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EP1527202A1 - Recognition layers made of hydrogel based on polyacrylamide for use in biosensor technology - Google Patents

Recognition layers made of hydrogel based on polyacrylamide for use in biosensor technology

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Publication number
EP1527202A1
EP1527202A1 EP03790648A EP03790648A EP1527202A1 EP 1527202 A1 EP1527202 A1 EP 1527202A1 EP 03790648 A EP03790648 A EP 03790648A EP 03790648 A EP03790648 A EP 03790648A EP 1527202 A1 EP1527202 A1 EP 1527202A1
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EP
European Patent Office
Prior art keywords
immobilization layer
layer according
hydrophilic
polyacrylamide
hydrophilic immobilization
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.)
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Application number
EP03790648A
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German (de)
French (fr)
Inventor
Hans-Dieter Feucht
Walter Gumbrecht
Manfred Stanzel
Heinrich Zeininger
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.)
Siemens AG
Siemens Corp
Original Assignee
Siemens AG
Siemens Corp
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 Siemens AG, Siemens Corp filed Critical Siemens AG
Publication of EP1527202A1 publication Critical patent/EP1527202A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes

Definitions

  • the present invention relates to an immobilization layer for biosensors and their use for the production of biosensory detection layers, in particular for the production of so-called DNA chips.
  • biosensors are increasingly used in which a biological detection system is linked to a physical transducer.
  • Recognition systems are understood to be biological recognition molecules, such as antibodies, enzymes, nucleic acids and the like, which are bound to a support (transducer) via a so-called immobilization layer.
  • Transducer a support
  • immobilization layer a support
  • Mainly calorimetric, piezoelectric, optical and electrochemical principles are used as transducers.
  • the detection systems or originally the immobilization layers, are usually immobilized in approximately two-dimensional layers on the transducer systems.
  • the recognition molecules can be immobilized by 5 covalent bonds, by affinity interaction, but also by hydrophilic / hydrophobic interactions. For reasons of stability, covalent bonds are preferred, but the formation of stable complexes, such as biotin / avidin, is also successfully used.
  • a good overview of the structure of almost two-dimensional biological recognition layers is given by I. Willner, E. Katz: "Redox protein layers on conductive supports - systems for bioelectronic applications" in Angew. Chem. 2000, 112, pp. 1230-69.
  • the biological function carriers ie the recognition molecules
  • the biological function carriers are often replaced by alkoxysilanes, which are known as contain linker groups, but are also immobilized with the aid of cyanuric chloride or carbodiimide.
  • recognition molecules labeled with tiolalkyl are used, which are immobilized on the transducer surface via sulfur-gold bonds in the form of so-called self-assembly layers.
  • WO 00/43539 describes the construction of a three-dimensional DNA recognition layer by immobilizing the DNA capture probes in the form of polymer brushes.
  • Timofeev et al. describes a chemically modified, radically crosslinked polyacrylamide which can be used, for example, for immobilizing capture oligos (EN Timofeev et al., Regioselective Immobilization of Short Oligonucleotides to Acrylic Copolymer Gels, Nucleic Acids Research, 1966, Vol. 24, 16, 3142-3148).
  • amino or aldehyde groups are used as coupling groups in the hydrogel.
  • Aldehyde- or amino-functionalized scavenger oligos can be covalently immobilized on these coupling groups under reductive reaction conditions.
  • the object of the present invention is to produce a hydrophilic immobilization layer for biosensor applications based on a hydrogel and to use such immobilization layers to generate recognition layers by covalently coupling biological recognition molecules.
  • the present invention solves this problem by using free-radically crosslinked or photo-structured hydrogels as the immobilization layer.
  • hydrogels are described in the applicant's German patent applications "Radically crosslinkable composition for producing a hydrogel layer” or "Photo-structurable composition for producing a hydrogel layer” (file number not yet known).
  • the present invention accordingly relates to a hydrophilic immobilization layer for biosensors made from a free-radically crosslinked hydrogel based on polyacrylamide, the starting composition comprising acrylamide, crosslinking agents, radical initiators, at least one comonomer with reactive linker groups and optionally plasticizers and other additives.
  • the subject of the present compound is also a hydrophilic immobilization layer made of a photostructured hydrogel based on polyacrylamide, the starting composition being acrylamide, crosslinking agent, photoinitiators, at least one film former, at least one comonomer with reactive linker groups and optionally plasticizers and other additives.
  • the systems according to the invention allow the construction of sensor arrays with biological recognition molecules in a three-dimensional matrix with a high integration density.
  • compositions which ensure the miscibility of the monomers involved and the initiators.
  • Commercial additives can be used to reduce the surface tension.
