EP1563086A1 - Device for determining the activity of enzymes - Google Patents

Device for determining the activity of enzymes

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Publication number
EP1563086A1
EP1563086A1 EP03789039A EP03789039A EP1563086A1 EP 1563086 A1 EP1563086 A1 EP 1563086A1 EP 03789039 A EP03789039 A EP 03789039A EP 03789039 A EP03789039 A EP 03789039A EP 1563086 A1 EP1563086 A1 EP 1563086A1
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EP
European Patent Office
Prior art keywords
activity
enzymes
electrode array
enzyme
substrate
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Withdrawn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
EP03789039A
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German (de)
French (fr)
Inventor
Uwe Trebbe
Nico KÖHLING
Uwe GLÄSEL
Frank HÜPPMEYER
Tilo Weiss
Michael Frank
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Henkel AG and Co KGaA
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Henkel AG and Co KGaA
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Filing date
Publication date
Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP1563086A1 publication Critical patent/EP1563086A1/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Withdrawn legal-status Critical Current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

Definitions

  • the invention relates to a device for determining the activity of enzymes.
  • Enzymes are used in technology for various purposes, for example in detergents or cleaning agents and in the food industry. Knowledge of the activity of the enzymes is essential, which is why the price for enzymes is not based on their weight or volume, but rather on their activity, i.e. their actual effectiveness. Knowledge of the activity of enzymes, e.g. in washing and cleaning liquors, and the resulting need-based dosing of other components can lead to increased product performance with reduced use of raw materials. Also in the food industry, knowledge of the actual effectiveness of the enzyme in manufacturing processes is a crucial factor in increasing the efficiency of production processes.
  • WO 00/75360 A2 discloses a method and an apparatus for enzyme detection in which the degradation of a biodegradable polymer by the enzyme to be detected is observed. The degradation is monitored by a quartz microbalance, surface plasmon resonance, ellipsometry, impedance spectroscopy or capacitive measurements. In all of these processes, the degradation of a biodegradable polymer is used as a signaling mechanism.
  • the object of the invention is to provide a solution with the simplest possible Enzyme activities can be determined quickly in solution without denvation or other sample preparation.
  • a device of the type mentioned at the outset by a support on which at least one electrode array is arranged, which is coated with a polymer film, self-assembled monolayers (SAM) or silanes, an enzyme to be determined on the coating being adsorptive degradable substrate is applied.
  • SAM self-assembled monolayers
  • silanes an enzyme to be determined on the coating being adsorptive degradable substrate is applied.
  • the surface of the transducer above the electrode array is modified so that a substrate can be bound by adsorption.
  • the modification can be achieved, inter alia, by chemical surface treatment, such as, for example, silanization, or by the deposition of a thin polymer membrane.
  • various potential substrates can be immobilized by adsorption. If a sensor manufactured in this way is immersed in a solution that contains an enzyme that is able to degrade the immobilized substrate, the impediometric properties of the membrane change, among other things. These can be easily converted into electrical signals and processed accordingly.
  • the degradation of preferably monomers or oligomers which are adsorptively bound to the sensor.
  • a special film must be immobilized on the transducer surface for each enzyme class whose activity is detected with a sensor based on the principle of substrate consumption. Since only the adsorbed substrates are adapted accordingly in the device according to the invention the effort to develop a new system is relatively low. Experimentally complex optical methods or other complex processes are not required.
  • the substrate consists of monomers or oligomers.
  • Adsorptively bound monomers can be used to detect the activity of enzymes that are reactive, e.g. Oxidases or reductases. The reactive conversion of the monomers changes their dielectric properties, which can also be demonstrated.
  • the electrode array is designed as an interdigital electrode array. With such comb-like electrode finger structures, impediometric effects can be determined with great sensitivity. This type of electrode arrangement is therefore particularly suitable for the devices.
  • the electrode fingers are preferably approximately 10 ⁇ m wide and are also arranged at a distance of approximately 10 ⁇ m from one another.
  • Polystyrene or polyvinyl chloride has been found to be particularly suitable as the material for the polymer film.
  • a suitable plasticizer is preferably added to these polymers.
  • the polymers are generally applied from solution, e.g. according to the established process of drop, dip or spin coating.
  • the thickness of the polymer layer is preferably 1-5 ⁇ m.
  • the layer thickness of the adsorptively bound monomers or oligomers is in the nanometer range.
  • the carrier of the sensor consists of glass
  • other materials can also be used for the carrier, for example plastic, SiO 2 on Si, Al 2 O 3 , SiC or SiN x .
  • Platinum is particularly suitable as the electrode material, but it is also all other electrical conductors, such as gold, silver, platinum-iridium or poly-silicon, which are established in thin-film technology, are possible.
