-electrode unit 225: reference impedance measuring unit
300: 차동증폭기300: differential amplifier
이하에서, 본 발명의 바람직한 실시예를 첨부한 도면들을 참조하여 상세히 설명한다. 아래의 실시예는 본 발명의 내용을 이해하기 위해 제시된 것일 뿐이며 당 분야에서 통상의 지식을 가진 자라면 본 발명의 기술적 사상 내에서 많은 변형이 가능할 것이다. 따라서 본 발명의 권리범위가 이러한 실시예에 한정되는 것으로 해석돼서는 안 된다. Hereinafter, with reference to the accompanying drawings, preferred embodiments of the present invention will be described in detail. The following examples are only presented to understand the content of the present invention, and those skilled in the art will be capable of many modifications within the technical spirit of the present invention. Therefore, the scope of the present invention should not be construed as limited to these embodiments.
도 1은 본 발명에 따른 교차전극 바이오센서의 원리를 설명하기 위한 도면이다. 도 1에 도시된 바와 같이 본 발명에 따른 교차전극 바이오센서는 기판(10) 상에 교차전극부(20)가 설치되어 이루어진다. 교차전극부(interdigitated microelectrode part, IME part, 20)는 빗 모양을 하는 두 전극(21, 22)이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 설치된다. 1 is a view for explaining the principle of the cross-electrode biosensor according to the present invention. As shown in FIG. 1, in the cross-electrode biosensor according to the present invention, the cross-electrode part 20 is installed on the substrate 10. The interdigitated microelectrode part (IME part) 20 is installed in a form in which two electrodes 21 and 22 having a comb shape are interlocked with each other at predetermined intervals.
이 때, 두 전극(21, 22) 사이의 임피던스는 다음과 같이 정리된다. At this time, the impedance between the two electrodes 21, 22 is arranged as follows.
Z = R + jX Z = R + jX
= R + j(XL-XC)= R + j (X L -X C )
= R - jXC = R-jX C
= R - j(1/wC)  = R-j (1 / wC)
여기서, Z는 임피던스(impedance), R은 저항(resistance), X는 리액턴스(reactance), C는 정전용량(capacitance), w는 각주파수(angular frequency)이다. 리액턴스 X는 인덕터 성분인 XL과 커패시터 성분인 XC 나뉘는데, 두 전극(21, 22) 사이는 전기적으로 직접 연결되어 있지 않기 때문에 인덕터 성분(XL)은 무시되고 커패시터 성분(XC)만 존재한다고 볼 수 있다. Where Z is impedance, R is resistance, X is reactance, C is capacitance, and w is angular frequency. The reactance X is divided into the inductor component X L and the capacitor component X C. Since the two electrodes 21 and 22 are not electrically connected directly, the inductor component X L is ignored and only the capacitor component X C is present. It can be said that.
두 전극(21, 22) 사이의 공간에 표적(target) 생체물질(32)에 특이적으로 반응하는 수용체(주로 항체, 압타머 등, 31)를 고정하고 표적 생체물질(32)이 수용체(31)에 반응 했을 때의 두 전극(21, 22) 사이에서의 임피던스 변화를 확인하면 표적 생체물질(32)의 정량분석이 가능하게 된다. A receptor (mainly an antibody, aptamer, etc.) 31 that specifically reacts with the target biomaterial 32 is fixed in the space between the two electrodes 21, 22 and the target biomaterial 32 is the receptor 31. When the impedance change between the two electrodes (21, 22) is confirmed when reacting to the) it is possible to quantitatively analyze the target biomaterial (32).
도 2는 수용체(31)와 표적 생체물질(32)의 반응에 의한 교차전극부(20)의 임피던스 변화를 설명하기 위한 그래프이다. 수용체(31)와 표적 생체물질(32)이 특이결합을 하게 되면 두 전극(21, 22) 사이에 존재하던 물(혹은 버퍼용액, 혈청, 혈액 등)을 밀어내고 표적 생체물질(32)이 위치하게 되므로 저항이 증가하게 된다. 또한, 리액턴스는 유전율이 물(혹은 버퍼용액, 혈청, 혈액 등)보다 작은 표적 생체물질(32)의 성질에 의해서 정전용량(C)의 값이 감소하게 되어 XC 값은 커지고 따라서 -XC 값은 감소하게 된다. 이러한 임피던스(저항과 리액턴스)의 변화량을 확인하여 표적 생체물질(32)의 양을 정확하게 검출 할 수 있다는 것이다. 2 is a graph for explaining the change in the impedance of the cross-electrode portion 20 by the reaction of the receptor 31 and the target biomaterial 32. When the receptor 31 and the target biomaterial 32 are specifically bound, the water (or buffer solution, serum, blood, etc.) existing between the two electrodes 21 and 22 is pushed out and the target biomaterial 32 is positioned. As a result, the resistance increases. Moreover, reactance is the dielectric constant of water (or buffer solutions, serum, blood, etc.) decreases the value of the capacitance (C) by the nature of the small target biological material (32) than the value X C is greater according -X value C Will decrease. By checking the amount of change in the impedance (resistance and reactance) it is possible to accurately detect the amount of the target biomaterial (32).