  • a water-swellable hydrogel is obtained, into which biological or chemical recognition molecules for analytical or diagnostic applications are coupled in using the linker groups while maintaining their functionality can.
  • the present invention accordingly also relates to the use of the immobilization layers for the production of biosensory recognition layers by (covalent) coupling or immobilization of chemical or biological recognition molecules, the recognition molecules preferably being scavenger oligonucleotides.
  • the starting composition for generating the hydrogel layer (immobilization layer) can be applied to the suitable carrier using all modern coating technologies. However, spin coating and dispensing are preferably used.
  • the properties of the hydrogel layer to be produced with regard to hydrophilicity, crosslinking density, swellability, etc. can be varied widely by the type of starting components used, their relationship to one another and ultimately the type of layer formation.
  • the hydrogel matrix can be adapted to the biological recognition molecules used, in particular with regard to the crosslinking density.
  • the crosslinking density will be controlled by the type and concentration of the crosslinking molecules used, such as acrylic and / or methacrylic compounds, in particular methylenebis (meth) acrylamide and / or dimethacrylic acid esters, such as tetraethylene glycol dimethacrylate.
  • the hydrogel mixture can also be adapted to the coating method preferred for the specific application.
  • a polymeric film former such as polyvinylpyrrolidone, polyacrylamide and / or polyhydroxymethacrylate
  • high-boiling solvents such as.
  • ethylene glycol can be used for the hydrogel mixture, which do not evaporate completely during spin coating and thus as a plasticizer in the
  • the residual solvent content can then be further reduced in a targeted manner by means of a prebake step before crosslinking, and thus the polymerization yield or the resulting layer thickness can be controlled. If necessary, additional plasticizer systems, such as di- and / or triethylene glycol, can be added.
  • the hydrogel mixture in solution is applied in drops, depending on the transducer dimensions, in sizes from a few microliters to one nanoliter.
  • the dispensing are high-boiling solvents, which have a long enough life for the drop have at the tip of the dispensing cannula used. This makes the dosing and settling of the drop reproducible.
  • the boiling point of the solvent must not be too high to allow the solvent to evaporate sufficiently quickly from the settled drop.
  • a tempering step to control the residual solvent content may be required. According to the invention, preference is given to using dimethylformamide and / or ethylene glycol for dispensing the hydrogel mixture.
  • the hydrogel mixture can be applied in layer or spot form on transducer or carrier surfaces made of metal, glass, silicon, silicon dioxide, silicon nitride or plastic.
  • Surfaces with topography which consist of different materials, such as.
  • B. Interdigital electrode arrays are coated on silicon nitride as passivation.
  • the coating of surfaces also includes the coating of inner surfaces of microchannels or nanotubes.
  • the surfaces to be coated are optionally coated with an adhesion promoter.
  • the hydrogel layer is polymerized and crosslinked by thermal or UV initiation.
  • the hydrogel layer can also be structured by contact or proximity exposure through a mask.
  • the hydrogel layer works like a negative resist. Polymerization and crosslinking take place in the irradiated area. There is no reaction in the darkened areas.
  • the hydrogel mixture found here is detached from the substrate in a development step. Auxiliary components such as polymeric film formers or plasticizers can be removed from the crosslinked hydrogel layer by extraction. Under certain circumstances, this step can take place at the same time as the actual equipment step.
  • the bioglogical or chemical detection systems are preferably made from aqueous solution, from aqueous buffer solution or applied to the immobilization layer from mixtures of polar solvents with water. It is applied by dripping on or spotting on / dispensing.
  • the solution with the biological or chemical recognition molecules can also be brought to the crosslinked hydrogel layer by transport through the fluidic system itself.
  • Cross-linked hydrogel spots which are surrounded by a protective ring, are advantageously used for the precise loading of measuring spots.
  • a tempering step may be required for the covalent coupling of the biological or chemical recognition molecules, which are provided with a coupling group that matches the linker group present in the crosslinked hydrogel.
  • a inoalkyl groups are particularly suitable for coupling to the linker groups epoxy and maleic anhydride.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The invention relates to a hydrophilic immobilization layer for biosensors made of a radically cross-linkable hydrogel based on polyacrylamide, whereby the starting composition comprises acrylamide, cross-linkers, radical initiator(s), at least one comonomer having reactive linker groups and optionally comprises softeners or the inventive layer is made of a photostructured hydrogel based on polyacrylamide, whereby the starting composition comprises acrylamide, cross-linkers, photoinitiators, at least one film former, at least one comonomer having reactive linker groups and optionally comprises softeners.