  • a plurality of electrode arrays are arranged next to one another on the carrier, on the respective polymer film of which different substrates are applied to determine different enzymes. It is thus possible to determine the activity of different enzymes contained in a solution at the same time with only one device.
  • the invention also proposes a method for determining the activity of enzymes with a device described above, which is characterized in that the device is immersed in an enzyme solution and the change over time in the impediometric properties of the system is measured and evaluated.
  • the immobilized substrate is broken down more or less quickly by the enzymes contained in the solution, which changes the impediometric properties or the capacitive resistance of the system, which can easily be converted into electrical signals and evaluated accordingly.
  • the method can be carried out several times with one device without any problems. Experiments have shown that a system can be used ten times until the immobilized substrate layer is completely degraded. The device can then be used again by adsorbing the enzyme substrates again. A device according to the invention can thus be easily regenerated and then used again.
  • FIG. 1 is a schematic view of a construction according to the invention on a greatly enlarged scale
  • Fig. 2 shows an interdigital electrode array in an enlarged again compared to Figure 1 Scale
  • Fig. 3 four measurement curves recorded with four different devices.
  • a device for determining the activity of enzymes is generally designated 1 in FIG.
  • This device 1 initially has a carrier 2 which e.g. is made of glass.
  • An interdigital electrode array (IDA) is arranged on this carrier 2, for example applied photolithographically, the comb-like interdigitated electrode fingers are denoted by 3. These electrode fingers 3 are alternately connected to an (electrical) lead 4 or lead 5, as can be seen more clearly from FIG.
  • the sensitive area of the interdigital electrode array is labeled F.
  • the distance between the electrode fingers in the exemplary embodiment is 10 ⁇ m
  • the width of an electrode finger is of the same order of magnitude, i.e. likewise at 10 ⁇ m
  • the length of an electrode finger is 1 mm
  • the width of the lead 4 or lead 5, on the other hand is of the order of magnitude of 200 ⁇ m.
  • the periodicity in which the electrode arrangement repeats is preferably 25.
  • the interdigital electrode array thus formed on the carrier 2 is coated with a polymer film 6 which is circular in the exemplary embodiment and which, for example, has a diameter of 2 mm and preferably consists of polystyrene or polyvinyl chloride.
  • a substrate of monomers or oligomers that is degradable by the enzyme to be determined is bound adsorptively to this polymer film 6; the thickness of this substrate layer, not shown in the drawing, is in the nanometer range.
  • the interdigital electrode array is preferably produced on the carrier 2 using the photolithographic method.
  • a laminating film is preferably glued on, which leaves a circular cutout above the interdigital electrode array.
  • a solution of the polymer and preferably a plasticizer in THF or a mixture of THF-cyclohexanone (7: 3) is dripped onto this section and the polymer film 6 is formed after the solvent has evaporated.
  • a substrate which can be degraded by the enzyme to be determined is then applied adsorptively to the polymer film 6.
  • a protein for example, is used as such a degradation adduct.
  • a Bovine Serum Albuminum BSA was specifically used as the protein.
  • Eppendorf pipette 0.5 to 10 ⁇ l
  • 4 ⁇ l of the BSA solution were added to the area coated with the polymer film 6. Until such a protease-sensitive activity sensor is used, it is stored dust-free and dry.
  • the protein dried on the polymer layer 6 is water-soluble, but a separate washing step can be dispensed with if the sensor is immersed in the stirred background solution for about 10 minutes. Before the protease is added, however, it is also possible to store the device after rinsing off the excess protein solution until use and to use it only later.
  • the polymer layer 6 is preferably applied from a solution, specifically with the addition of a plasticizer, for example of 30% by weight TBTC.
  • the application is preferably carried out according to the established methods of drop, dip or spin coating.
  • FIG. 3 shows four different measurement curves from four different devices, the AC conductivity being reproduced over time at a frequency of 10 kHz. This frequency has proven to be particularly effective, the arrow on the uppermost measurement curve in FIG. 3 shows the point in time at which protease was added to the solution.
  • Such devices can in principle be used several times. Of course, they are not completely reversible since the absorbed enzyme substrates are consumed. However, studies have shown that devices can be used several times, for example up to 10 times, before the enzyme substrates have to be adsorptively bound again to the polymer layer. Such regeneration is possible without any problems.

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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)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a device for determining the activity of enzymes. The aim of the invention is to determine enzyme activity directly in solution in the simplest and most rapid manner possible, without derivatisation or the preparation of other samples. To achieve this, at least one electrode array (3), which is coated with a polymer film (6), self-assembled monolayers (SAM), or silanes, is located on a support (2) and a substrate is applied to said coating by adsorption, said substrate being degradable by the enzyme whose activity is to be determined.

Description

Vorrichtung zur Bestimmung der Aktivität von Enzymen Device for determining the activity of enzymes
Die Erfindung betrifft eine Vorrichtung zur Bestimmung der Aktivität von Enzymen.The invention relates to a device for determining the activity of enzymes.