위와 같이 인덕터 성분은 무시되고 커패시터 성분의 리액턴스만 주로 고려될 경우에는, 구동주파수가 높아야 임피던스의 변화를 확인하기가 용이하고, 구동 주파수가 낮으면 임피던스의 변화가 미미하여 그 변화를 확인하기가 어려운 것이 일반적이다. 따라서 미량의 표적 생체물질(32)을 검출하기 위해서는 높은 구동주파수를 사용할 수밖에 없다. When the inductor component is ignored and the reactance of the capacitor component is mainly taken into consideration as described above, it is easy to check the change of impedance when the driving frequency is high, and when the driving frequency is low, the change of impedance is small and it is difficult to check the change. It is common. Therefore, in order to detect a small amount of the target biomaterial 32, a high driving frequency may be used.
그러나 구동주파수의 주파수가 높으면, 특이적으로 결합된 표적 생체물질(32)의 윗 공간을 통해서, 즉 루트 A를 통해서 전류가 주로 흐르게 되므로 표적 생체물질(32)의 검출이 제대로 이루어지지 않게 된다. 뿐만 아니라 주파수가 높으면, 표적 생체물질(32)이 고주파에 의해 손상되어 제대로 검출되지 않을 우려가 있다. However, if the frequency of the driving frequency is high, the current mainly flows through the space above the specifically coupled target biomaterial 32, that is, root A, so that detection of the target biomaterial 32 is not properly performed. In addition, if the frequency is high, the target biomaterial 32 may be damaged by high frequency and may not be detected properly.
표적 생체물질(32)의 검출이 이루어지기 위해서는 전류가 루트 B를 통해서 흐르는 것이 바람직하므로, 본 발명은 전류가 루트 B를 통해서 흐를 수 있도록 10Hz ~100Hz의 낮은 구동주파수를 사용하는 것을 특징으로 한다. 그러면 주파수가 낮기 때문에 표적 생체물질(32)이 손상되는 것도 방지되어 바람직하다. 물론, 이 경우 주파수가 낮기 때문에 루트 B에서의 미세 임피던스 변화를 검출하기 어렵다는 단점이 있지만, 이러한 단점은 후술하는 바와 같이 차동증폭기를 이용하여 극복할 수 있다. Since the current flows through the root B in order to detect the target biomaterial 32, the present invention is characterized by using a low driving frequency of 10 Hz to 100 Hz so that the current can flow through the root B. Since the frequency is low, the target biomaterial 32 is also prevented from being damaged. Of course, in this case, since the frequency is low, there is a disadvantage in that it is difficult to detect a change in the fine impedance at the root B, but this disadvantage can be overcome by using a differential amplifier as described below.
본 발명에서와 같이 10Hz ~100Hz의 낮은 구동주파수를 사용하면서 루트 B 상에 있는 생체물질(32)을 검출코자 하는 경우에는 두 전극(21, 22)의 간극이 3~7㎛인 것이 바람직하다. 왜냐하면, 간극이 너무 작아 3㎛보다 작으면 검출신호의 편차가 너무 커서 신뢰성 있는 테스트가 이루어지 못하고, 간극이 너무 커서 7㎛보다 크면 민감도가 떨어져서 소량의 생체물질(32)을 검출하는데 부족함이 있기 때문이다. 편차와 민감도를 고려할 때에 5㎛인 경우가 가장 바람직하다. When detecting the biomaterial 32 on the root B while using a low driving frequency of 10 Hz to 100 Hz as in the present invention, the gap between the two electrodes 21 and 22 is preferably 3 to 7 μm. If the gap is too small and less than 3 μm, the deviation of the detection signal is too large to perform a reliable test. If the gap is too large and larger than 7 μm, the sensitivity is insufficient to detect a small amount of the biomaterial 32. Because. Considering the deviation and sensitivity, the case of 5 μm is most preferable.