Description

Beschreibung description

E KENNUNGSSSCHICHTEN AUS HYDROGEL AUF DER BASIS VON POLYACRYLAMID FÜR DIE BIOSENSORIKE HYDROGEL IDENTIFICATION LAYERS BASED ON POLYACRYLAMIDE FOR BIOSENSORICS

Die vorliegende Erfindung betrifft eine Immobilisierungsschicht für Biosensoren sowie ihre Verwendung zur Erzeugung von biosensorischen Erkennungsschichten, insbesondere zur Erzeugung von sogenannten DNA-Chips .The present invention relates to an immobilization layer for biosensors and their use for the production of biosensory detection layers, in particular for the production of so-called DNA chips.

In der modernen biologischen Analysentechnik, aber auch in der medizinischen Diagnostik, werden in zunehmendem Maße Biosensoren eingesetzt, bei denen ein biologisches Erkennungssystem mit einem physikalischen Transducer verknüpft ist. Unter Erkennungssystemen versteht man biologische Erkennungsmo- leküle, wie Antikörper, Enzyme, Nucleinsäuren und dergleichen, welche über eine sogenannte Immobilisierungsschicht an einem Träger (Transducer) gebunden sind. Als Transducer werden hauptsächlich kalorimetrische, piezoelektrische, optische und elektrochemische Prinzipien verwendet. 0In modern biological analysis technology, but also in medical diagnostics, biosensors are increasingly used in which a biological detection system is linked to a physical transducer. Recognition systems are understood to be biological recognition molecules, such as antibodies, enzymes, nucleic acids and the like, which are bound to a support (transducer) via a so-called immobilization layer. Mainly calorimetric, piezoelectric, optical and electrochemical principles are used as transducers. 0

Die Erkennungssysteme, respektive ursprünglich die Immobilisierungsschichten, werden dabei meist in annähernd zwei- di ensionalen Schichten auf den Transducersystemen immobilisiert. Die Immobilisierung der Erkennungsmoleküle kann durch 5 kovalente Bindungen, durch Affinitätswechselwirkung aber auch durch hydrophil/hydrophobe Wechselwirkungen erfolgen. Aus Stabilitätsgründen werden kovalente Bindungen bevorzugt, jedoch kommt auch die Bildung stabiler Komplexe, wie zum Beispiel Biotin/Avidin, erfolgreich zum Einsatz. Einen guten 0 Überblick über den Aufbau annähernd zwei-dimensionaler biologischer Erkennungsschichten geben I. Willner, E. Katz: "Re- doxproteinschichten auf leitenden Trägern - Systeme für bioelektronische Anwendungen" in Angew. Chem. 2000, 112, S. 1230-69. 5 Bei Transducer-Oberflächen, welche NH- oder OH-Gruppen enthalten, werden die biologischen Funktionsträger, d.h. die Erkennungsmoleküle, häufig durch Alkoxysilane, welche sogenann- te Linkergruppen enthalten, aber auch mit Hilfe von Cyanurch- lorid oder Carbodiimid immobilisiert. Zur Ausrüstung goldhaltiger Transduceroberflächen werden mit T iolalkyl gelabelte Erkennungsmoleküle eingesetzt, die über Schwefel-Gold- Bindungen in Form von sogenannten Self-Assembly-Schichten auf der Transduceroberflache immobilisiert werden. Ein interessanter Ansatz zur Immobilisierung von Nucleinsäuren auf Transduceroberflächen ist die photochemisch unterstützte Synthese von Affymetrix (Light-directed spatially addressable parallel chemical synthesis, S.P.A. Fodor et al . , Science 251, 767-773 (1991) ) .The detection systems, or originally the immobilization layers, are usually immobilized in approximately two-dimensional layers on the transducer systems. The recognition molecules can be immobilized by 5 covalent bonds, by affinity interaction, but also by hydrophilic / hydrophobic interactions. For reasons of stability, covalent bonds are preferred, but the formation of stable complexes, such as biotin / avidin, is also successfully used. A good overview of the structure of almost two-dimensional biological recognition layers is given by I. Willner, E. Katz: "Redox protein layers on conductive supports - systems for bioelectronic applications" in Angew. Chem. 2000, 112, pp. 1230-69. 5 In the case of transducer surfaces that contain NH or OH groups, the biological function carriers, ie the recognition molecules, are often replaced by alkoxysilanes, which are known as contain linker groups, but are also immobilized with the aid of cyanuric chloride or carbodiimide. To equip gold-containing transducer surfaces, recognition molecules labeled with tiolalkyl are used, which are immobilized on the transducer surface via sulfur-gold bonds in the form of so-called self-assembly layers. An interesting approach to immobilizing nucleic acids on transducer surfaces is the photochemically supported synthesis of Affymetrix (Light-directed spatially addressable parallel chemical synthesis, SPA Fodor et al., Science 251, 767-773 (1991)).

