EP1604198A2 - Substrat electrique destine a etre employe en tant que support de biomolecules - Google Patents

Substrat electrique destine a etre employe en tant que support de biomolecules

Info

Publication number
EP1604198A2
EP1604198A2 EP03799443A EP03799443A EP1604198A2 EP 1604198 A2 EP1604198 A2 EP 1604198A2 EP 03799443 A EP03799443 A EP 03799443A EP 03799443 A EP03799443 A EP 03799443A EP 1604198 A2 EP1604198 A2 EP 1604198A2
Authority
EP
European Patent Office
Prior art keywords
substrate according
electrical substrate
approximately
layer
metal core
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03799443A
Other languages
German (de)
English (en)
Inventor
Gerhard Hartwich
Harald Lossau
Herbert Wieder
Norbert Persike
Christian Musewald
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
fidicula GmbH
Original Assignee
Friz Biochem Gesellschaft fuer Bioanalytik mbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Friz Biochem Gesellschaft fuer Bioanalytik mbH filed Critical Friz Biochem Gesellschaft fuer Bioanalytik mbH
Publication of EP1604198A2 publication Critical patent/EP1604198A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • 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
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors

Definitions

  • the invention relates to an electrical substrate for use as a carrier of biomolecules in a method for electrochemical detection in an electrolyte solution.
  • the invention also relates to the use of such a substrate in an electrochemical method for the detection of biomolecules.
  • a measuring cell with a passage chamber is arranged behind the separation column, into which a working electrode and a counter electrode protrude, over which the electrolyte solution flows.
  • a potential is applied between the working electrode and the counter electrode, which oxidizes or reduces the search substance.
  • the electron flow is measured as current flow at the working electrode and is a measure of that Content of the search substance in the sample.
  • association events on the basis of the change in the electrochemical properties of the probe oligonucleotides associated with the association, cf. such as WO 97/46568, WO 99/51778, WO 00/31101 or WO 00/42217.
  • the object of the invention is to increase the detection accuracy of an electrochemical detection method of the type mentioned at the outset.
  • This object is achieved according to the invention by the electrical substrate according to claim 1 and the use according to claim 26. Further advantageous details, aspects and configurations of the present invention result from the dependent claims, the description, the figures and the examples.
  • the electrical substrate according to the invention contains an insulating carrier plate which bears a conductor pattern with conductor tracks and connection contact surfaces, and test points arranged on the conductor tracks for the application of biomolecules.
  • the conductor tracks have a metal core made of a highly conductive base metal and a gold layer surrounding the metal core.
  • the conductor tracks are also continuously provided with a diffusion barrier layer, which prevents direct contact of the electrolyte solution with the metal core when an electrochemical detection method is carried out.
  • the invention is based on the knowledge of the present inventors that the base metal core, typically copper, provided on electrical substrates can strongly influence the measurement signal during electrochemical detection.
  • the base metal core typically copper
  • copper oxidation leads to a signal peak at a potential of 250 mV relative to an Ag / AgCI reference electrode.
  • Many of the electrochemical detection methods indicated as preferred are also carried out in this potential range. Particularly when very small amounts of a test substance are to be detected, a comparatively small number of copper atoms can lead to a falsification or an undesirable influence on the measurement signal.
  • Electrochemical detection also offers a number of other advantages which only come into play when the disruptive, electrochemical influence of the base metal is reduced or completely eliminated by the diffusion barrier layer. This includes, for example, the significantly higher sensitivity of the electrochemical readout process compared to conventional processes due to the direct connection of the capture molecules to the subsequent electronics. This also greatly reduces the evaluation time required. The comparatively simple preparation also leads to a reduction in the total time required for a measurement. In contrast to conventional methods, the substance to be examined does not have to be modified by a special marker or brought to a detectable amount of substance by means of faulty amplifications (multiplicative methods).
  • the metal core of the substrate according to the invention comprises copper, tungsten and / or aluminum.
  • the metal core can advantageously be formed from copper.
  • the diffusion barrier layer comprises an intermediate layer made of nickel, titanium and / or platinum arranged between the metal core and the outer gold layer.
  • an intermediate layer effectively prevents the diffusion of atoms from the base of the noble metal into the electrolyte solution and thus enables highly sensitive electrochemical detection methods.
  • the intermediate layer expediently has a thickness of approximately 2 ⁇ m to approximately 10 ⁇ m, preferably approximately 3 ⁇ m to approximately 8 ⁇ m, particularly preferably approximately 4 ⁇ m to approximately 6 ⁇ m.
  • the diffusion barrier layer comprises a lacquer layer applied to the gold layer.
  • the diffusion barrier layer comprises a gold layer arranged on the metal core, the pores of which are essentially closed by melting a surface area of the gold layer, so that the migration of atoms from the metal core is practically prevented.
  • the diffusion barrier layer can also be formed by a combination of several of the measures described.
  • the diffusion barrier layer can be formed only in partial areas by a lacquer layer applied to the gold layer. In areas without an applied lacquer layer, such as the test points, the gold layer can be melted by laser bombardment, so that the gold layer itself forms a diffusion barrier layer in these areas.