도 3은 차동증폭기가 적용된 본 발명에 따른 교차전극 바이오센서를 설명하기 위한 도면이다. 도 3을 참조하면, 기판(1) 상에 신호교차전극부(120)와 기준교차전극부(220)가 설치되며, 신호교차전극부(120)와 기준교차전극부(220) 각각은 도 1의 교차전극부(20)와 같이 빗 모양을 하는 두 전극(21, 22)이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 배치된다. 3 is a view illustrating a cross-electrode biosensor according to the present invention to which a differential amplifier is applied. Referring to FIG. 3, the signal cross-electrode portion 120 and the reference cross-electrode portion 220 are installed on the substrate 1, and each of the signal cross-electrode portion 120 and the reference cross-electrode portion 220 is illustrated in FIG. 1. Two electrodes 21 and 22 having a comb-like shape, such as the cross-electrode portion 20 of, are arranged in a staggered manner while being spaced apart by a predetermined interval.
신호교차전극부(120)에서는 두 전극(21, 22) 사이에 설치되는 수용체(31)에 의하여 표적 생체물질(32)의 포획이 이루어지는 반면에 기준교차전극(220)에서는 이러한 포획이 이루어지지 않는다. The signal cross-electrode unit 120 captures the target biomaterial 32 by the receptor 31 disposed between the two electrodes 21 and 22, while the capture cross-electrode unit 220 does not capture the target biomaterial 32. .
이는 신호교차전극부(120)와 기준교차전극부(220) 모두에 수용체(31)를 설치한 후에 신호교차전극부(120)에만 표적 생체물질(32)을 제공함으로써 이루어질 수도 있고, 신호교차전극부(120)에만 수용체(31)를 설치하고 신호교차전극부(120)와 기준교차전극부(220) 모두에 표적 생체물질(32)을 제공함으로써 이루어질 수도 있다. This may be achieved by providing the target biomaterial 32 only to the signal cross-electrode portion 120 after installing the receptor 31 in both the signal cross-electrode portion 120 and the reference cross-electrode portion 220. The receiver 31 may be provided only in the unit 120, and the target biomaterial 32 may be provided to both the signal cross-electrode unit 120 and the reference cross-electrode unit 220.
신호교차전극부(120)와 기준교차전극부(220)에는 구동신호 인가부(1)를 통하여 구동주파수가 인가된다. 그러면 신호임피던스 측정부(125)에서는 신호교차전극부(120)에서의 임피던스가 신호임피던스로서 측정되고, 기준임피던스 측정부(225)에서는 기준교차전극부(220)에서의 임피던스가 기준임피던스로서 측정된다. The driving frequency is applied to the signal cross electrode unit 120 and the reference cross electrode unit 220 through the driving signal applying unit 1. Then, the impedance at the signal cross-electrode unit 120 is measured as the signal impedance in the signal impedance measuring unit 125, and the impedance at the reference cross-electrode unit 220 is measured as the reference impedance in the reference impedance measuring unit 225. .
따라서 신호임피던스는 두 전극(21, 22) 사이에서 표적 생체물질(32)이 포획되는 경우에 대한 두 전극(21, 22) 사이의 임피던스가 될 것이고, 기준임피던스는 두 전극(21, 22) 사이에서 표적 생체물질(32)이 포획되지 않은 경우에 대한 두 전극(21, 22) 사이의 임피던스가 될 것이다. Therefore, the signal impedance will be the impedance between the two electrodes 21 and 22 for the case where the target biomaterial 32 is captured between the two electrodes 21 and 22, and the reference impedance will be between the two electrodes 21 and 22. Will be the impedance between the two electrodes 21, 22 for the case where the target biomaterial 32 is not captured.
차동증폭기(300)는 상기 신호임피던스와 기준임피던스를 각각 입력받아 상기 신호임피던스를 차동증폭하면서 결과신호를 출력한다. The differential amplifier 300 receives the signal impedance and the reference impedance, respectively, and differentially amplifies the signal impedance and outputs a result signal.