Zur Steigerung der Empfindlichkeit von Biosensoren sowie zur Optimierung der Reproduzierbarkeit der damit erhaltenen Mess- ergebnisse ist der Einsatz dreidimensionaler Immobilisierungsschichten für die biologischen Erkennungsmoleküle sinnvoll. Die Firma Schleicher & Schüll GmbH bietet unter dem Namen FAST™ Südes DNA-Chips an, in welchen die Fänger-Oligos in einer drei-dimensionalen Nitrocellulose-Membran immobili- siert sind (BioMolecular Screening, Catalog 2001, intern. Edit. Fa. Schleicher & Schüll) .To increase the sensitivity of biosensors and to optimize the reproducibility of the measurement results obtained, the use of three-dimensional immobilization layers for the biological recognition molecules makes sense. The company Schleicher & Schüll GmbH offers under the name FAST ™ Südes DNA chips in which the catcher oligos are immobilized in a three-dimensional nitrocellulose membrane (BioMolecular Screening, Catalog 2001, intern. Edit. Schleicher & Schüll).

In der WO 00/43539 ist der Aufbau einer dreidimensionalen DNA-Erkennungsschicht durch Immobilisierung der DNA-Fänger- Sonden in Form von Polymer-Brushes beschrieben.WO 00/43539 describes the construction of a three-dimensional DNA recognition layer by immobilizing the DNA capture probes in the form of polymer brushes.

Von Timofeev et al . wird ein chemisch modifiziertes, radikalisch vernetztes Polyacrylamid beschrieben, das zum Beispiel für die Immobilisierung von Fänger-Oligos eingesetzt werden kann (E. N. Timofeev et al . , Regioselective Immobilization of Short Oligonucleotides to Acrylic Copolymer Gels, Nucleic Acids Research, 1966, Vol.24, No . 16, 3142-3148). Hier werden als Kopplungsgruppen im Hydrogel Amino- oder Aldehydgruppen verwendet. Aldehyd- bzw. Amino-funktionalisierte Fänger- Oligos können an diese Kopplungsgruppen unter reduktiven Reaktionsbedingungen kovalent immobilisiert werden. Das bedeutet aber, dass neben der eigentlichen Kopplungsreaktion zwi- sehen A ino- und Aldehydgruppe, bzw. umgekehrt, ein zusätzlicher Reduktionsschritt unter Einsatz von Reduktionsmitteln erforderlich ist. Weitere von Timofeev et al . beschriebene Methoden zur chemischen Aktivierung des vernetzten Polyacryl- a ids erfordern ebenfalls zusätzliche Reaktionsschritte in der Polymermatrix.By Timofeev et al. describes a chemically modified, radically crosslinked polyacrylamide which can be used, for example, for immobilizing capture oligos (EN Timofeev et al., Regioselective Immobilization of Short Oligonucleotides to Acrylic Copolymer Gels, Nucleic Acids Research, 1966, Vol. 24, 16, 3142-3148). Here, amino or aldehyde groups are used as coupling groups in the hydrogel. Aldehyde- or amino-functionalized scavenger oligos can be covalently immobilized on these coupling groups under reductive reaction conditions. However, this means that in addition to the actual coupling reaction between see A ino- and aldehyde group, or vice versa, an additional reduction step using reducing agents is required. More from Timofeev et al. Methods described for the chemical activation of the crosslinked polyacrylic acid also require additional reaction steps in the polymer matrix.

Aufgabe der vorliegenden Erfindung ist die Erzeugung einer hydrophilen ImmobilisierungsSchicht für biosensorische Anwen- düngen auf Basis eines Hydrogels sowie die Verwendung solcher Immobilisierungsschichten zur Erzeugung von Erkennungsschichten durch kovalente Einkopplung biologischer Erkennungsmoleküle.The object of the present invention is to produce a hydrophilic immobilization layer for biosensor applications based on a hydrogel and to use such immobilization layers to generate recognition layers by covalently coupling biological recognition molecules.

Die vorliegende Erfindung löst diese Aufgabe unter Verwendung von radikalisch vernetzten oder fotostruktruierten Hydrogelen als Immobilisierungsschicht. Solche Hydrogele sind in den deutschen Patentanmeldungen "Radikalisch vernetzbare Zusammensetzung zur Erzeugung einer Hydrogelschicht" bzw. "Foto- strukturierbare Zusammensetzung zur Erzeugung einer Hydrogelschicht" (Aktenzeichen noch nicht bekannt) der Anmelderin beschrieben.The present invention solves this problem by using free-radically crosslinked or photo-structured hydrogels as the immobilization layer. Such hydrogels are described in the applicant's German patent applications "Radically crosslinkable composition for producing a hydrogel layer" or "Photo-structurable composition for producing a hydrogel layer" (file number not yet known).