Enzyme werden in der Technik für verschiedene Zwecke eingesetzt, beispielsweise in Waschmitteln oder Reinigungsmitteln sowie in der Lebensmittelindustrie. Wesentlich ist dabei die Kenntnis der Aktivität der Enzyme, deshalb richtet sich der Preis für Enzyme auch nicht nach deren Gewicht bzw. Volumen, sondern nach deren Aktivität, d.h. ihrer tatsächlichen Wirksamkeit. Die Kenntnis der Aktivität von Enzymen, z.B. in Wasch- und Reinigungslaugen, und die daraus resultierende bedarfsgerechte Dosierung von anderen Komponenten können zu einer gesteigerten Leistungsfähigkeit des Produktes bei vermindertem Rohstoffeinsatz führen. Auch in der Lebensmittelindustrie macht die Kenntnis um die tatsächliche Wirksamkeit des Enzyms in Herstellungsprozessen einen entscheidenden Faktor zur Steigerung der Effizienz von Produktionsabläufen aus.Enzymes are used in technology for various purposes, for example in detergents or cleaning agents and in the food industry. Knowledge of the activity of the enzymes is essential, which is why the price for enzymes is not based on their weight or volume, but rather on their activity, i.e. their actual effectiveness. Knowledge of the activity of enzymes, e.g. in washing and cleaning liquors, and the resulting need-based dosing of other components can lead to increased product performance with reduced use of raw materials. Also in the food industry, knowledge of the actual effectiveness of the enzyme in manufacturing processes is a crucial factor in increasing the efficiency of production processes.
Es sind verschiedene Lösungen zur Bestimmung der Enzymaktivität bekannt geworden, die sich im großtechnischen Maßstab jedoch bisher nicht durchgesetzt haben.Various solutions for determining the enzyme activity have become known, but have not yet become established on an industrial scale.
So ist aus WO 00/75360 A2 ein Verfahren und eine Apparatur zur Enzymdetektion bekannt, bei welchem der Abbau eines bioabbaubaren Polymers durch das zu detektierende Enzym beobachtet wird. Der Abbau wird dabei durch eine Quarzmikrowaage, Oberflächenplasmonenresonanz, Ellipsometrie, Impedanzspektroskopie oder kapazitive Messungen verfolgt. Bei all diesen Verfahren wird der Abbau eines bioabbaubaren Polymers als signalgebender Mechanismus verwandt.WO 00/75360 A2 discloses a method and an apparatus for enzyme detection in which the degradation of a biodegradable polymer by the enzyme to be detected is observed. The degradation is monitored by a quartz microbalance, surface plasmon resonance, ellipsometry, impedance spectroscopy or capacitive measurements. In all of these processes, the degradation of a biodegradable polymer is used as a signaling mechanism.
Die Verwendung solcher abbaubarer Biopolymere ist jedoch mit verschiedenen Nachteilen verbunden. Vor allem sind solche Sensoren aufgrund der aufwendigen Präparationen geeignet dünner Filme auf den entsprechenden Transducerstrukturen nicht praxistauglich.However, the use of such degradable biopolymers has various disadvantages. Above all, such sensors are not suitable for practical use due to the complex preparations of suitably thin films on the corresponding transducer structures.
Aufgabe der Erfindung ist es, eine Lösung zu schaffen, mit der möglichst einfach und schnell Enzymaktivitäten direkt in Lösung ohne Denvatisierung oder andere Probenvorbereitung bestimmt werden können.The object of the invention is to provide a solution with the simplest possible Enzyme activities can be determined quickly in solution without denvation or other sample preparation.
Diese Aufgabe wird mit einer Vorrichtung der eingangs bezeichneten Art gelöst durch einen Träger, auf dem wenigstens ein Elektrodenarray angeordnet ist, welches mit einem Polymerfilm, self-assembled monolayers (SAM) oder Silanen beschichtet ist, wobei auf die Beschichtung adsorptiv ein vom zu bestimmenden Enzym abbaubares Substrat aufgebracht ist.This object is achieved with a device of the type mentioned at the outset by a support on which at least one electrode array is arranged, which is coated with a polymer film, self-assembled monolayers (SAM) or silanes, an enzyme to be determined on the coating being adsorptive degradable substrate is applied.