도 1에서와 같이 차동증폭 없이 교차전극부(20)만을 사용하는 경우에는 10Hz ~ 100Hz 의 낮은 주파수 범위에서 루트 B에서의 임피던스 변화가 미미하여 미소량의 표적 생체물질(32)를 검출하기 어렵지만, 도 3에서와 같이 신호교차전극부(120)와 기준교차전극부(220)를 두어 기준교차전극부(220)의 임피던스를 기준으로 하여 신호교차전극부(120)의 임피던스를 차동증폭하면 이들 사이의 임피던스 차이가 명확하게 나타나기 때문에 표적 생체물질(32)이 미량이더라도 그 양을 정량적으로 정밀하게 검출할 수 있게 된다. When only the cross-electrode portion 20 is used without differential amplification as shown in FIG. 1, it is difficult to detect a small amount of the target biomaterial 32 due to a small change in impedance at the root B in a low frequency range of 10 Hz to 100 Hz. As shown in FIG. 3, when the signal cross-electrode portion 120 and the reference cross-electrode portion 220 are provided to differentially amplify the impedance of the signal cross-electrode portion 120 based on the impedance of the reference cross-electrode portion 220. Since the impedance difference is clearly shown, even if the amount of the target biomaterial 32 is small, the amount can be detected quantitatively and precisely.
도 4는 도 1의 교차전극부(20)의 제조과정 및 표적 생체물질(32)의 특이적 결합을 설명하기 위한 도면이다. 4 is a view for explaining the manufacturing process and the specific binding of the target biomaterial 32 of the cross-electrode portion 20 of FIG.
먼저, 도 4a에 도시된 바와 같이, 실리콘 기판(10) 상에 열산화법(thermal oxidation)으로 500nm 두께의 실리콘 산화막(SiO2, 11)을 형성한 다음에, 실리콘 산화막(11) 상에 스퍼터링(sputtering)법으로 Ti 30nm, Pt 150nm를 순차적으로 적층하여 금속층(20a)을 형성한다. Ti층은 Pt층과 실리콘 산화막(11)의 결합력을 증가시키기 위한 접착층(adhesion layer)로서 사용된 것이다. 그 다음에 금속층(20a) 상에 감광막을 도포하고 포토리소그래프 공정으로 상기 감광막을 패터닝하여 감광막 패턴(40)을 형성한다. First, as shown in FIG. 4A, a 500 nm thick silicon oxide film SiO 2 , 11 is formed on the silicon substrate 10 by thermal oxidation, and then sputtering on the silicon oxide film 11 is performed. A metal layer 20a is formed by sequentially stacking Ti 30 nm and Pt 150 nm by sputtering. The Ti layer is used as an adhesion layer for increasing the bonding force between the Pt layer and the silicon oxide film 11. Next, a photoresist film is coated on the metal layer 20a and the photoresist film is patterned by a photolithography process to form a photoresist pattern 40.
이어서, 도 4b에 도시된 바와 같이 감광막 패턴(40)을 식각 마스크로 하여 실리콘 산화막(11)이 노출될 때까지 ICP-RIE(inductively coupled plasma reactive ion etcher)를 이용하여 금속층(20)을 식각하여 두 전극(21, 22)을 형성한 후, 도 4c에 도시된 바와 같이 감광막 패턴(40)을 제거한다. Subsequently, as shown in FIG. 4B, the metal layer 20 is etched using inductively coupled plasma reactive ion etcher (ICP-RIE) until the silicon oxide film 11 is exposed using the photoresist pattern 40 as an etch mask. After the two electrodes 21 and 22 are formed, the photoresist pattern 40 is removed as shown in FIG. 4C.
표적 생체물질(32)의 특이적 결합은 다음과 같이 이루어진다. Specific binding of the target biomaterial 32 is performed as follows.
도 4d에 도시된 바와 같이, 두 전극(21, 22) 사이의 실리콘 산화막(11) 표면에 베타아밀로이드 항체를 선택적으로 고정하기 위한 연결분자층(33)으로서 Calixcrown SAM(Self-Assembled Monolayer)을 형성한 후에, 연결분자층(33)에 수용체(31)로서 베타아밀로이드 항체를 고정시킨다. 그러면 도 4e에서와 같이 표적 생체물질(32)인 베타아밀로이드가 수용체(31)에 선택적으로 특이 결합된다. As shown in FIG. 4D, a Calixcrown Self-Assembled Monolayer (SAM) is formed as a connecting molecule layer 33 for selectively fixing the beta amyloid antibody on the surface of the silicon oxide film 11 between the two electrodes 21 and 22. After that, the beta amyloid antibody is fixed to the linking molecule layer 33 as the receptor 31. Then, as shown in FIG. 4E, the beta amyloid, which is the target biomaterial 32, is selectively bound to the receptor 31.