Gegenstand der vorliegenden Erfindung ist demnach einmal eine hydrophile ImmobilisierungsSchicht für Biosensoren aus einem radikalisch vernetzten Hydrogel auf Basis von Polyacrylamid, wobei die AusgangsZusammensetzung Acrylamid, Vernetzungsmittel, Radikalinitiatoren, wenigstens ein Comonomer mit reaktiven Linkergruppen und gegebenenfalls Weichmacher sowie sons- tige Additive umfasst.The present invention accordingly relates to a hydrophilic immobilization layer for biosensors made from a free-radically crosslinked hydrogel based on polyacrylamide, the starting composition comprising acrylamide, crosslinking agents, radical initiators, at least one comonomer with reactive linker groups and optionally plasticizers and other additives.

Gegenstand der vorliegenden Verbindung ist auch eine hydrophile Immobilisierungsschicht aus einem fotostrukturierten Hydrogel auf Basis von Polyacrylamid, wobei die Ausgangs- Zusammensetzung Acrylamid, Vernetzungsmittel, Fotoinitiatoren, wenigstens einen Filmbildner, wenigstens ein Comonomer mit reaktiven Linkergruppen und gegebenenfalls Weichmacher sowie andere Additive umfasst.The subject of the present compound is also a hydrophilic immobilization layer made of a photostructured hydrogel based on polyacrylamide, the starting composition being acrylamide, crosslinking agent, photoinitiators, at least one film former, at least one comonomer with reactive linker groups and optionally plasticizers and other additives.

Die erfindungsgemäßen Systeme erlauben den Aufbau von Sensor- arrays mit biologischen Erkennungsmolekülen in einer dreidimensionalen Matrix in hoher Integrationsdichte.The systems according to the invention allow the construction of sensor arrays with biological recognition molecules in a three-dimensional matrix with a high integration density.

Bevorzugte Ausführungsformen bzw. Zusammensetzungen der erfindungsgemäßen hydrophilen Immobilisierungsschichten ergeben sich aus den Unteransprüchen 3 bis 10.Preferred embodiments or compositions of the hydrophilic immobilization layers according to the invention result from subclaims 3 to 10.

Den Zusammensetzungen können gegebenenfalls weitere Komponenten beigefügt werden, welche die Mischbarkeit der beteiligten Monomere und der Initiatoren gewährleisten. Zur Herabsetzung der Oberflächenspannung können handelsübliche Additive verwendet werden.If necessary, further components can be added to the compositions, which ensure the miscibility of the monomers involved and the initiators. Commercial additives can be used to reduce the surface tension.

Nach Schichtherstellung auf einem Transducersystem und thermischer bzw. Fotovernetzung oder Fotopolymerisation oder Fo- tostrukturierung bzw. Polymerisationsstrukturierung wird ein mit Wasser quellbares Hydrogel erhalten, in das unter Verwendung der Linkergruppen biologische oder chemische Erkennungs- molküle für analytische oder diagnostische Anwendungen unter Erhalt ihrer Funktionsfähigkeit eingekoppelt werden können. Gegenstand der vorliegenden Erfindung ist demzufolge auch die Verwendung der Immobilisierungsschichten zur Herstellung von biosensorischen Erkennungsschichten durch (kovalentes) Einkuppeln bzw. Immobilisieren von chemischen oder biologischen Erkennungsmolekülen, wobei die Erkennungsmoleküle vorzugswei- se Fänger-Oligonukleotide sind.After layer production on a transducer system and thermal or photo crosslinking or photo polymerization or photo structuring or polymerization structuring, a water-swellable hydrogel is obtained, into which biological or chemical recognition molecules for analytical or diagnostic applications are coupled in using the linker groups while maintaining their functionality can. The present invention accordingly also relates to the use of the immobilization layers for the production of biosensory recognition layers by (covalent) coupling or immobilization of chemical or biological recognition molecules, the recognition molecules preferably being scavenger oligonucleotides.

Grundsätzlich kann die AusgangsZusammensetzung zur Erzeugung der Hydrogelschicht (Immobilisierungsschicht) mit allen modernen Beschichtungstechnologien auf die geeigneten Träger aufgebracht werden. Bevorzugt werden jedoch Spin-Coating sowie Dispensieren angewendet. Die Eigenschaften der zu erzeugenden Hydrogelschicht bezüglich Hydrophile, Vernetzungsdichte, Quellbarkeit, etc. lassen sich in weiten Bereichen durch die Art der verwendeten Ausgangskomponenten, deren Verhältnis zueinander und letztend- lieh der Art der Schichtbildung variieren.Basically, the starting composition for generating the hydrogel layer (immobilization layer) can be applied to the suitable carrier using all modern coating technologies. However, spin coating and dispensing are preferably used. The properties of the hydrogel layer to be produced with regard to hydrophilicity, crosslinking density, swellability, etc. can be varied widely by the type of starting components used, their relationship to one another and ultimately the type of layer formation.

