(19)
(11)EP 3 399 305 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
07.04.2021 Bulletin 2021/14

(21)Application number: 16881941.5

(22)Date of filing:  20.10.2016
(51)International Patent Classification (IPC): 
G01N 27/327(2006.01)
G01N 33/68(2006.01)
G01N 33/543(2006.01)
(86)International application number:
PCT/KR2016/011788
(87)International publication number:
WO 2017/115988 (06.07.2017 Gazette  2017/27)

(54)

INTERDIGITATED ELECTRODE BIOSENSOR USING REACTION BETWEEN RECEPTOR AND TARGET BIOMATERIAL

BIOSENSOR MIT INEINANDERGREIFENDEN ELEKTRODEN MIT VERWENDUNG EINER REAKTION ZWISCHEN REZEPTOR UND ZIELBIOMATERIAL

BIO-CAPTEUR À ÉLECTRODE INTER-DIGITÉE UTILISANT UNE RÉACTION ENTRE UN RÉCEPTEUR ET UN BIOMATÉRIAU CIBLE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 28.12.2015 KR 20150187388

(43)Date of publication of application:
07.11.2018 Bulletin 2018/45

(73)Proprietor: Korea Institute of Science and Technology
Seoul 02792 (KR)

(72)Inventors:
  • HWANG, Kyo Seon
    Seoul 02792 (KR)
  • KIM, Young Soo
    Seoul 02792 (KR)
  • KIM, Jin Sik
    Seoul 02792 (KR)
  • YOO, Yong Kyoung
    Seoul 02792 (KR)

(74)Representative: Walaski, Jan Filip et al
Venner Shipley LLP 200 Aldersgate
London EC1A 4HD
London EC1A 4HD (GB)


(56)References cited: : 
WO-A1-2016/043402
KR-A- 20030 038 084
KR-A- 20090 101 764
KR-A- 20150 089 226
US-A1- 2009 084 686
US-A1- 2013 143 775
WO-A1-2016/129894
KR-A- 20050 103 824
KR-A- 20120 067 967
US-A1- 2007 117 221
US-A1- 2011 312 518
  
  • HONGXIA CHEN ET AL: "Molecular Recognition of Arginine by Supramolecular Complexation with Calixarene Crown Ether Based on Surface Plasmon Resonance", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 12, no. 4, 4 April 2011 (2011-04-04), pages 2315-2324, XP055607977, DOI: 10.3390/ijms12042315
  • RÓISE E. MCGOVERN ET AL: "Microscale Crystals of Cytochrome? c and Calixarene on Electrodes: Interprotein Electron Transfer between Defined Sites", ANGEWANDTE CHEMIE, INTERNATIONAL EDITION, vol. 54, no. 21, 1 April 2015 (2015-04-01) , pages 6356-6359, XP055607980, DE ISSN: 1433-7851, DOI: 10.1002/anie.201500191
  • YONG KYOUNG YOO ET AL: "Ultra-sensitive detection of brain-derived neurotrophic factor (BDNF) in the brain of freely moving mice using an interdigitated microelectrode (IME) biosensor", SCIENTIFIC REPORTS, vol. 6, no. 1, 1 September 2016 (2016-09-01), XP055608003, DOI: 10.1038/srep33694
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

[Technical Field]



[0001] The present invention relates to an interdigitated microelectrode biosensor, and more particularly, to an interdigitated microelectrode biosensor using the reaction between receptors and target biomaterials, wherein the receptors reacting specifically with the target biomaterials are located between interdigitated microelectrodes to increase the width of impedance detection and the limit of impedance detection and also to improve the accuracy of detection according to the characteristics of monomers and oligomers of the target biomaterials.

[Background Art]



[0002] In recent years, many biosensors have been developed for detecting the presence and concentration of a variety of biological substances, such as genes and proteins, by electrical methods. One example is to use interdigitated microelectrodes. Since biosensors using interdigitated microelectrodes have a very substantially broad region in a zigzag configuration where receptors capable of binding specifically to biological substances are immobilized, they are praised for their ability to measure even a low concentration of the biological substances.

[0003] Such a biosensor using interdigitated microelectrodes is disclosed in Korean Patent No.777973 (published on Nov. 29, 2007). According to this patent, since the concentration of a biological substance is measured based on an electric current flowing between the electrodes, it is necessary to use conductive particles for the flow of electric current between flowing between the electrodes, it is necessary to use conductive particles for the flow of electric current between the electrodes. However, the use of the conductive particles is troublesome.