도 5는 채널(55)을 설명하기 위한 도면이다. 도 4e에서와 같이 표적 생체물질(32)이 특이적으로 결합되는 영역이 외부에 완전 노출되어 버리면 검출 에러가 발생할 수 있으므로 이 부분을 덮어줄 필요가 있다. 이를 위해 두 전극(21, 22)이 채널(55) 안에 놓이도록 두 전극(21, 22) 상에 보호캡(50)이 설치되는 것이 바람직하다. 보호캡(50)에 의한 채널(55)은 시료가 특이 결합 영역으로 유입되는 것을 도와주는 역할도 한다. 보호캡(50)은 PDMS(Polydimethylsiloxane) 재질의 것을 사용하는 것이 바람직하다.5 is a diagram for describing the channel 55. As shown in FIG. 4E, when a region where the target biomaterial 32 is specifically bound is completely exposed to the outside, a detection error may occur, so it is necessary to cover this portion. For this purpose, it is preferable that the protective cap 50 is installed on the two electrodes 21 and 22 so that the two electrodes 21 and 22 are placed in the channel 55. The channel 55 by the protective cap 50 also serves to help the sample enter the specific binding region. The protective cap 50 is preferably made of a polydimethylsiloxane (PDMS) material.
여러 성분이 포함되어 있는 시료를 채널(55) 내에 투입하면 수용체(31)에 특이 반응하는 표적 생체물질(32) 만이 수용체(31)에 결합된다. 위의 경우 표적 생체물질(32)이 베타아밀로이드가 될 것이다. 이 때, 베타아밀로이드 이외의 다른 물질들이 채널(55) 내의 다른 부분에 비특이적으로 결합되어 버리는 것은 바람직하지 않으므로 이를 방지하기 위하여, 수용체(31)가 고정되지 않은 부분을 제외한 보호캡(50) 내벽 및 전극(21, 22)의 표면에 흡착방지층(51)이 코팅되는 것이 바람직하다. 여기서의 흡착방지층(51)은 BSA (Bovine Serum Albumin)으로 이루어지는 것이 바람직하다. When a sample containing various components is introduced into the channel 55, only the target biomaterial 32 that specifically reacts with the receptor 31 is bound to the receptor 31. In the above case, the target biomaterial 32 will be beta amyloid. At this time, it is not desirable that other materials other than beta amyloid are non-specifically bound to other parts in the channel 55, and to prevent this, the inner wall of the protective cap 50 except for the part where the receptor 31 is not fixed and It is preferable that the adsorption preventing layer 51 is coated on the surfaces of the electrodes 21 and 22. In this case, the adsorption preventing layer 51 is preferably made of BSA (Bovine Serum Albumin).
도 6 및 도 7은 도 3의 교차전극 바이오센서의 검출능력을 설명하기 위한 도면들로서, 표적 생체물질(32)의 특이결합에 대한 기여를 살펴본 것이다. 6 and 7 illustrate the detection capability of the cross-electrode biosensor of FIG. 3 and illustrate the contribution to specific binding of the target biomaterial 32.
도 6에 도시된 바와 같이, 실험부(100)와 대조부(200)로 구분하고 실험부(100)와 대조부(200) 각각에 도 3에서와 같이 신호교차전극부(120)와 기준교차전극부(220)를 한 쌍씩 형성하였다. 그리고 실험부(100)의 신호교차전극부(120)에는 베타아밀로이드 항체를 고정시키고, 대조부(200)의 신호교차전극부(120)에는 선택성 확인(negative control)을 위하여 PSA(Prostate-specific antigen) 항체를 고정하였다. 또한 실험부(100)와 대조부(200) 각각에 두 개의 마이크로 채널을 가지는 PDMS 칩을 부착시켰다.As shown in FIG. 6, the test unit 100 and the control unit 200 are divided into the test unit 100 and the control unit 200, respectively, as shown in FIG. 3. The electrode portions 220 were formed in pairs. In addition, the beta amyloid antibody is fixed to the signal cross-electrode unit 120 of the experiment unit 100, and the PSA (prostate-specific antigen) for selectivity control (negative control) to the signal cross-electrode unit 120 of the control unit 200. ) Antibodies were fixed. In addition, a PDMS chip having two microchannels was attached to each of the experiment unit 100 and the control unit 200.