  • the gold layer in the above-mentioned configurations has a thickness of approximately 0.15 ⁇ m to approximately 10 ⁇ m, preferably approximately 1 ⁇ m to approximately 5 ⁇ m, particularly preferably approximately 2 ⁇ m to approximately 3 ⁇ m.
  • the diffusion barrier layer is formed by a gold layer arranged on the metal core, the thickness of which is chosen so large that it prevents direct contact of the electrolyte solution with the metal core.
  • the insulating carrier plate is expediently a one-sided rigid carrier plate, a double-sided rigid carrier plate or a rigid multilayer carrier plate.
  • the insulating carrier plate can be a one-sided or double-sided flexible carrier plate, in particular made of a polyimide film, or a rigidly flexible carrier plate.
  • a base material selected from the group BT (bismaleimide triazine resin with quartz glass), CE (cyanate ester with quartz glass), CEM1 (hard paper core with FR4 outer layers), CEM3 (glass fleece core with FR4 outer layers), FR2 ( Phenolic resin paper), FR3 (hard paper), FR4 (epoxy glass hard fabric), FR5 (epoxy glass hard fabric with cross-linked resin system), PD (polyimide resin with aramid reinforcement), PTFE (polytetrafluoroethylene with glass or ceramic), CHn (highly cross-linked hydrocarbons with ceramic) and glass ,
  • BT bismaleimide triazine resin with quartz glass
  • CE cyanate ester with quartz glass
  • CEM1 hard paper core with FR4 outer layers
  • CEM3 glass fleece core with FR4 outer layers
  • FR2 Phenolic resin paper
  • FR3 hard paper
  • FR4 epoxy glass hard fabric
  • FR5 epoxy glass hard
  • the insulating carrier plate is formed by a semiconductor plate or a semiconductor plate provided with a carrier plate insulation layer.
  • the insulating carrier plate of the electrical substrate can advantageously be formed by a silicon plate provided with a SiNx insulation layer.
  • Embodiment of the invention on a width of 50 microns to 250 microns, in particular from 80 microns to 200 microns.
  • the conductor tracks are formed on a semiconductor substrate, such as the SiN x -coated Si plate mentioned, they can also be made considerably narrower in accordance with the conventional processes of semiconductor technology and have a width of a few ⁇ m or even below a micrometer. If the conductor tracks are made very narrow, they advantageously have widenings in the area of the test sites in order to provide a sufficiently large area for the absorption of biomolecules. Furthermore, it can advantageously be provided according to the invention that an insulation layer is applied to the outer gold layer in partial areas.
  • the insulation layer can advantageously be formed by a thermally and / or optically curable, structurable lacquer. In an expedient embodiment, the insulation layer is formed by a parylene layer.
  • the insulation layer preferably has a thickness of approximately 1 ⁇ m to approximately 30 ⁇ m, particularly preferably approximately 5 ⁇ m to approximately 20 ⁇ m.
  • the insulation layer expediently has cutouts on part of the conductor tracks as far as the gold layer underneath which form test sites for applying the biomolecules.
  • the conductor pattern contains one or more plated-through holes which have a metal core made of a highly conductive base metal arranged on their peripheral edge surface and a gold layer surrounding the metal core.
  • the plated-through holes are continuously provided with a diffusion barrier layer, which prevents direct contact of the electrolyte solution with the metal core in the electrochemical detection method.
  • the metal core of the vias is preferably formed from tungsten or aluminum.
  • the diffusion barrier layer is expediently formed by an intermediate layer of nickel, titanium and / or platinum arranged between the metal core of the plated-through holes and the outer gold layer.
  • the thickness of the intermediate layer of the plated-through holes is advantageously approximately 0.01 ⁇ m to approximately 1 ⁇ m, preferably approximately 0.05 ⁇ m to approximately 0.5 ⁇ m, particularly preferably approximately 0.1 ⁇ m to approximately 0.2 ⁇ m.
  • the gold layer of the plated-through holes advantageously has a thickness of approximately 0.05 ⁇ m to approximately 0.75 ⁇ m, preferably from approximately 0.15 ⁇ m to approximately 0.5 ⁇ m, particularly preferably from approximately 0.3 ⁇ m.
  • the invention also includes the use of an electrical substrate of the type described in an electrochemical detection method selected from the group chronoamperometry (CA), chronocoulometry (CC), linear sweep voltammetry (LSV), cyclic voltammetry (CSV), AC voltammetry, voltammetry techniques with different pulse shapes , in particular square wave voltammetry (SWV), differential pulse voltammetry (DPV), or normal pulse voltammetry (NPV), AC or DC impedance spectroscopy, chronopotentiometry and cyclic chronopotentiometry.
  • CA chronoamperometry
  • CC chronocoulometry
  • LSV linear sweep voltammetry
  • CSV cyclic voltammetry
  • AC voltammetry techniques with different pulse shapes in particular square wave voltammetry (SWV), differential pulse voltammetry (DPV), or normal pulse voltammetry (NPV), AC or DC impedance spectroscopy, chronopotentiometry and cyclic chronopotentiometry.
  • FIG. 1 shows a section of an electrical substrate according to an embodiment of the invention in a schematic representation
  • Figure 2 is a section through the electrical substrate of Figure 1 along the line A-A;
  • FIG. 3 shows a section as in FIG. 2 through an electrical substrate according to another exemplary embodiment of the invention. Ways of Carrying Out the Invention
  • 10 denotes an electrical substrate which is used as a carrier of biomolecules in a method for electrochemical detection in an electrolyte solution, as is described, for example, in the publication WO 00/42217.
  • the electrical substrate 10 comprises an insulating carrier plate 12 made of the epoxy glass fiber fabric FR4, on which a conductor pattern with a plurality, in the exemplary embodiment fifty, parallel conductor tracks is arranged. Of the majority of the conductor tracks, only a part of the counterelectrode 28 and three of forty-eight parallel working electrodes, which are designated 20A to 20C, are shown in the detail of FIG. 1.