[0004] Further, the biosensor has the problem that a larger amount of an electric field having an influence on the impedance between the electrodes escapes upward from the electrodes than the amount generated between the electrodes. That is to say, the impedance variation is more affected by changes generated above the electrodes than by reactions generated between the electrodes. As a result, a narrow width and a low limit of impedance detection as well as a low accuracy of impedance detection are obtained, thereby implying poor reliability and availability of the biosensor.

[Prior art document]



[0005] (Patent document 1) Korean Patent No.777973 (published on Nov. 29, 2007)

[0006] US 2007/117221 A1 relates to an immunoassay apparatus on a chip.

[0007] US 2009/084686 A1 relates to a biosensor for detecting presence and concentration of various bio-materials.

[0008] HONGXIA CHEN ET AL: "Molecular Recognition of Arginine by Supramolecular Complexation with Calixarene Crown Ether Based on Surface Plasmon Resonance", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 12, no. 4, 4 April 2011 (2011-04-04), pages 2315-2324, XP055607977, DOI: 10.3390/ijms12042315 relates to a method by which arginine can be identified using an artificial monolayer based on surface plasmon resonance (SPR) .

[0009] ROISE E. MCGOVERN ET AL: "Microscale Crystals of Cytochrome c and Calixarene on Electrodes: Interprotein electron Transfer between Defined Site", ANGEWANDTE CHEMIE, INTERNATIONAL EDITION, vol. 54, no. 21, 1 April 2015 (2015-04-01), pages 6356-6359, XP055607980, DE ISSN: 1433-7851, DOI: 10.1002/anie.201500191 relates to a study on electroactivity accompanied by fast interprotein electron transfer in crystals.

[0010] US 2013/143775 A1 relates to a method for detecting the presence and/or the reaction of a biomolecule by monitoring changes of electrical property.

[0011] US 2011/312518 A1 relates to microfluidic devices for measurement or detection involving cells or biomolecules.

[0012] According to an aspect of the present invention, there is provided an interdigitated microelectrode biosensor according to claim 1.

[0013] Accordingly, the present invention has been made in view of the above-mentioned problems occurring in the prior art, and it is an object of the present invention to provide an interdigitated microelectrode biosensor using the reaction between receptors and target biomaterials, wherein the receptors reacting specifically with the target biomaterials are located between interdigitated microelectrodes, without having any conductive particles adapted to allow electric current to flow between the interdigitated microelectrodes, thereby increasing the width of impedance detection and the limit of impedance detection and also improving the accuracy of detection according to the characteristics of monomers and oligomers of the target biomaterials.

[Technical Solution]



[0014] To accomplish the above-mentioned object, according to the present disclosure, there is provided an interdigitated microelectrode biosensor including: an insulating layer adapted to cover all of a biosensor formation region of a substrate; a first interdigitated microelectrode having a plurality of first protrusion electrodes arranged in a comb-like shape on the insulating layer of the substrate; a second interdigitated microelectrode facing the first interdigitated microelectrode and having a plurality of second protrusion electrodes arranged in a comb-like shape on the insulating layer of the substrate in such a manner as to be interdigitated with the first protrusion electrodes of the first interdigitated microelectrode; and a plurality of receptors arranged in the space between the first interdigitated microelectrode and the second interdigitated microelectrode arranged interdigitatedly with each other so as to react specifically with target biomaterials.

[Advantageous Effects]



[0015] According to the present disclosure, the interdigitated microelectrode biosensor using the reaction between the receptors and the target biomaterials is configured to locate the receptors reacting specifically with the target biomaterials between the interdigitated microelectrodes, without having any conductive particles adapted to allow electric current to flow between the interdigitated microelectrodes, and configured to permit the adjacent interdigitated microelectrodes to face each other, so that the electric field is prevented from escaping upward from the interdigitated microelectrodes, thereby increasing the width of impedance detection by tens to hundreds of times and improving the accuracy of the detection.

[0016] In addition, the interdigitated microelectrode biosensor according to the present disclosure is configured to locate the receptors reacting specifically with the target biomaterials on the insulating layer between the respective interdigitated microelectrodes, so that the accuracy of detection can be improved according to the characteristics of the monomers and oligomers of the target biomaterials.

[Description of Drawings]



[0017] 

FIG.1 illustrates a configuration of an interdigitated microelectrode biosensor using the reaction between receptors and target biomaterials according to the present invention.

FIG.2 is a sectional view taken along the line A-A of FIG.1, which illustrates an interdigitated microelectrode biosensor according to a first embodiment of the present invention.

FIG.3 shows detailed configurations and actual shapes of the interdigitated microelectrodes of FIG.2.

FIG.4 is a graph showing a variation in the impedance of the interdigitated microelectrode biosensor through the reaction between antibodies and target biomaterials.