Die Hydrogelmatrix kann an die zum Einsatz kommenden biologischen Erkennungsmoleküle, insbesondere im Hinblick auf die Vernetzungsdichte, angepasst werden. Die Vernetzungsdichte wird durch Art und Konzentration der verwendeten Vernetzermoleküle, wie Acryl- und/oder MethacrylVerbindungen, insbesondere Methylenbis (meth) acrylamid und/oder Dimethacrylsäu- reester, wie Tetraethylenglycoldimethacrylat, gesteuert werden.The hydrogel matrix can be adapted to the biological recognition molecules used, in particular with regard to the crosslinking density. The crosslinking density will be controlled by the type and concentration of the crosslinking molecules used, such as acrylic and / or methacrylic compounds, in particular methylenebis (meth) acrylamide and / or dimethacrylic acid esters, such as tetraethylene glycol dimethacrylate.

Die Hydrogelmischung kann auch an das für den speziellen Anwendungszweck bevorzugte Beschichtungsverfahren angepasst werden.The hydrogel mixture can also be adapted to the coating method preferred for the specific application.

Für Spin-Coating kommt zum Einen die Verwendung eines polyme- ren Filmbildners, wie Polyvinylpyrrolidon, Polyacrylamid und/oder Polyhydroxymethacrylat, in Frage. Zum anderen können hochsiedende Lösungsmittel, wie z. B. Ethylenglykol, für die Hydrogelmischung verwendet werden, die beim Spin-Coating nicht vollständig verdampfen und so als Weichmacher in derFor spin coating, the use of a polymeric film former, such as polyvinylpyrrolidone, polyacrylamide and / or polyhydroxymethacrylate, is suitable. On the other hand, high-boiling solvents, such as. As ethylene glycol, can be used for the hydrogel mixture, which do not evaporate completely during spin coating and thus as a plasticizer in the

Schicht verbleiben. Der Restlösemittelgehalt kann dann durch einen Prebake-Schritt vor der Vernetzung gezielt weiter reduziert und somit u.a. die Polymerisationsausbeute bzw. die resultierende Schichtdicke gesteuert werden. Gegebenenfalls können zusätzliche Weichmachersysteme, wie Di- und/oder Triethylenglykol, zugesetzt werden.Layer remain. The residual solvent content can then be further reduced in a targeted manner by means of a prebake step before crosslinking, and thus the polymerization yield or the resulting layer thickness can be controlled. If necessary, additional plasticizer systems, such as di- and / or triethylene glycol, can be added.

Bei der Schichtbildung durch Dispensieren wird die Hydrogelmischung in Lösung je nach Transducerdimensionen in Tropfen in einer Größe von einigen Mikrolitern bis zu einem Nanoliter aufgebracht. Für das Dispensieren werden hochsiedenden Lösemittel, die eine ausreichend lange Lebensdauer des Tropfens an der Spitze der Dispensierkanüle aufweisen, verwendet. Damit wird das Dosieren und Absetzen des Tropfens reproduzierbar. Andererseits darf der Siedepunkt des Lösungsmittels nicht zu hoch sein, um ein ausreichend rasches Abdampfen des Lösungsmittels aus dem abgesetzten Tropfen zu ermöglichen. Gegebenenfalls ist hier ein Temperschritt zur Steuerung des Gehalts an Restlösemittel erforderlich. Erfindungsgemäß kommen für das Dispensieren der Hydrogelmischung bevorzugt Dirnethylformamid und/oder Ethylenglykol zum Einsatz.When the layer is formed by dispensing, the hydrogel mixture in solution is applied in drops, depending on the transducer dimensions, in sizes from a few microliters to one nanoliter. For the dispensing are high-boiling solvents, which have a long enough life for the drop have at the tip of the dispensing cannula used. This makes the dosing and settling of the drop reproducible. On the other hand, the boiling point of the solvent must not be too high to allow the solvent to evaporate sufficiently quickly from the settled drop. A tempering step to control the residual solvent content may be required. According to the invention, preference is given to using dimethylformamide and / or ethylene glycol for dispensing the hydrogel mixture.