Mit einer derartigen Vorrichtung ist eine einfache und schnelle Bestimmung von Enzymaktivitäten direkt in Lösung ohne Denvatisierung oder andere Probenvorbereitung möglich, so dass bisher aufwendige Verfahren zur Bestimmung der Enzymaktivität erheblich vereinfacht werden. Die Oberfläche des Transducers über dem Ektrodenarray wird so modifiziert, dass ein Substrat adsorptiv gebunden werden kann. Die Modifikation kann unter anderem durch chemische Oberflächenbehandlung, wie beispielsweise eine Silanisierung, oder durch die Abscheidung einer dünnen Polymermembran erreicht werden. Je nach Oberfläche des Transducers können verschiedene potentielle Substrate adsorptiv immobilisiert werden. Taucht ein so hergestellter Sensor in eine Lösung, die ein Enzym enthält, das in der Lage ist, das immobilisierte Substrat abzubauen, ändern sich unter anderem die impediometrischen Eigenschaften der Membran. Diese können sehr einfach in elektrische Signale umgewandelt und entsprechend verarbeitet werden.With such a device, simple and quick determination of enzyme activities directly in solution without denvation or other sample preparation is possible, so that previously complicated methods for determining enzyme activity are considerably simplified. The surface of the transducer above the electrode array is modified so that a substrate can be bound by adsorption. The modification can be achieved, inter alia, by chemical surface treatment, such as, for example, silanization, or by the deposition of a thin polymer membrane. Depending on the surface of the transducer, various potential substrates can be immobilized by adsorption. If a sensor manufactured in this way is immersed in a solution that contains an enzyme that is able to degrade the immobilized substrate, the impediometric properties of the membrane change, among other things. These can be easily converted into electrical signals and processed accordingly.
Im Gegensatz zum Abbau eines Polymers wird erfindungsgemäß der Abbau von vorzugsweise Monomeren bzw. Oligomeren, die adsorptiv an den Sensor gebunden sind. Wesentliche Vorteile sind eine einfachere, sehr günstige und reproduzierbare Herstellung der Substratfilme auf den Transduceroberflächen und die Möglichkeit, die Vorrichtung in situ zu regenerieren.In contrast to the degradation of a polymer, according to the invention the degradation of preferably monomers or oligomers, which are adsorptively bound to the sensor. Significant advantages are a simpler, very cheap and reproducible production of the substrate films on the transducer surfaces and the possibility to regenerate the device in situ.
Für jede Enzymklasse, deren Aktivität mit einem Sensor nach dem Prinzip des Substratverzehrs nachgewiesen wird, muss ein spezieller Film auf der Transduceroberfläche immobilisiert werden. Da bei der erfindungsgemäßen Vorrichtung lediglich die adsorbierten Substrate entsprechend angepasst werden müssen, ist der Aufwand für die Entwicklung eines neuen Systems relativ gering. Experimentell aufwendige optische Methoden oder andere aufwendige Verfahren werden nicht benötigt.A special film must be immobilized on the transducer surface for each enzyme class whose activity is detected with a sensor based on the principle of substrate consumption. Since only the adsorbed substrates are adapted accordingly in the device according to the invention the effort to develop a new system is relatively low. Experimentally complex optical methods or other complex processes are not required.
In besonders vorteilhafter Ausgestaltung ist vorgesehen, dass das Substrat aus Monomeren oder Oligomeren besteht. Adsorptiv gebundene Monomere können zum Nachweis der Aktivität von Enzymen eingesetzt werden, die reaktiv wirken, wie z.B. Oxidasen oder Reduktasen. Durch die reaktive Umwandlung der Monomere ändern sich ihre dielektrischen Eigenschaften, die ebenfalls nachgewiesen werden können.In a particularly advantageous embodiment, it is provided that the substrate consists of monomers or oligomers. Adsorptively bound monomers can be used to detect the activity of enzymes that are reactive, e.g. Oxidases or reductases. The reactive conversion of the monomers changes their dielectric properties, which can also be demonstrated.
In besonders vorteilhafter Ausgestaltung ist vorgesehen, dass das Elektrodenarray als Interdigitalelektrodenarray ausgebildet ist. Mit solchen, kammartige Elektrodenfinger aufweisenden Strukturen können impediometrische Effekte mit großer Empfindlichkeit bestimmt werden. Diese Art der Elektrodenanordnung ist deshalb für die Vorrichtungen besonders geeignet.In a particularly advantageous embodiment, it is provided that the electrode array is designed as an interdigital electrode array. With such comb-like electrode finger structures, impediometric effects can be determined with great sensitivity. This type of electrode arrangement is therefore particularly suitable for the devices.
Bei Verwendung eines Interdigitalelektrodenarrays sind die Elektrodenfinger bevorzugt etwa 10 μm breit und in einem Abstand von ebenfalls etwa 10 μm zueinander angeordnet.When using an interdigital electrode array, the electrode fingers are preferably approximately 10 μm wide and are also arranged at a distance of approximately 10 μm from one another.