  • the forty-eight parallel working electrodes each have, as shown by way of example for the working electrodes 20A to 20C, an essentially rectangular test point 24, on which biomolecules 26 are applied for carrying out an electrochemical detection method.
  • FIG. 2 shows a section along the line A-A of FIG. 1 through the conductor tracks 20A to 20C.
  • Each of the conductor tracks 20 consists of a copper core 14 which is continuously covered by a nickel barrier layer 16 and a gold layer 18.
  • the copper core 14 has a thickness of approximately 28 ⁇ m. It represents an inexpensive and highly conductive basic component of the conductor tracks 20.
  • the copper cores 14 are continuously coated with the approximately 2 ⁇ m thick gold layer 18. Between the copper core 14 and the gold layer 18 is arranged as a diffusion barrier in each case an approximately 6 ⁇ m thick, continuous nickel layer 16.
  • the entire conductor pattern is covered with a 15 ⁇ m to 20 ⁇ m thick insulation layer 22, in the exemplary embodiment made of a structurable, optically curable lacquer. Rectangular recesses 24 are made in this insulation layer 22, for example by laser bombardment of the insulation layer 22 with high-energy pulses from an excimer laser. The recesses 24 form the test sites for receiving the biomolecules 26.
  • the conductor tracks 20 of the exemplary embodiment in FIGS. 1 and 2 are approximately 100 ⁇ m wide and are arranged on the carrier plate 12 at a distance of approximately 200 ⁇ m (center-center).
  • the square test points 24 have an extent of approximately 60 ⁇ m ⁇ 60 ⁇ m.
  • the working electrodes 20A-20C, the counter electrode 28 and a reference electrode, which may also be provided, are each connected to connection contact surfaces (not shown) of the electrical substrate 10 for contacting.
  • charge-time curves are recorded, for example, in chronocoulometry, as in the
  • test sites 24 of the forty-eight working electrodes 20A, 20B, 20C, ... are selectively occupied with probe biomolecules, for example 20 nucleotide ligate oligonucleotides.
  • the test sites 24 are then brought into contact with a signal oligonucleotide solution, for example a 12 nucleotide signal nucleic acid oligomer ligand, and measured after a predetermined incubation period.
  • the signal nucleic acid oligomer ligands carry one or more redox labels and are complementary to a region of the ligate oligonucleotide near the surface, so that an association between ligate oligonucleotide and redox-labeled signal nucleic acid oligomer complexing agent can take place.
  • the working electrodes individually or in groups, are set to a first potential by means of a potentiostat, at which little to no electrolysis (electrochemical change in the redox state) of the redox marking can take place.
  • the working electrode in ferrocene-modified ligate oligonucleotides is set to a potential of approximately 100 mV in relation to the reference electrode, in the exemplary embodiment (Ag / AgCI (KCI)).
  • the working electrode (s) is or are set to a second higher potential by a jump in potential, at which the electrolysis of the redox marking takes place in the diffusion-limited limit case.
  • the working electrode is set to about 500 mV against Ag / AgCI (KCI). The transferred charges are recorded as a measurement signal depending on the time.
  • This chronocoulometry measurement signal is composed of three components: a diffusive component, which is caused by the redox-active components in the volume phase and has a t 1 2 dependency, a first instantaneous Part that results from the charge redistribution in the double layer on the electrode surface and a second instantaneous part that is caused by the conversion of redox-active components that are immobilized on the electrode surface.
  • the sample solution is added which should or may contain the ligand nucleic acid oligomer (target) which has a nucleotide sequence which is complementary to the 20 nucleotide of the ligate oligonucleotides in a region.
  • target the ligand nucleic acid oligomer
  • a second electrochemical measurement is carried out.
  • the change in the instantaneous charge signal is proportional to the number of signal oligonucleotide ligands displaced and is therefore pro- proportional to the number of target oligonucleotides present in the test solution.
  • each of the conductor tracks 20 contains a copper core 14.
  • the copper core 14 was coated directly with an approximately 7 ⁇ m thick gold layer 18 and the conductor pattern is coated with a 15 ⁇ m thick parylene. Lacquer layer 22 coated.
  • a recess is made in the lacquer layer 22 by excimer laser bombardment for each conductor track 20. Laser energy and the number of laser pulses are selected such that after removal of the lacquer layer 22, the gold layer 18 lying beneath the lacquer layer melts in a surface region 26. As a result, the surface pores of the gold layer 18 in the area of the test sites 24 are closed, so that the gold layer 18 forms a barrier layer impermeable to diffusing copper atoms there. In the other areas, the lacquer layer 22 prevents the copper atoms from coming into contact with the electrolyte solution.
  • each conductor track 20 contains an approximately 2 ⁇ m thick metal core made of tungsten.
  • the tungsten core is continuously coated with a diffusion barrier layer, which is formed from 2 ⁇ m thick layers of titanium and platinum.
  • An approximately 2 ⁇ m thick gold layer is continuously applied to this diffusion barrier layer, on which a number of test sites for receiving biomolecules are defined in the manner described above.
  • An electrical substrate designed in this way also allows highly sensitive electrochemical detection methods to be carried out.
  • a silicon carrier plate coated with SiN x contains a plurality of circular vias, in which a tungsten core running around the edge of the vias with an approximately 0.1 ⁇ m thick titanium and an approximately 0.1 ⁇ m thick platinum layer is coated. A 0.3 ⁇ m thick gold layer is applied to this barrier layer. This creates an electrical semiconductor substrate suitable for highly sensitive electrochemical detection methods.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Urology & Nephrology (AREA)
  • Electrochemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