Die Hydrogelmischung kann in Schicht- oder Spotform auf Transducer- oder Trägeroberflächen aus Metall, Glas, Silici- um, Siliciumdioxid, Siliciumnitrid oder Kunststoff aufgebracht werden. Es können auch Oberflächen mit Topographie, die aus unterschiedlichen Materialien bestehen, wie z. B. In- terdigitalelektrodenarrays auf Siliciumnitrid als Passivie- rung, beschichtet werden. Die Beschichtung von Flächen schließt auch die Beschichtung innerer Oberflächen von Mikro- kanälen oder Nanotubes ein. Die zu beschichtenden Oberflächen sind gegebenenfalls mit einem Haftvermittler beschichtet.The hydrogel mixture can be applied in layer or spot form on transducer or carrier surfaces made of metal, glass, silicon, silicon dioxide, silicon nitride or plastic. Surfaces with topography, which consist of different materials, such as. B. Interdigital electrode arrays are coated on silicon nitride as passivation. The coating of surfaces also includes the coating of inner surfaces of microchannels or nanotubes. The surfaces to be coated are optionally coated with an adhesion promoter.

Die Polymerisation und Vernetzung der Hydrogelschicht erfolgt durch thermische oder UV-Initiierung. Bei UV-Initiierung kann auch eine Strukturierung der Hydrogelschicht durch Kontakt- bzw. Proximitybelichtung durch eine Maske erfolgen. DieThe hydrogel layer is polymerized and crosslinked by thermal or UV initiation. In the case of UV initiation, the hydrogel layer can also be structured by contact or proximity exposure through a mask. The

Hydrogelschicht arbeitet hier wie ein Negativ-Resist . Im bestrahlten Bereich wird polymerisiert und vernetzt. In den abgedunkelten Bereichen findet keine Reaktion statt. Die hier befindliche Hydrogelmischung wird in einem Entwicklungs- schritt wieder vom Substrat abgelöst. Hilfskomponenten wie polymere Filmbildner oder Weichmacher können durch Extraktion aus der vernetzten Hydrogelschicht entfernt werden. Dieser Schritt kann unter Umständen zeitgleich mit dem eigentlichen Ausrüstungsschritt erfolgen.The hydrogel layer works like a negative resist. Polymerization and crosslinking take place in the irradiated area. There is no reaction in the darkened areas. The hydrogel mixture found here is detached from the substrate in a development step. Auxiliary components such as polymeric film formers or plasticizers can be removed from the crosslinked hydrogel layer by extraction. Under certain circumstances, this step can take place at the same time as the actual equipment step.

Die bioglogischen oder chemischen Erkennungssysteme werden vorzugsweise aus wässriger Lösung, aus wässriger Pufferlösung oder aus Gemischen polarer Lösungsmittel mit Wasser auf die Immobilisierungsschicht aufgebracht. Das Aufbringen erfolgt durch Auftropfen oder Aufspotten/Aufdispensieren. In Nanotu- bes oder Mikrokanäle kann das Heranbringen der Lösung mit den biologischen oder chemischen Erkennungsmolekülen an die vernetzte Hydrogelschicht auch durch den Transport durch das fluidische System selbst erfolgen. Für die zielgenaue Beladung von Messspots werden vorteilhafterweise vernetzte Hydro- gelspots verwendet, die von einem Schutzring umgeben sind.The bioglogical or chemical detection systems are preferably made from aqueous solution, from aqueous buffer solution or applied to the immobilization layer from mixtures of polar solvents with water. It is applied by dripping on or spotting on / dispensing. In nanotubes or microchannels, the solution with the biological or chemical recognition molecules can also be brought to the crosslinked hydrogel layer by transport through the fluidic system itself. Cross-linked hydrogel spots, which are surrounded by a protective ring, are advantageously used for the precise loading of measuring spots.

Für das kovalente Ankoppeln der biologischen oder chemischen Erkennungsmoleküle, die mit einer zur im vernetzten Hydrogel vorhandenen Linkergruppe passenden Kopplungsgruppe versehen sind, kann je nach Reaktivität ein Temperschritt erforderlich sein. Um das Austrocknen der Hydrogelschicht während derDepending on the reactivity, a tempering step may be required for the covalent coupling of the biological or chemical recognition molecules, which are provided with a coupling group that matches the linker group present in the crosslinked hydrogel. To dry out the hydrogel layer during the

Kopplungsreaktion zu verhindern, kann in einer Klimakammer gearbeitet werden. Besonders geeignet für die Ankopplung an die Linkergruppen Epoxyd und Maleinsäureanhydrid sind A ino- alkylgruppen. Preventing the coupling reaction can be worked in a climatic chamber. A inoalkyl groups are particularly suitable for coupling to the linker groups epoxy and maleic anhydride.