Als Material für den Polymerfilm hat sich als besonders geeignet Polystyrol oder Polyvinylchlorid herausgestellt. Zur Verbesserung der Filmbildung wird diesen Polymeren jeweils bevorzugt noch ein geeigneter Weichmacher zugesetzt. Die Polymere werden allgemein aus Lösung aufgebracht, z.B. nach dem etablierten Verfahren des Drop-, Dip- oder Spin-Coatings.Polystyrene or polyvinyl chloride has been found to be particularly suitable as the material for the polymer film. To improve the film formation, a suitable plasticizer is preferably added to these polymers. The polymers are generally applied from solution, e.g. according to the established process of drop, dip or spin coating.
Die Dicke der Polymerschicht beträgt bevorzugt 1-5 μm. Die Schichtdicke der adsorptiv gebundenen Monomere oder Oligomere liegt im Nanometerbereich.The thickness of the polymer layer is preferably 1-5 μm. The layer thickness of the adsorptively bound monomers or oligomers is in the nanometer range.
In weiterer vorteilhafter Ausgestaltung ist vorgesehen, dass der Träger des Sensors aus Glas besteht, alternativ können auch andere Materialien für den Träger eingesetzt werden, beispielsweise Kunststoff, SiO2 auf Si, AI2O3, SiC oder SiNx. Als Elektrodenmaterial ist Platin besonders gut geeignet, es sind aber auch alle anderen, in der Dünnschichttechnologie etablierten elektrischen Leiter, wie Gold, Silber, Platin-Iridium oder auch Poly-Silizium möglich.In a further advantageous embodiment it is provided that the carrier of the sensor consists of glass, alternatively other materials can also be used for the carrier, for example plastic, SiO 2 on Si, Al 2 O 3 , SiC or SiN x . Platinum is particularly suitable as the electrode material, but it is also all other electrical conductors, such as gold, silver, platinum-iridium or poly-silicon, which are established in thin-film technology, are possible.
In einer weiteren besonders bevorzugten Ausgestaltung ist vorgesehen, dass auf dem Träger nebeneinander mehrere Elektrodenarrays angeordnet sind, auf deren jeweiligen Polymerfilm zur Bestimmung unterschiedlicher Enzyme unterschiedliche Substrate aufgebracht sind. Es ist somit möglich, mit nur einer Vorrichtung gleichzeitig die Aktivität verschiedener in einer Lösung enthaltenen Enzyme zu bestimmen.In a further particularly preferred embodiment, it is provided that a plurality of electrode arrays are arranged next to one another on the carrier, on the respective polymer film of which different substrates are applied to determine different enzymes. It is thus possible to determine the activity of different enzymes contained in a solution at the same time with only one device.
Die Erfindung schlägt auch ein Verfahren zur Bestimmung der Aktivität von Enzymen mit einer vorbeschriebenen Vorrichtung vor, das sich dadurch auszeichnet, dass die Vorrichtung in eine Enzymiösung eingetaucht und die zeitliche Änderung der impediometrischen Eigenschaften des Systems gemessen und ausgewertet wird.The invention also proposes a method for determining the activity of enzymes with a device described above, which is characterized in that the device is immersed in an enzyme solution and the change over time in the impediometric properties of the system is measured and evaluated.
Durch die in der Lösung enthaltenen Enzyme wird das immobilisierte Substrat mehr oder weniger schnell abgebaut, wodurch sich die impediometrischen Eigenschaften bzw. der kapazitive Widerstand des Systems verändert, was leicht in elektrische Signale umgewandelt und entsprechend ausgewertet werden kann. Das Verfahren kann mit einer Vorrichtung problemlos mehrfach ausgeführt werden, Versuche haben ergeben, dass durchaus ein System zehnmal eingesetzt werden kann, bis die immobilisierte Substratschicht vollständig abgebaut ist. Anschließend kann die Vorrichtung erneut eingesetzt werden, indem erneut die Enzymsubstrate adsorptiv angebunden werden. Eine erfindungsgemäße Vorrichtung kann so ohne weiteres regeneriert und dann erneut verwendet werden.The immobilized substrate is broken down more or less quickly by the enzymes contained in the solution, which changes the impediometric properties or the capacitive resistance of the system, which can easily be converted into electrical signals and evaluated accordingly. The method can be carried out several times with one device without any problems. Experiments have shown that a system can be used ten times until the immobilized substrate layer is completely degraded. The device can then be used again by adsorbing the enzyme substrates again. A device according to the invention can thus be easily regenerated and then used again.
Die Erfindung ist nachstehend anhand der Zeichnung beispielhaft näher erläutert. Diese zeigt in:The invention is explained in more detail below with reference to the drawing. This shows in:
Fig. 1 eine schematische Ansicht eines erfindungsgemäßen Aufbaus im stark vergrößerten Maßstab,1 is a schematic view of a construction according to the invention on a greatly enlarged scale,
Fig. 2 ein Interdigitalelektrodenarray in gegenüber Figur 1 nochmals vergrößertem Maßstab undFig. 2 shows an interdigital electrode array in an enlarged again compared to Figure 1 Scale and
Fig. 3 vier mit vier verschiedenen Vorrichtungen aufgenommenen Messkurven.Fig. 3 four measurement curves recorded with four different devices.