L'invention concerne un substrat électrique destiné à être employé en tant que support de biomolécules au cours d'un procédé de détection électrochimique dans une solution électrolytique. Ledit substrat est composé d'une plaque support isolante (12) portant un motif conducteur présentant des tracés conducteurs (20; 20A-20C, 28) et des surfaces de contact de connexion, ainsi que de zones de test (24) disposées sur les tracés conducteurs (20; 20A-20C, 28), destinées à l'application de biomolécules (26). Les tracés conducteurs (20; 20A-20C, 28) comportent un noyau métallique (14) réalisé dans un métal commun conducteur, et une couche d'or extérieure (18). Par ailleurs, les tracés conducteurs (20; 20A-20C, 28) sont pourvus d'une couche de blocage de diffusion (16) empêchant le contact direct entre la solution électrolytique et le noyau métallique (14) au cours du procédé de détection électrochimique.
EP03799443A 2002-12-23 2003-12-23 Substrat electrique destine a etre employe en tant que support de biomolecules Withdrawn EP1604198A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10261528A DE10261528B4 (de) 2002-12-23 2002-12-23 Elektrisches Substrat zum Einsatz als Träger von Biomolekülen
DE10261528 2002-12-23
PCT/DE2003/004259 WO2004059305A2 (fr) 2002-12-23 2003-12-23 Substrat electrique destine a etre employe en tant que support de biomolecules