Claims

Patentansprüche claims 1.. Hydrophile Immobilisierungsschicht für Biosensoren aus einem radikalisch vernetzten Hydrogel auf Basis von Po- lyacrylamid, wobei die Ausgangszusammensetzung Acrylamid, Nernetzungsmittel, Radikalinitiator (en) , wenigstens ein Comonomer mit reaktiven Linkergruppen und gegebenenfalls Weichmacher umfasst.1 .. Hydrophilic immobilization layer for biosensors made of a free-radically crosslinked hydrogel based on polyacrylamide, the starting composition comprising acrylamide, wetting agent, free radical initiator (s), at least one comonomer with reactive linker groups and optionally plasticizers. 2. Hydrophile Immobilisierungsschicht aus einem fotostrukturierten Hydrogel auf Basis von Polyacrylamid, wobei die AusgangsZusammensetzung Acrylamid, Vernetzungsmittel, Fotoinitiator (en) , wenigstens einen Filmbildner, wenigstens ein Comonomer mit reaktiven Linkergruppen und gegebenenfalls Weichmacher umfasst.2. Hydrophilic immobilization layer made of a photostructured hydrogel based on polyacrylamide, the starting composition comprising acrylamide, crosslinking agent, photoinitiator (s), at least one film former, at least one comonomer with reactive linker groups and optionally plasticizers. 3. Hydrophile Immobilisierungsschicht nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass das Vernetzungsmittel eine Acryl- und/oder Methacrylverbindung ist.3. Hydrophilic immobilization layer according to claim 1 or 2, characterized in that the crosslinking agent is an acrylic and / or methacrylic compound. 4. Hydrophile Immobilisierungsschicht nach Anspruch 3, dadurch gekennzeichnet, dass das Vernetzungsmittel Methylenbis (meth) acrylamid und/oder Dimethacryl- säureester ist.4. Hydrophilic immobilization layer according to claim 3, characterized in that the crosslinking agent is methylenebis (meth) acrylamide and / or dimethacrylic acid ester. 5. Hydrophile Immobilisierungsschicht nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass das Comonomer mit reaktiven Linkergruppen Maleinsäurean- hydrid und/oder Glycidyl (meth) acrylat ist.5. Hydrophilic immobilization layer according to one of claims 1 to 4, characterized in that the comonomer with reactive linker groups is maleic anhydride and / or glycidyl (meth) acrylate. 6. Hydrophile Immobilisierungsschicht nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, dass der Weichmacher Mono-, Di- und/oder Triethylenglycol ist. 6. Hydrophilic immobilization layer according to one of claims 1 to 5, characterized in that the plasticizer is mono-, di- and / or triethylene glycol. 7. Hydrophile Immobilisierungsschicht nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass die Ausgangszusammensetzung, in einem polaren, mit Wasser mischbaren Lösungsmittel vorliegt.7. Hydrophilic immobilization layer according to one of claims 1 to 6, characterized in that the starting composition is present in a polar, water-miscible solvent. 8. Hydrophile Immobilisierungsschicht nach Anspruch 7, dadurch gekennzeichnet, dass das Lösungsmittel Dirnethylformamid ist.8. Hydrophilic immobilization layer according to claim 7, characterized in that the solvent is dirnethylformamide. 9. Hydrophile Immobilisierungsschicht nach einem der Ansprüche 2 bis 8, dadurch gekennzeichnet, dass der Filmbildner Polyvinylpyrolidon, Polyacrylamid und/oder Polyhydroxymethacrylat ist.9. Hydrophilic immobilization layer according to one of claims 2 to 8, characterized in that the film former is polyvinyl pyrolidone, polyacrylamide and / or polyhydroxymethacrylate. 10. Hydrophile Immobilisierungsschicht nach einem der Ansprüche 1 bis 9, dadurch gekennzeichnet, dass sie auf Transducer- oder Trägeroberflächen aus Metall, Glas, Silicium, Siliciumdioxid, Siliciumnitrid, Kunststoff oder auf Oberflächen mit Topographie erzeugt ist.10. Hydrophilic immobilization layer according to one of claims 1 to 9, characterized in that it is produced on transducer or carrier surfaces made of metal, glass, silicon, silicon dioxide, silicon nitride, plastic or on surfaces with topography. 11. Verwendung der Immobilisierungsschicht nach einem der vorhergehenden Ansprüche zur Herstellung von biosensori- schen Erkennungsschichten durch (kovalentes) Einkuppeln bzw. Immobilisieren von chemischen oder biologischen Er- kennungsmolekülen.11. Use of the immobilization layer according to one of the preceding claims for the production of biosensory recognition layers by (covalent) coupling or immobilization of chemical or biological recognition molecules. 12. Verwendung nach Anspruch 11, dadurch gekennzeichnet, dass die Erkennungsmoleküle Fänger- Oligonukleotide sind. 12. Use according to claim 11, characterized in that the recognition molecules are capture oligonucleotides.
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