Eine Vorrichtung zur Bestimmung der Aktivität von Enzymen ist in Figur 1 allgemein mit l bezeichnet. Diese Vorrichtung 1 weist zunächst einen Träger 2 auf, der z.B. aus Glas besteht. Auf diesem Träger 2 ist ein Interdigitalelektrodenarray (IDA) angeordnet, beispielsweise photolitographisch aufgebracht, die kammartig ineinandergreifenden Elektrodenfinger sind mit 3 bezeichnet. Diese Elektrodenfinger 3 sind abwechselnd mit einer (elektrischen) Zuleitung 4 oder Ableitung 5 verbunden, wie dies genauer aus Figur 2 zu erkennen ist. Die sensitive Fläche des Interdigitalelektrodenarrays ist mit F bezeichnet.A device for determining the activity of enzymes is generally designated 1 in FIG. This device 1 initially has a carrier 2 which e.g. is made of glass. An interdigital electrode array (IDA) is arranged on this carrier 2, for example applied photolithographically, the comb-like interdigitated electrode fingers are denoted by 3. These electrode fingers 3 are alternately connected to an (electrical) lead 4 or lead 5, as can be seen more clearly from FIG. The sensitive area of the interdigital electrode array is labeled F.
Der Abstand der Elektrodenfinger zueinander beträgt beim Ausführungsbeispiel 10 μm, die Breite eines Elektrodenfingers liegt in derselben Größenordnung, d.h. ebenfalls bei 10μm, die Länge eines Elektrodenfingers beträgt 1 mm, die Breite der Zuleitung 4 bzw. Ableitung 5 liegt demgegenüber in einer Größenordnung von 200 μm. Die Periodizität, in der sich die Elektrodenanordnung wiederholt, liegt vorzugsweise bei 25.The distance between the electrode fingers in the exemplary embodiment is 10 μm, the width of an electrode finger is of the same order of magnitude, i.e. likewise at 10 μm, the length of an electrode finger is 1 mm, the width of the lead 4 or lead 5, on the other hand, is of the order of magnitude of 200 μm. The periodicity in which the electrode arrangement repeats is preferably 25.
Das so auf dem Träger 2 gebildete Interdigitalelektrodenarray ist mit einem beim Ausführungsbeispiel kreisförmigen Polymerfilm 6 beschichtet, welcher beispielsweise einen Durchmesser von 2 mm aufweist und vorzugsweise aus Polystyrol oder Polyvinylchlorid besteht. Außenseitig ist an diesen Polymerfilm 6 adsorptiv ein vom zu bestimmenden Enzym abbaubares Substrat aus Monomeren oder Oligomeren gebunden, die Dicke dieser zeichnerisch nicht dargestellten Substratschicht liegt im Nanometerbereich.The interdigital electrode array thus formed on the carrier 2 is coated with a polymer film 6 which is circular in the exemplary embodiment and which, for example, has a diameter of 2 mm and preferably consists of polystyrene or polyvinyl chloride. On the outside, a substrate of monomers or oligomers that is degradable by the enzyme to be determined is bound adsorptively to this polymer film 6; the thickness of this substrate layer, not shown in the drawing, is in the nanometer range.
Anders als in Figur 1 dargestellt, kann auf einem Träger 2 nicht nur ein einziger Transducer realisiert werden, sondern es können nebeneinander auf dem gleichen Träger mehrere Transducer verwirklicht werden, in dem dementsprechend nebeneinander mehrere Interdigitalelektrodenarrays mit Filmbeschichtung vorgesehen werden. Zur Herstellung eines solchen Transducers 1 wird das Interdigitalelektrodenarray bevorzugt im photolitographischen Verfahren auf dem Träger 2 hergestellt. Anschließend wird vorzugsweise eine Laminierfolie aufgeklebt, die über dem Interdigitalelektrodenarray einen kreisrunden Ausschnitt freiläßt. In diesen Ausschnitt wird eine Lösung des Polymers und vorzugsweise eines Weichmachers in THF bzw. einer Mischung aus THF-Cyclohexanon (7:3) aufgetropft und der Polymerfilm 6 bildet sich nach der Verdunstung des Lösungsmittels aus. Anschließend wird adsorptiv an den Polymerfilm 6 ein vom zu bestimmenden Enzym abbaubares Substrat aufgebracht.In contrast to that shown in FIG. 1, not only a single transducer can be realized on a carrier 2, but a plurality of transducers can be realized next to one another on the same carrier, in which a plurality of interdigital electrode arrays with film coating are accordingly provided next to one another. To produce such a transducer 1, the interdigital electrode array is preferably produced on the carrier 2 using the photolithographic method. Subsequently, a laminating film is preferably glued on, which leaves a circular cutout above the interdigital electrode array. A solution of the polymer and preferably a plasticizer in THF or a mixture of THF-cyclohexanone (7: 3) is dripped onto this section and the polymer film 6 is formed after the solvent has evaporated. A substrate which can be degraded by the enzyme to be determined is then applied adsorptively to the polymer film 6.