FIGS.5a to 5c are sectional views showing a method for manufacturing the interdigitated microelectrode biosensor illustrated in FIGS.1 to 3 according to the first embodiment of the present invention.

FIG.6 is a graph showing signal sizes of the interdigitated microelectrode biosensor when beta-amyloid monomers, beta-amyloid oligomers, and beta-amyloid monomers monomerized from beta-amyloid oligomers, which have the same concentrations as each other, react.

FIG.7 is a graph showing sizes of target detection signals according to the functionalization zone of the interdigitated microelectrode biosensor illustrated in FIGS.1 to 3.

FIG.8 is a sectional view taken along the line A-A of FIG.1, which illustrates an interdigitated microelectrode biosensor according to a second embodiment of the present invention.

FIGS.9a to 9c are sectional views showing a method for manufacturing the interdigitated microelectrode biosensor illustrated in FIGS.1 to 3 according to the second embodiment of the present invention.


[Best Mode for Invention]



[0018] Embodiments of the present disclosure will now be described in more detail with reference to the attached drawings.

[0019] FIG.1 illustrates a configuration of an interdigitated microelectrode biosensor using the reaction between receptors and a target biomaterial according to the present disclosure, and FIG.2 is a sectional view taken along the line A-A of FIG.1, which illustrates an interdigitated microelectrode biosensor according to a first embodiment of the present disclosure. Further, FIG.3 shows detailed configurations and actual shapes of the interdigitated microelectrodes of FIG.2.

[0020] As shown in FIGS.1 to 3, an interdigitated microelectrode biosensor according to the present disclosure includes: a first interdigitated microelectrode 100 having a plurality of first protrusion electrodes arranged in a comb-like shape on a substrate PL; a second interdigitated microelectrode 200 facing the first interdigitated microelectrode 100 and having a plurality of second protrusion electrodes arranged in a comb-like shape on the substrate PL in such a manner as to be interdigitated with the first protrusion electrodes of the first interdigitated microelectrode 100; and a plurality of receptors 231 arranged in the space between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 interdigitatedly arranged with each other so as to react specifically with target biomaterials 232. In this case, the receptors 231 include at least one of beta-amyloid antibodies, aptamers, and peptides.

[0021] First, the detection of impedance through the interdigitated microelectrode biosensor using the reaction with the target biomaterials 232 will be explained. The impedance between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 is summarized as follows:

wherein Z is impedance, R is resistance, X is reactance, C is capacitance, and w is angular frequency. The reactance X is divided into inductor component XL and capacitor component XC. The inductor component XL is ignored and only the capacitor component XC remains because the first interdigitated microelectrode 100 is not directly connected electrically to the second interdigitated microelectrode 200.

[0022] Accordingly, the plurality of receptors 231 are immobilized in the space between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200, and at the time when the target biomaterials 232 react with the receptors 231, if the variation of impedance in the space between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200, that is, in the space between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 facing each other, quantitative analysis of the target biomaterials 232 can be obtained.

[0023] As shown in FIGS.1 and 2, if the plurality of receptors 231 are immobilizedly arranged in the space between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200, generally, variations in electric field and impedance occur in a horizontal direction along which the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 are arranged in the state of placing the plurality of receptors 231 therebetween.

[0024] FIG.4 is a graph showing a variation in the impedance of the interdigitated microelectrode biosensor through the reaction between antibodies and target biomaterials.

[0025] As shown in FIG.4, if the plurality of receptors 231 bind specifically to the target biomaterials 232, a variation in resistance occurs between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 because the target biomaterials 232 are located therebetween. Further, the capacitance C decreases due to the properties of the target biomaterials, resulting in an increase in reactance Xc and a decrease in -Xc. The amount of the target biomaterials 232 can be exactly detected by measuring the resistance and reactance variations.

[0026] If the inductor component is ignored and only the reactance having the capacitor component is considered, like this, it is easy to check the variation in impedance only when a driving frequency is high, and contrarily, if the driving frequency is low, it is hard to check the variation in impedance because the variation is very weak. So as to detect a small amount of target biomaterials 232, therefore, high driving frequency should be used.

[0027] If the driving frequency is high, however, electric current generally flows through the space above the target biomaterials 232 binding specifically to the receptors 231, so that the detection of the target biomaterials 232 cannot be detected well. If the driving frequency is high, in addition, the target biomaterials 232 may be damaged by the high driving frequency, so that they are not detected well.

[0028] So as to detect the target biomaterials 232 well, accordingly, a low driving frequency in a range of 10 to 100 Hz is used. Because the driving frequency is low, accordingly, the damage on the target biomaterials 232 can be desirably prevented. Of course, it is hard to detect minute impedance variations because of the low frequency, but such a difficulty can be solved by means of the adoption of a differential amplifier.