두 채널 모두에 0.1X PBS 버퍼 용액을 주입하였고, 각 전극부에서 출력되는 임피던스 신호가 안정적으로 일정하게 유지되는 시점까지 신호를 관찰하며 안정화 작업을 진행하였다. 그리고 베타이밀로이드를 채널에 주입하여 임피던스 신호의 변화량을 관찰하여 베타아밀로이드의 항원항체 반응을 확인하였다. 0.1X PBS buffer solution was injected into both channels, and the stabilization was performed by observing the signal until the impedance signal output from each electrode part remained stable and stable. In addition, the beta amyloid was injected into the channel to observe the change in the impedance signal to confirm the antigen-antibody reaction of the beta amyloid.
도 7은 10, 100, 1000 pg/mL의 베타아밀로이드를 순차적으로 주입하였을 때의 실험부(100)와 대조부(200)에서의 임피던스 변화를 나타내는 그래프이다. 도 7을 참조하면, 실험부(100)의 임피던스 변화량은 베타아밀로이드의 농도가 증가할수록 더욱 증가하나(초록색), 비특이적 항체가 고정되어 있는 대조부(200)의 경우에는 베타아밀로이드를 주입하였을 때 그 신호의 변화가 매우 미비함을 알 수 있다(파란색). 이는 실험부(100)에서는 표적 생체물질(32)인 베타아밀로이드의 특이적 결합이 이루어지는 반면에 대조부(200)에서는 이러한 특이적 결합이 이루어지지 않고 있음을 반영하는 결과이다. FIG. 7 is a graph showing impedance changes in the test unit 100 and the control unit 200 when 10, 100, and 1000 pg / mL of beta amyloid was sequentially injected. Referring to FIG. 7, the impedance change amount of the experiment unit 100 increases as the beta amyloid concentration increases (green), but in the case of the control unit 200 in which the non-specific antibody is immobilized, the beta amyloid is injected. It can be seen that the signal change is very insignificant (blue). This is a result reflecting that the specific binding of the beta amyloid that is the target biomaterial 32 is made in the experiment unit 100, while the control unit 200 does not perform such specific binding.
도 8은 도 3의 교차전극 바이오센서의 검출능력을 설명하기 위한 100pg/mL 농도의 베타아밀로리이드 검출실험 결과 그래프로서, 차동증폭기(300)의 기여를 살펴본 것이다. FIG. 8 is a graph illustrating a beta amyloid detection test result at a concentration of 100 pg / mL for explaining the detection capability of the cross-electrode biosensor of FIG. 3, and looks at the contribution of the differential amplifier 300.
도 8a는 신호교차전극부(120)와 기준교차전극부(220)에서 출력되는 임피던스를 차동 증폭하지 않고 그대로 검출한 것이다. 도 8a에서 알 수 있듯이 신호교차전극부(120)와 기준교차전극부(220)의 임피던스 변화가 제대로 구별되지 않기 때문에 표적 생체물질(32)의 양을 정량적으로 검출하기가 매우 어렵다. 8A illustrates the impedance output from the signal cross electrode unit 120 and the reference cross electrode unit 220 without being differentially amplified. As can be seen in FIG. 8A, it is very difficult to quantitatively detect the amount of the target biomaterial 32 because impedance changes of the signal cross-electrode portion 120 and the reference cross-electrode portion 220 are not properly distinguished.
반면에, 도 8b는 신호교차전극부(120)에서 출력되는 신호임피던스(빨간색)를 기준교차전극부(220)에서 출력되는 기준임피던스(파란색)에 대해 차동증폭하여 그 결과를 출력한 것이다. 도 8b에서 알 수 있듯이 신호임피던스와 기준임피던스의 구별이 명확하게 이루어지기 때문에 100pg/mL 만큼 소량의 경우라도 제대로 측정할 수 있음을 확인할 수 있다. On the other hand, FIG. 8B differentially amplifies the signal impedance (red) output from the signal cross electrode unit 120 with respect to the reference impedance (blue) output from the reference cross electrode unit 220 and outputs the result. As can be seen in Figure 8b it can be seen that even if a small amount of 100pg / mL can be properly measured because the distinction between the signal impedance and the reference impedance.