Als ein solches Abbauedukt wird beispielsweise ein Protein eingesetzt. So wurde z.B. speziell ein Bovines Serum Albuminum BSA als Protein verwendet. Dazu wurde eine 1 Gew.%-ige Lösung von BSA in 60 mM TRIS, 0,5 mM MgCI2, pH-Wert = 6,7 hergestellt und die einsetzende Schaumbildung abgewartet. Mittels einer Eppendorfpipette (0,5 bis 10 μl) wurden je 4 μl der BSA-Lösung in den mit dem Polymerfilm 6 beschichteten Bereich gegeben. Bis zum Einsatz eines derartig Protease-sensitiven Aktivitätssensors wird dieser staubfrei und trocken gelagert. Das auf der Polymerschicht 6 eingetrocknete Protein ist wasserlöslich, auf einen separaten Abwaschschritt kann aber verzichtet werden, wenn der Sensor in der gerührten Hintergrundlösung für ca. 10 min eintaucht. Bevor die Proteasezugabe erfolgt, ist es aber auch möglich, die Vorrichtung nach dem Abspülen der überstehenden Proteinlösung bis zum Einsatz zu lagern und erst später einzusetzen.A protein, for example, is used as such a degradation adduct. For example, a Bovine Serum Albuminum BSA was specifically used as the protein. For this purpose, a 1% by weight solution of BSA in 60 mM TRIS, 0.5 mM MgCl 2 , pH = 6.7 was prepared and the onset of foam formation. Using an Eppendorf pipette (0.5 to 10 μl), 4 μl of the BSA solution were added to the area coated with the polymer film 6. Until such a protease-sensitive activity sensor is used, it is stored dust-free and dry. The protein dried on the polymer layer 6 is water-soluble, but a separate washing step can be dispensed with if the sensor is immersed in the stirred background solution for about 10 minutes. Before the protease is added, however, it is also possible to store the device after rinsing off the excess protein solution until use and to use it only later.
Die Polymerschicht 6 wird bevorzugt aus einer Lösung aufgebracht, und zwar unter Zugabe eines Weichmachers, beispielsweise von 30 Gew.% TBTC, das Aufbringen erfolgt vorzugsweise nach den etablierten Verfahren des Drop-, Dip- oder Spin- Coatings.The polymer layer 6 is preferably applied from a solution, specifically with the addition of a plasticizer, for example of 30% by weight TBTC. The application is preferably carried out according to the established methods of drop, dip or spin coating.
Taucht man eine vorbeschriebene Vorrichtung 1 in eine Lösung, die ein Enzym enthält, das in der Lage ist, das immobilisierte Substrat abzubauen, ändern sich die impediometrischen Eigenschaften des Systems, was sehr einfach in elektrische Signale umgewandelt und entsprechend verarbeitet werden kann. Figur 3 zeigt vier verschiedene Messkurven von vier verschiedenen Vorrichtungen, wobei die Wechselstromleitfähigkeit über der Zeit bei einer Frequenz von 10 kHz wiedergegeben ist. Diese Frequenz hat sich als besonders effektiv herausgestellt, der Pfeil an der obersten Messkurve in Figur 3 zeigt den Zeitpunkt, zu dem der Lösung Protease zugegeben wurde.If a device 1 described above is immersed in a solution which contains an enzyme which is able to degrade the immobilized substrate, the impediometric properties of the system change, which can very easily be converted into electrical signals and processed accordingly. FIG. 3 shows four different measurement curves from four different devices, the AC conductivity being reproduced over time at a frequency of 10 kHz. This frequency has proven to be particularly effective, the arrow on the uppermost measurement curve in FIG. 3 shows the point in time at which protease was added to the solution.
Derartige Vorrichtungen können grundsätzlich mehrfach verwandt werden. Sie sind selbstverständlich nicht vollständig reversibel, da die absorbierten Enzymsubstrate verbraucht werden. In Untersuchungen hat sich jedoch gezeigt, dass Vorrichtungen durchaus mehrere Male, beispielsweise bis zu 10 Male, verwendet werden können, bevor die Enzymsubstrate erneut adsorptiv an die Polymerschicht gebunden werden müssen. Eine solche Regeneration ist problemlos möglich.Such devices can in principle be used several times. Of course, they are not completely reversible since the absorbed enzyme substrates are consumed. However, studies have shown that devices can be used several times, for example up to 10 times, before the enzyme substrates have to be adsorptively bound again to the polymer layer. Such regeneration is possible without any problems.