[0029] In case where the conventional interdigitated microelectrode biosensor is used to detect biomaterials, antibodies are immobilized on tops and sides of the microelectrodes and the surrounding portions of the microelectrodes, and next, an impedance variation at the time when the antibodies bind to target molecules is observed. In this case, the antibodies are immobilized two-dimensionally only on the surfaces of the microelectrodes. According to an embodiment of the present invention, however, the receptors 231 and the antibodies are disposed only between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200, and in this case, an amount of electric field discharged to the outside can be reduced. Further, the receptors 231 and the antibodies are immobilized on a region where the electric field is concentrated, thereby extending the accuracy and dynamic range of the biosensor. According to the present invention, particularly, in case where the target biomaterials 232 are detected with the low driving frequency in the range of 10 to 100 Hz, a gap between the two microelectrodes 100 and 200 is desirably in a range of 3 to 7 µm. If the gap is less than 3 µm, a deviation in detected signals is too high, thereby failing to provide a reliable test, and if the gap is greater than 7 µm, sensitivity decreases to cause a difficulty in detecting a small amount of target biomaterials 232. When considering the deviation and sensitivity, most desirably, the gap is 5 µm.

[0030] FIGS.5a to 5c are sectional views showing a method for manufacturing the interdigitated microelectrode biosensor illustrated in FIGS.1 to 3 according to the first embodiment of the present disclosure.

[0031] So as to form an insulating layer 201, as shown in FIG.5a, a 500 nm thick silicon dioxide (SiO2) is formed on the substrate PL by means of thermal oxidation, and next, titanium (Ti) having a thickness in the range of 30 to 50 nm and platinum (Pt) having a thickness in the range of 150 to 200 nm are sequentially deposited on the silicon dioxide layer by means of sputtering, thereby forming metal layers. The titanium (Ti) layer and the platinum (Pt) layer are used as adhesion layers to increase the bonding strength of the silicon dioxide layer. The substrate on which Si02/Ti/Pt are deposited in this order is provided with a photoresist micropatterned through photolithography.

[0032] Subsequently, the multi-layer thin film deposition substrate having the micropatterned photoresist is allowed to sequentially etch the titanium (Ti) layer and the platinum (Pt) layer through inductively coupled plasma reactive ion etcher (ICP-RIE) to form the two microelectrodes 100 and 200 with the metal patterns, and after that, the photosensitive film patterns formed thereon are removed.

[0033] As shown in FIG.5b, next, a surface treatment process is performed, and at the surface treatment process, a calixcrown self-assembled monolayer or a polyvinylpyrrolodone (PVP) surface modification layer, as a connection molecular layer 233 adapted to selectively immobilize beta-amyloid antibodies, is formed on the surface of the insulating layer 201 between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200. After that, the beta-amyloid antibodies as the receptors 231 are immobilized onto the connection molecular layer 233. If so, the beta-amyloids as the target biomaterials 232 selectively bind specifically to the receptors 231.

[0034] In this case, a reference electrode for the signal comparison of the interdigitated microelectrode biosensor on which the beta-amyloid antibodies are immobilized and an interdigitated microelectrode biosensor on which prostate-specific antigen (PSA) antibodies are immobilized for negative control are provided.

[0035] Subsequently, if a region where the target biomaterials 232 bind specifically is completely exposed to the outside, detection errors may happen, and accordingly, there is a need to cover the region. To do this, a protection cap 250 is desirably disposed above the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200. Further, a polydimethylsiloxane (PDMS) chip having two microchannels is attached to prevent non-specific binding to other materials except the beta-amyloid, and an absorption prevention layer (bovine serum albumin) 235 is coated on the entire portion except the microchannels and a portion where the antibodies of the interdigitated microelectrode biosensor are immobilized, that is, on the inner wall of the protection cap 250 and the surfaces of the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 except the portion where the receptors 231 are not immobilized.

[0036] So as to perform stabilization, additionally, 0.1X phosphate-buffered saline (PBS) is injected into the two microchannels, and while an impedance signal of the interdigitated microelectrode biosensor is being observed until it is maintained stably and constantly, the stabilization is desirably performed. Initial stabilization time is given for five minutes to the biosensor whose stabilization is finished, and 10 pg/ml beta-amyloid is injected into the microchannels to observe the impedance signal for about 15 minutes, thereby checking the antigen-antibody reaction of the beta-amyloid. So as to minimize the non-specific binding or the influence of the electrical signal caused by the biomaterials existing in the PBS solution, after that, a clean PBS solution is injected to perform a solution change. Next, the impedance variation is observed for five minutes, thereby checking the size of a final signal through the specific reaction between the beta-amyloid and the antibodies.