본 발명에 의하면, 종래와 같이 도전성 입자를 사용하지 않고서도 임피던스 측정을 통해서 생체 물질의 존재 유무 및 그 농도를 정밀하게 검출할 수 있다. 또한 표적 생체물질(32)이 변성 또는 손상되지 않도록 10Hz ~100Hz의 낮은 주파수를 사용하면서도 정밀 검출이 이루어질 수 있다는 장점이 있다. 그리고 차동증폭을 이용함으로써 소량의 생체물질에 대해서도 정밀 검출이 가능하다는 장점이 있다. According to the present invention, the presence or absence of a biological substance and its concentration can be accurately detected through impedance measurement without using conductive particles as in the prior art. In addition, there is an advantage that the precise detection can be made while using a low frequency of 10Hz ~ 100Hz so that the target biomaterial 32 is not denatured or damaged. In addition, the use of differential amplification has the advantage of enabling precise detection of small amounts of biomaterials.

Claims (11)

  1. 빗 모양을 하는 두 전극이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 기판 상에 설치되며, 표적물질의 포획을 위하여 상기 두 전극 사이에 수용체가 설치되는 교차전극부;Comb-shaped two electrodes are installed on the substrate in the form of interlocking staggered spaced apart by a predetermined interval, the cross-electrode portion is installed between the two electrodes to capture the target material;
    상기 두 전극 사이에 구동주파수를 인가하는 구동신호 인가부; 및A driving signal applying unit applying a driving frequency between the two electrodes; And
    상기 두 전극 사이의 임피던스를 측정하는 임피던스 측정부; 를 포함하여 이루어짐으로써, An impedance measuring unit measuring an impedance between the two electrodes; By being made, including
    상기 표적물질의 포획 여부에 따라 나타나는 상기 임피던스의 변화를 측정하여 상기 표적물질을 분석하는 것을 특징으로 하는 교차전극 바이오센서. Cross-electrode biosensor, characterized in that for analyzing the target material by measuring the change in the impedance appearing according to the capture of the target material.
  2. 제1항에 있어서, 상기 구동주파수가 10Hz ~100Hz 의 주파수 범위를 갖는 것을 특징으로 하는 교차전극 바이오센서. The cross-electrode biosensor of claim 1, wherein the driving frequency has a frequency range of 10 Hz to 100 Hz.
  3. 제2항에 있어서, 상기 두 전극 사이의 간극이 3~7㎛ 인 것을 특징으로 하는 교차전극 바이오센서. The cross-electrode biosensor of claim 2, wherein a gap between the two electrodes is 3 to 7 µm.
  4. 제1항에 있어서, 상기 교차전극부 상에 채널이 형성되도록 상기 교차전극부 상에 보호캡이 설치되고, 상기 수용체가 설치되지 않는 상기 채널 내부의 면에 흡착방지층이 코팅되는 것을 특징으로 하는 교차전극 바이오센서. The crossover of claim 1, wherein a protective cap is installed on the crossover electrode portion to form a channel on the crossover electrode portion, and an adsorption prevention layer is coated on a surface inside the channel where the receptor is not installed. Electrode Biosensor.
  5. 제4항에 있어서, 상기 흡착방지층이 BSA(Bovine Serum Albumin)로 이루어지는 것을 특징으로 하는 교차전극 바이오센서. The cross-electrode biosensor of claim 4, wherein the adsorption prevention layer is made of BSA (Bovine Serum Albumin).