Publications (1)

Publication Number Publication Date
EP1604198A2 true EP1604198A2 (fr) 2005-12-14

Family

ID=32478079

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03799443A Withdrawn EP1604198A2 (fr) 2002-12-23 2003-12-23 Substrat electrique destine a etre employe en tant que support de biomolecules

Country Status (6)

Country Link
US (1) US20060275925A1 (fr)
EP (1) EP1604198A2 (fr)
JP (1) JP2006519975A (fr)
AU (1) AU2003299274A1 (fr)
DE (1) DE10261528B4 (fr)
WO (1) WO2004059305A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006063604A1 (fr) * 2004-12-14 2006-06-22 Friz Biochem Gesellschaft Für Bioanalytik Mbh Substrat de realisation controlee de reactions de liaison ligat/ligand et procede de fabrication
WO2006098813A1 (fr) * 2005-02-01 2006-09-21 Second Sight Medical Products, Inc. Dispositif implantable revetu microminiaturise
JP6149480B2 (ja) * 2013-04-16 2017-06-21 大日本印刷株式会社 バイオセンサ用電極、バイオセンサ用電極部材およびバイオセンサ

Family Cites Families (8)

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Publication number Priority date Publication date Assignee Title
US2090649A (en) * 1936-09-16 1937-08-24 Tetreault Amos Indexing attachment for slotting machines
JP2886317B2 (ja) * 1990-10-05 1999-04-26 富士通株式会社 配線基板およびその製造方法
US6281006B1 (en) * 1998-08-24 2001-08-28 Therasense, Inc. Electrochemical affinity assay
US6180523B1 (en) * 1998-10-13 2001-01-30 Industrial Technology Research Institute Copper metallization of USLI by electroless process
ES2319380T3 (es) * 1999-07-26 2009-05-07 Clinical Micro Sensors, Inc. Determinacion de secuencias de acido nucleico usando la deteccion electronica.
US20020090649A1 (en) * 1999-12-15 2002-07-11 Tony Chan High density column and row addressable electrode arrays
DE10156433A1 (de) * 2001-04-27 2002-10-31 Febit Ag Verfahren und Vorrichtungen zur elektronischen Bestimmung von Analyten
US7473398B2 (en) * 2001-05-25 2009-01-06 Roche Diagnostics Operations, Inc. Biosensor

Non-Patent Citations (3)

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Title
BALCH M: "Just For Starters. What is a PCB?", NUTS & VOLTS, vol. 24, no. 12, December 2003 (2003-12-01), pages 17 - 20 *
See also references of WO2004059305A3 *
WIDMANN D ET AL: "Technologie hochintegrierter Schaltungen", 1996, SPRINGER, BERLIN *

Also Published As

Publication number Publication date
US20060275925A1 (en) 2006-12-07
DE10261528A1 (de) 2004-07-08
AU2003299274A8 (en) 2004-07-22
AU2003299274A1 (en) 2004-07-22
JP2006519975A (ja) 2006-08-31
WO2004059305A2 (fr) 2004-07-15
DE10261528B4 (de) 2006-10-05
WO2004059305A3 (fr) 2004-10-14

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