[0037] FIG.6 is a graph showing signal sizes of the interdigitated microelectrode biosensor when a beta-amyloid monomer, a beta-amyloid oligomer, and a beta-amyloid monomer monomerized from beta-amyloid oligomer, which have the same concentrations as each other, react.

[0038] As shown in FIGS.1 to 3, the interdigitated microelectrode biosensor can distinguish the monomers of protein and the oligomers of protein from each other. As shown in FIG.6, a 100 pg/mL beta-amyloid monomer and a 100 pg/mL beta-amyloid oligomer are provided to have the same concentration as each other. Further, the 100 pg/mL beta-amyloid monomer is left at a room temperature for six hours and thus oligomerized, and next, when the oligomerized beta-amyloid monomer is injected into the interdigitated microelectrode biosensor where the beta-amyloid antibodies are formed, it is checked that the signal size of the monomer is greater by about 30 times than the oligomer. This is because the detection principle of the interdigitated microelectrode biosensor is determined upon the number of proteins binding to the antibodies and the sizes occupied by the proteins. After the oligomerized sample is monomerized through the injection of a detergent (e.g., epps) thereinto, the reaction is detected, and in this case, the monomerized sample has a very similar value to the value detected by using the sample only with the monomer, so that it is checked that the oligomer is generally monomerized.

[0039] FIG.7 is a graph showing sizes of target detection signals according to the functionalization zone of the interdigitated microelectrode biosensor illustrated in FIGS.1 to 3.

[0040] The interdigitated microelectrode biosensor as shown in FIGS.1 to 3 has a principle wherein the plurality of receptors 232 formed of the antibodies or aptamers are immobilized on the surface of the silicon dioxide (SiO2) layer as the insulating layer between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 so as to detect target molecules existing in the sample. In this case, when compared with the existing detection through functionalization above the electrodes, the detection according to the present invention is more excellent in sensitivity, and as shown in FIG.7, when compared with the detection signals at the time when the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 are functionalized, the detection signals according to the present invention are more increased by about 60%.

[0041] FIG.8 is a sectional view taken along the line A-A of FIG.1, which illustrates an interdigitated microelectrode biosensor according to a second embodiment of the present invention.

[0042] As shown in FIG.8, an interdigitated microelectrode biosensor according to a second embodiment of the present invention includes: a first interdigitated microelectrode 100 having a plurality of first protrusion electrodes arranged in a comb-like shape on a substrate PL; a second interdigitated microelectrode 200 facing the first interdigitated microelectrode 100 and having a plurality of second protrusion electrodes arranged in a comb-like shape on the substrate PL in such a manner as to be interdigitated with the plurality of first protrusion electrodes arranged in the first interdigitated microelectrode 100; and a plurality of receptors 231 immobilized in the space between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 arranged interdigitatedly with each other so as to react specifically with target biomaterials.

[0043] In detail, pattern of the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 are formed in comb-like shapes on the substrate PL, by a photolithography process using a photoresist, polymer, or silicon structure, and next, patterning for them is also carried out by the photolithography process to form metal patterns 210a surrounding both sides of the patterns of the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200.

[0044] The formation of the plurality of receptors 231 in the space between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 allows electric field and impedance variations to occur predominantly in the horizontal direction in which the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 are arranged in the state of placing the plurality of receptors 231 therebetween. With this arrangement, an electric field and an impedance are prevented from escaping upward from or perpendicularly to the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200, and they are generated in the horizontal direction, so that the reaction efficiencies of the plurality of receptors 231 immobilized between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 can be enhanced and the width of impedance detection can be improved by tens to hundreds of times.

[0045] FIGS.9a to 9c are sectional views showing a method for manufacturing the interdigitated microelectrode biosensor illustrated in FIGS.1 to 3 according to the second embodiment of the present disclosure.

[0046] According to the patterning method using the photoresist, polymer, and the silicon structure, as shown in FIG.9a, a 300 nm thick silicon dioxide (SiO2) is deposited on the substrate (Si wafer) PL made of silicon by means of PECVD, thereby forming an insulating layer 201. So as to form the insulating layer 201, otherwise, a 500 nm thick silicon dioxide (SiO2) is deposited on the substrate (Si wafer) PL made of silicon by means of thermal oxidation.

[0047] So as to form patterns of the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200, further, a photoresist PR is micropatterned. So as to form the microelectrodes with metal patterns 210, after that, titanium (Ti) having a thickness in the range of 30 to 50 nm and platinum (Pt) having a thickness in the range of 150 to 200 nm are sequentially deposited on the silicon dioxide layer by means of sputtering. In this case, the titanium (Ti) layer and the platinum (Pt) layer are used as adhesion layers to increase the bonding strength of the silicon dioxide layer. The substrate on which Si02/Ti/Pt are deposited in this order is provided with the photoresist micropatterned through photolithography.