  6. 빗 모양을 하는 두 전극이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 기판 상에 설치되며, 표적물질의 포획이 이루어지지 않는 기준교차전극부;Two electrodes having a comb shape are installed on the substrate in an interlocking manner while being spaced apart from each other by a predetermined interval, and the reference cross-electrode portion does not capture the target material;
    빗 모양을 하는 두 전극이 소정간격 이격되면서 엇갈리게 맞물리는 형태로 상기 기판 상에 설치되며, 두 전극 사이에 설치되는 수용체에 의하여 표적물질의 포획이 이루어지는 신호교차전극부;A signal cross-electrode unit disposed on the substrate in a shape in which two comb-shaped electrodes are alternately engaged with each other at a predetermined interval, and the target material is captured by a receptor provided between the two electrodes;
    상기 기준교차전극부와 신호교차전극부에 구동주파수를 각각 인가하도록 설치되는 구동신호 인가부;A driving signal applying unit installed to apply a driving frequency to the reference crossing electrode unit and the signal crossing electrode unit, respectively;
    상기 기준교차전극부에 있는 두 전극 사이의 임피던스를 기준임피던스로서 측정하는 기준임피던스 측정부;A reference impedance measuring unit which measures an impedance between two electrodes of the reference cross-electrode unit as a reference impedance;
    상기 신호교차전극부에 있는 두 전극 사이의 임피던스를 신호임피던스로서 측정하는 신호임피던스 측정부; 및A signal impedance measuring unit which measures an impedance between two electrodes in the signal crossing electrode unit as a signal impedance; And
    상기 기준임피던스 측정부 및 신호임피던스 측정부로부터 상기 기준임피던스와 신호임피던스를 각각 입력받아 차동 증폭하도록 설치되는 차동증폭기; 를 포함하여 이루어짐으로써, A differential amplifier installed to differentially amplify the reference impedance and the signal impedance from the reference impedance measuring unit and the signal impedance measuring unit; By being made, including
    상기 기준임피던스에 대한 상기 신호임피던스의 상대적 변화를 통하여 상기 표적물질을 분석하는 것을 특징으로 하는 교차전극 바이오센서. Cross-electrode biosensor, characterized in that for analyzing the target material through the relative change of the signal impedance with respect to the reference impedance.
  7. 제6항에 있어서, 상기 구동신호 인가부에서 인가되는 구동주파수가 10Hz ~100Hz의 주파수 범위를 갖는 것을 특징으로 하는 교차전극 바이오센서. The cross-electrode biosensor of claim 6, wherein the driving frequency applied by the driving signal applying unit has a frequency range of 10 Hz to 100 Hz.
  8. 제7항에 있어서, 상기 기준교차전극부를 이루는 두 전극 사이의 간극이 3~7㎛ 이고, 상기 신호교차전극부를 이루는 두 전극 사이의 간극이 이 3~7㎛ 인 것을 특징으로 하는 교차전극 바이오센서. 8. The cross-electrode biosensor according to claim 7, wherein a gap between the two electrodes forming the reference cross electrode is 3 to 7 µm, and a gap between the two electrodes forming the signal cross electrode is 3 to 7 µm. .
  9. 제6항에 있어서, 상기 기준교차전극부와 신호교차전극부 상에 채널이 각각 형성되도록 상기 기준교차전극부와 신호교차전극부 상에 보호캡이 각각 설치되고, 상기 수용체가 설치되지 않은 상기 채널 내부의 면에 흡착방지층이 코팅되는 것을 특징으로 하는 교차전극 바이오센서. The channel of claim 6, wherein protective caps are respectively provided on the reference cross-electrode portion and the signal cross-electrode portion so that channels are formed on the reference cross-electrode portion and the signal cross-electrode portion, respectively. Cross-electrode biosensor, characterized in that the adsorption prevention layer is coated on the inner surface.
  10. 제9항에 있어서, 상기 흡착방지층이 BSA(Bovine Serum Albumin)로 이루어지는 것을 특징으로 하는 교차전극 바이오센서. 10. The cross-electrode biosensor of claim 9, wherein the adsorption prevention layer is made of BSA (Bovine Serum Albumin).
  11. 제6항에 있어서, 상기 기준교차전극부와 상기 신호교차전극부 모두에 상기 수용체를 설치한 후에 상기 신호교차전극부에만 상기 표적물질을 제공함으로써 상기 표적물질의 포획이 상기 신호교차전극부에서만 일어나도록 하거나, 상기 신호교차전극부에만 상기 수용체를 설치하고 상기 기준교차전극부와 신호교차전극부 모두에 상기 표적물질을 제공함으로써 상기 표적물질의 포획이 상기 신호교차전극부에서만 일어나도록 하는 것을 특징으로 하는 교차전극 바이오센서. 7. The method of claim 6, wherein after the receptor is provided in both the reference cross-electrode portion and the signal cross-electrode portion, the capture of the target material occurs only at the signal cross-electrode portion by providing the target material only to the signal cross-electrode portion. Or by installing the receptor only in the signal cross-electrode portion and providing the target material to both the reference cross-electrode portion and the signal cross-electrode portion so that the capture of the target material occurs only at the signal cross-electrode portion. Cross-electrode biosensor.
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