Mit einer derartigen Vorrichtung ist grundsätzlich eine Online-Bestimmung der Enzymaktivität möglich. Somit kann beispielsweise die bedarfsgerechte Zugabe von oberflächenaktiven Substanzen in Geschirr- und Waschmaschinen gewährleistet werden. Einerseits kann man somit die notwendige Menge an Enzymiösung genau dosieren, andererseits können Substanzen, die das Enzym in seiner Funktionsweise beeinträchtigen (z.B. aufgrund ihrer Basizität), erst nach dem enzymatischen Reaktionsschritt zudosiert werden. With such a device, online determination of enzyme activity is fundamentally possible. This ensures, for example, the appropriate addition of surface-active substances in dishwashers and washing machines. On the one hand, you can precisely dose the necessary amount of enzyme solution, on the other hand, substances that impair the enzyme's functionality (e.g. due to its basicity) can only be added after the enzymatic reaction step.

Claims

Patentansprüche: claims:
1. Vorrichtung zur Bestimmung der Aktivität von Enzymen, gekennzeichnet durch einen Träger (2), auf dem wenigstens ein Elektrodenarray (3) angeordnet ist, welches mit einem Polymerfilm (6), self-assembled monolayers (SAM) oder Silanen beschichtet ist, wobei auf die Beschichtung adsorptiv ein vom zu bestimmenden Enzym abbaubares Substrat aufgebracht ist.1. Device for determining the activity of enzymes, characterized by a carrier (2) on which at least one electrode array (3) is arranged, which is coated with a polymer film (6), self-assembled monolayers (SAM) or silanes, wherein a substrate which is degradable by the enzyme to be determined is applied adsorptively on the coating.
2. Vorrichtung nach Anspruch 1 , dadurch gekennzeichnet, dass das Substrat aus Monomeren oder Oligomeren besteht.2. Device according to claim 1, characterized in that the substrate consists of monomers or oligomers.
3. Vorrichtung nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass das Elektrodenarray als Interdigitalelektrodenarray (3) ausgebildet ist.3. Device according to claim 1 or 2, characterized in that the electrode array is designed as an interdigital electrode array (3).
4. Vorrichtung nach Anspruch 3, dadurch gekennzeichnet, dass die Elektrodenfinger (3) des Interdigitalelektrodenarrays etwa 10 μm breit sind und in einem Abstand von etwa 10 μm zueinander angeordnet sind.4. The device according to claim 3, characterized in that the electrode fingers (3) of the interdigital electrode array are about 10 microns wide and are arranged at a distance of about 10 microns from each other.
5. Vorrichtung nach Anspruch 1 oder einem der folgenden, dadurch gekennzeichnet, dass der Polymerfilm (6) aus Polystyrol oder Polyvinylchlorid besteht.5. The device according to claim 1 or one of the following, characterized in that the polymer film (6) consists of polystyrene or polyvinyl chloride.
6. Vorrichtung nach Anspruch 5, dadurch gekennzeichnet, dass die Polymerschicht (6) eine Dicke von 1-5 μm aufweist.6. The device according to claim 5, characterized in that the polymer layer (6) has a thickness of 1-5 microns.
7. Vorrichtung nach Anspruch 1 oder einem der folgenden, dadurch gekennzeichnet, dass der Träger (2) aus Glas besteht.7. The device according to claim 1 or one of the following, characterized in that the carrier (2) consists of glass.
8. Vorrichtung nach Anspruch 1 oder einem der folgenden, dadurch gekennzeichnet, dass auf dem Träger (2) nebeneinander mehrere Mikroelektrodenarrays angeordnet sind, auf deren Polymerfilm zur Bestimmung unterschiedlicher Enzyme unterschiedliche Substrate aufgebracht sind . erfahren zur Bestimmung der Aktivität von Enzymen mit einer Vorrichtung nach einem oder mehreren der Patentansprüche 1 bis 8, dadurch gekennzeichnet, dass die Vorrichtung in eine Enzymiösung eingetaucht und die zeitliche Änderung der impediometrischen Eigenschaften des Systems gemessen und ausgewertet wird. 8. The device according to claim 1 or one of the following, characterized in that a plurality of microelectrode arrays are arranged side by side on the carrier (2), on the polymer film of which different substrates are applied to determine different enzymes. experienced in determining the activity of enzymes with a device according to one or more of claims 1 to 8, characterized in that the device is immersed in an enzyme solution and the temporal change in the impediometric properties of the system is measured and evaluated.
EP03789039A 2002-11-23 2003-11-14 Device for determining the activity of enzymes Withdrawn EP1563086A1 (en)

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