[0048] Subsequently, as shown in FIG.9b, the multi-layer thin film deposition substrate having the micropatterned photoresist is allowed to sequentially etch the titanium (Ti) layer and the platinum (Pt) layer through inductively coupled plasma reactive ion etcher (ICP-RIE) to form the two microelectrodes 100 and 200 with the metal patterns 210.

[0049] As shown in FIG.9c, next, a surface treatment process is performed, and at the surface treatment process, a calixcrown self-assembled monolayer or a polyvinylpyrrolodone (PVP) surface modification layer, as a connection molecular layer 233 adapted to selectively immobilize the beta-amyloid antibodies, is formed on the surface of the insulating layer 201 between the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200. After that, the beta-amyloid antibodies as the receptors 231 are immobilized onto the connection molecular layer 233. If so, the beta-amyloids as the target biomaterials 232 selectively bind specifically to the receptors 231. In this case, a reference electrode for the signal comparison of the interdigitated microelectrode biosensor on which the beta-amyloid antibodies are immobilized and an interdigitated microelectrode biosensor on which prostate-specific antigen (PSA) antibodies are immobilized for negative control are provided.

[0050] Subsequently, if a region where the target biomaterials 232 bind specifically is completely exposed to the outside, detection errors may happen, and accordingly, there is a need to cover the region. To do this, a protection cap 250 is desirably disposed above the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200. Further, a polydimethylsiloxane (PDMS) chip having two microchannels is attached to prevent non-specific binding to other materials except the beta-amyloids, and an absorption prevention layer (bovine serum albumin) 235 is coated on the entire portion except the microchannels and a portion where the antibodies of the interdigitated microelectrode biosensor are immobilized, that is, on the inner wall of the protection cap 250 and the surfaces of the first interdigitated microelectrode 100 and the second interdigitated microelectrode 200 except the portion where the receptors 231 are not immobilized.

[0051] So as to perform stabilization, additionally, 0.1X phosphate-buffered saline (PBS) is injected into the two microchannels, and while an impedance signal of the interdigitated microelectrode biosensor is being observed until it is maintained stably and constantly, the stabilization is desirably performed. Initial stabilization time is given for five minutes to the biosensor whose stabilization is finished, and 10 pg/ml beta-amyloid is injected into the microchannels to observe the impedance signal for about 15 minutes, thereby checking the antigen-antibody reaction of the beta-amyloid. So as to minimize the non-specific binding or the influence of the electrical signal caused by the biomaterials existing in the PBS solution, after that, a clean PBS solution is injected to perform a solution change. Next, the impedance variation is observed for five minutes, thereby checking the size of a final signal through the specific reaction between the beta-amyloids and the antibodies.

[0052] As set forth in the foregoing, the interdigitated microelectrode biosensor using the reaction between the receptors and the target biomaterials is configured to locate the receptors reacting specifically with the target biomaterials between the interdigitated microelectrodes, without having any conductive particles adapted to allow electric current to flow between the interdigitated microelectrodes, and configured to permit the adjacent interdigitated microelectrodes to face each other, so that the electric field is prevented from escaping upward from the interdigitated microelectrodes, thereby increasing the width of impedance detection by tens to hundreds of times and improving the accuracy of the detection.

[0053] In addition, the interdigitated microelectrode biosensor according to the present invention is configured to locate the receptors specifically reacting with the target biomaterials on the insulating layer between the respective interdigitated microelectrodes, so that the accuracy of detection can be improved according to the characteristics of the monomers and oligomers of the target biomaterials.


Claims

1. An interdigitated microelectrode biosensor comprising:

an insulating layer adapted to cover all of a biosensor formation region of a substrate (201);

a first interdigitated microelectrode (100) having a plurality of first protrusion electrodes arranged in a comb-like shape on the insulating layer of the substrate (201);

a second interdigitated microelectrode (200) facing the first interdigitated microelectrode and having a plurality of second protrusion electrodes arranged in a comb-like shape on the substrate in such a manner as to be interdigitated with the first protrusion electrodes of the first interdigitated microelectrode;

a plurality of receptors (231) arranged in the space between the first interdigitated microelectrode and the second interdigitated microelectrode arranged interdigitatedly with each other so as to react specifically with target biomaterials (232);

a protection cap adapted to cover the insulating layer, the first interdigitated microelectrode, and the second interdigitated microelectrode;

a polydimethylsiloxane, PDMS, chip attached to prevent non-specific binding to other materials except beta-amyloids and having two microchannels; and

an absorption prevention layer, bovine serum albumin, coated on the inner wall of the protection cap and the surfaces of the first interdigitated microelectrode and the second interdigitated microelectrode except the portion where the receptors are not immobilized,

wherein the first and second interdigitated microelectrodes are configured such that resistance and reactance between the first interdigitated microelectrode and the second interdigitated microelectrode increase in response to the target biomaterials binding specifically to the plurality of receptors.


 
2. The interdigitated microelectrode biosensor according to claim 1,
wherein the first interdigitated microelectrode and the second interdigitated microelectrode comprise:

a first interdigitated microelectrode pattern and a second interdigitated microelectrode pattern formed by patterning at least one of a photoresist, polymer and a silicon structure on the substrate; and

metal patterns formed to surround both sides of the first interdigitated microelectrode pattern and the second interdigitated microelectrode pattern in such a manner as to face each other.


 
3. The interdigitated microelectrode biosensor according to claim 1,
wherein on the surface of the insulating layer between the first interdigitated microelectrode and the second interdigitated microelectrode is formed a calixcrown self-assembled monolayer or a polyvinylpyrrolodone (PVP) surface modification layer, as a connection molecular layer adapted to selectively immobilize beta-amyloid antibodies, and the receptors comprise at least one of beta-amyloid antibodies, aptamers, and peptides.
 
4. The interdigitated microelectrode biosensor according to claim 1,
wherein the receptors comprise at least one of a beta-amyloid antibodies, aptamers, and peptides and are immobilized on the top of the insulating layer exposed to the space between the first interdigitated microelectrode and the second interdigitated microelectrode arranged interdigitatedly with each other in such a manner as to react specifically with the target biomaterials.
 


Ansprüche

1. Biosensor mit interdigitalen Mikroelektroden, der Folgendes umfasst:

eine Isolierschicht, die zum Abdecken eines gesamten Biosensorbildungsbereichs eines Substrats (201) ausgelegt ist;

eine erste interdigitale Mikroelektrode (100) mit mehreren ersten Vorsprungselektroden, die in einer kammartigen Form auf der Isolierschicht des Substrats (201) angeordnet sind;

eine zweite interdigitale Mikroelektrode (200), die der ersten interdigitalen Mikroelektrode zugewandt ist und mehrere zweite Vorsprungselektroden aufweist, die in einer kammartigen Form auf dem Substrat derart angeordnet sind, dass sie mit den ersten Vorsprungselektroden der ersten interdigitalen Mikroelektrode interdigital sind;

mehrere Rezeptoren (231), die in dem Raum zwischen der ersten interdigitalen Mikroelektrode und der zweiten interdigitalen Mikroelektrode, die miteinander interdigital angeordnet sind, angeordnet sind, um spezifisch mit Zielbiomaterialien (232) zu reagieren;

eine Schutzkappe, die zum Abdecken der Isolierschicht, der ersten interdigitalen Mikroelektrode und der zweiten interdigitalen Mikroelektrode ausgelegt ist;

einen Polydimethylsiloxan-(PDMS)-Chip, der zum Verhindern einer unspezifischen Bindung an andere Materialien außer Beta-Amyloiden angebracht ist und zwei Mikrokanäle aufweist; und

eine Absorptionsverhütungsschicht, Rinderserumalbumin, mit der die Innenwand der Schutzkappe und die Oberflächen der ersten interdigitalen Mikroelektrode und der zweiten interdigitalen Mikroelektrode mit Ausnahme des Bereichs, in dem die Rezeptoren nicht immobilisiert sind, beschichtet sind,

wobei die erste und die zweite interdigitale Mikroelektrode so konfiguriert sind, dass Widerstand und Reaktanz zwischen der ersten interdigitalen Mikroelektrode und der zweiten interdigitalen Mikroelektrode als Reaktion auf die Ziel-Biomaterialien, die spezifisch an die mehreren Rezeptoren binden, zunehmen.


 
2. Biosensor mit interdigitalen Mikroelektroden nach Anspruch 1,
wobei die erste interdigitale Mikroelektrode und die zweite interdigitale Mikroelektrode Folgendes umfassen:

ein erstes interdigitales Mikroelektrodenmuster und ein zweites interdigitales Mikroelektrodenmuster, die durch Strukturierung von mindestens einem aus einem Photoresist, einem Polymer und einer Siliciumstruktur auf dem Substrat gebildet werden; und

Metallmuster, die so ausgebildet sind, dass sie beide Seiten des ersten interdigitalen Mikroelektrodenmusters und des zweiten interdigitalen Mikroelektrodenmusters so umgeben, dass sie einander zugewandt sind.


 
3. Biosensor mit interdigitalen Mikroelektroden nach Anspruch 1,
wobei auf der Oberfläche der Isolierschicht zwischen der ersten interdigitalen Mikroelektrode und der zweiten interdigitalen Mikroelektrode eine selbstassemblierte Calix-Kronen-Monoschicht oder eine Polyvinylpyrrolodon-(PVP)-Oberflächenmodifikationsschicht als molekulare Verbindungsschicht ausgebildet ist, die Beta-Amyloid-Antikörper selektiv immobilisieren kann, und die Rezeptoren mindestens eines aus Beta-Amyloid-Antikörpern, Aptameren und Peptiden umfassen.
 
4. Biosensor mit interdigitalen Mikroelektroden nach Anspruch 1,
wobei die Rezeptoren mindestens eines aus Beta-Amyloid-Antikörpern, Aptameren und Peptiden umfassen und auf der Oberseite der Isolierschicht, die dem Raum zwischen der ersten interdigitalen Mikroelektrode und der zweiten interdigitalen Mikroelektrode, die interdigital zueinander angeordnet sind, ausgesetzt ist, so immobilisiert sind, dass sie spezifisch mit den Ziel-Biomaterialien reagieren.
 


Revendications

1. Biocapteur à microélectrodes interdigitées, comprenant :

une couche isolante conçue pour recouvrir la totalité d'une région de formation de biocapteur d'un substrat (201) ;

une première microélectrode interdigitée (100) ayant une pluralité de premières électrodes saillantes agencées en forme de peigne sur la couche isolante du substrat (201) ;

une seconde microélectrode interdigitée (200) en regard de la première microélectrode interdigitée et ayant une pluralité de secondes électrodes saillantes agencées en forme de peigne sur le substrat de manière à être interdigitées avec les premières électrodes saillantes de la première microélectrode interdigitée ;

une pluralité de récepteurs (231) agencés dans l'espace entre la première microélectrode interdigitée et la seconde microélectrode interdigitée agencées mutuellement de façon interdigitée afin de réagir de façon spécifique avec des biomatériaux cibles (232) ;

un couvercle de protection conçu pour recouvrir la couche isolante, la première microélectrode interdigitée et la seconde microélectrode interdigitée ;

une puce en polydiméthylsiloxane (PDMS) fixée pour empêcher une liaison non spécifique à d'autres matériaux, à l'exception de bêta-amyloïdes, et ayant deux microcanaux ; et

une couche de prévention d'absorption, à base d'albumine de sérum bovin, revêtue sur la paroi interne du couvercle de protection et les surfaces de la première microélectrode interdigitée et de la seconde microélectrode interdigitée, à l'exception de la partie où les récepteurs ne sont pas immobilisés,

les première et seconde microélectrodes interdigitées étant configurées de sorte que la résistance et la réactance entre la première microélectrode interdigitée et la seconde microélectrode interdigitée augmentent en réponse à la liaison spécifique des biomatériaux cibles à la pluralité de récepteurs.


 
2. Biocapteur à microélectrodes interdigitées selon la revendication 1,
dans lequel la première microélectrode interdigitée et la seconde microélectrode interdigitée comprennent :

une première sculpture de microélectrode interdigitée et une seconde sculpture de microélectrode interdigitée formées en sculptant au moins une structure parmi une résine photosensible, un polymère et une structure de silicium sur le substrat ; et

des sculptures métalliques formées pour entourer les deux côtés de la première sculpture de microélectrode interdigitée et de la seconde sculpture de microélectrode interdigitée de sorte qu'elles soient face à face.


 
3. Biocapteur à microélectrodes interdigitées selon la revendication 1,
dans lequel, sur la surface de la couche isolante entre la première microélectrode interdigitée et la seconde microélectrode interdigitée, est formée une monocouche autoassemblée de calix-couronne ou une couche de modification de surface polyvinylpyrrolodone (PVP), en guise de couche moléculaire de connexion conçue pour immobiliser de manière sélective des anticorps de bêta-amyloïde, et les récepteurs comprennent au moins un récepteur parmi des anticorps de bêta-amyloïde, des aptamères et des peptides.
 
4. Biocapteur à microélectrodes interdigitées selon la revendication 1,
dans lequel les récepteurs comprennent au moins un récepteur parmi des anticorps de bêta-amyloïde, des aptamères et des peptides et sont immobilisés sur le dessus de la couche isolante exposée à l'espace entre la première microélectrode interdigitée et la seconde microélectrode interdigitée agencées mutuellement de façon interdigitée de manière à réagir de façon spécifique avec les biomatériaux cibles.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description