DE10226945A1 - Field effect transistor for identifying biochemical reaction or bonding in diagnostics or other clinical applications or as a label-free biosensor, has collector molecules coupled to an organic semiconductor on a channel region - Google Patents

Abstract

In a field effect transistor (FET) (10), comprising a substrate (20), a drain region (30), a source region (40), a channel region (70) and a gate region, the channel region has a layer based on an organic semiconductor material and, coupled to this by physical or chemical interactions, collector molecules selected from nucleic acids, proteins, enzymes, peptides, low-molecular organic ligands and supramolecular systems.

Description

The present invention relates to a field effect transistor based on an organic semiconductor material, the one to carry out of methods for the detection of biochemical (binding) reactions as well therefor especially for the investigation of enzymatic reactions, nucleic acid hybridizations, Protein-protein interactions and other binding reactions in the field of genome, proteome or drug research in biology and Medicine or in diagnostics and other clinical applications capable and its use as a label-free biosensor.

For the biosciences and medical diagnostics is detection (bio) chemical reactions, i.e. the detection of biologically relevant molecules in a defined Examination material of outstanding importance. In this frame the development of so-called biochips or biosensors is steadily increasing promoted. Such biochips are usually around miniaturized hybrid functional elements with biological and technical Components, in particular immobilized on a surface (outer surface and / or inner surface) biomolecules that serve as specific interaction partners. Often points the structure of these functional elements rows and columns. you then speaks of so-called "chip arrays". Because thousands of biological or biochemical functional elements on a chip can be arranged these are usually made using microtechnical methods. In particular come as biological and biochemical functional elements DNA, RNA, LNA, PNA, (for nucleic acids and their chemical derivatives can e.g. Single strands Triplex structures or combinations thereof), saccharides, Peptides, proteins (e.g. antibodies, Antigens, receptors), organic molecules that come from compound libraries, based on combinatorial chemistry methods, cell components (e.g. organelles), single cells, multicellular organisms as well cell aggregates in question.

As part of the use of such biochips in Corresponding analysis methods are today mostly optical methods used largely on procedures with previous labeling (Labeling) one or more analytes (Technology Standards for Microarray Research, M. Schena, R. W. Davis in Microarray Biochip Technology, Eaton Publishing, 2000). Doing so will provide proof corresponding biological or biochemical reactions, for example small amounts of different capture molecules point-like and matrix-like, so-called Spots, on a surface made of glass or gold, for example. Then will an analyte to be examined, which is usually fluorescence-labeled Contains target molecules through this surface pumped. When the corresponding molecules of the fluorescent labeled Analytes with or on those immobilized on the surface of the carrier substrate Capture molecules or Implement or bind probes, for example, by optical excitation a laser and measuring the corresponding fluorescence signal Reaction or bond can be demonstrated. The signal transmission thus takes place by means of optical detection, e.g. after a fluorescent label, using passive chip material, usually glass or silicon.

A disadvantage of such an optical However, the method consists in marking the analyte or must be labeled, i.e. with appropriate fluorescence molecules, for example Cy3, Cy5, or the like, must be provided. For one, there is one chemical reaction between the analyte molecule and the fluorescent dye molecule necessary. To the others leave the emissivity of the fluorescent molecules with longer or repeated measurements after, reducing the intensity of the measurement signal decreases. Aside from the use of such fluorescent markers costs incurred such fluorescent labeling methods are generally only semi-quantitative Evaluation. In addition, it is usually not a satisfactory one receive linear evaluation behavior.

For qualitative and quantitative Analysis of such biomolecules, such as. Nucleic acids Therefore, the aim is to have marking-free procedures, i.e. Method, the one without complex chemical Modification of the biomolecules to be examined can be carried out. Label-free analytical procedures offer the prerequisite for inexpensive online detection using microelectronic components, to that extent the otherwise necessary amplification or sample preparation reactions can be avoided.

Newer methods also use electronic or electrochemical signals for detection, which are generated based on a previous redox reaction. The Redox reaction is there carried out by an enzyme which to the. before the hybridization event to be detected nucleic acid must be bound. The active chip is based on CMOS technology. However, even with such a method, one is prior to the analysis Processing over a labeling reaction with the addition of enzyme and substrate or amplification the analyte or analytes required.

The present invention is therefore based on the object of providing a simple, flexible and inexpensive method and a device for the detection of biochemical reactions without the analyte, ie the target molecules to be investigated, having to be labeled and thus in native form can be used.

This task is carried out in the claims featured embodiments solved.

In particular, a field effect transistor that is suitable for the detection of biochemical reactions is provided, comprising
a substrate,
a drain area,
a source area
a channel area as well
a gate area
wherein the channel region has at least one layer based on an organic semiconductor material and capture molecules, selected from the group consisting of nucleic acids, proteins, enzymes, peptides, low molecular weight organic ligands and supramolecular systems, are coupled to this layer via physical or chemical interactions.

Both the drain area as well the source area can a metal electrode, for example platinum, palladium, gold or Silver included. Between the drain area and the source area a channel area is provided, the channel area at least has a layer based on an organic semiconductor material. Forms the layer based on an organic semiconductor material thus the channel area required in a field effect transistor for transporting electrical charge carriers from the source area in the drain area. Furthermore, a gate area is provided over which the electrical conductivity the channel area is controllable. The gate area can be a gate electrode, for example made of nickel, and comprise a separating layer. The gate area is through the separation layer from the drain region, the source region and the Channel area so electrically separated that the conductivity or resistance (Impedance) of the channel area above the gate area is controllable. The separation layer can be electrical insulating separating layer and / or a layer with a large dielectric constant. The separating layer is preferably formed from silicon dioxide.

The channel region is formed by a layer based on an organic semiconductor material. The charge carrier density of the channel region can be controlled via the gate region via the field effect occurring in such an arrangement, in particular via the electrical potential difference between the gate electrode and the channel region. Preferably, the organic semiconductor material is composed of (i) conjugated polymeric hydrocarbons, including polyacetylene, polyphenylene, poly (phenylene vinylene) and their derivatives, including oligomers of these conjugated polymeric hydrocarbons, (ii) conjugated heterocyclic aromatics, including polyaniline, polypyrrole, polythiophene, polyfuran and poly (thiophene vinylene), including oligomers thereof, or (iii) selected from ortho or ortho and peri-fused polycyclic aromatic hydrocarbons having 4 to 20 fused rings. Derivatives of the present invention will be understood in the context of particular derivatives thereof with (C ₁ -C ₆₎ alkyl, (C ₃ -C- ₇₎ - cycloalkyl which may have one or more hetero atoms, (C ₁ -C ₆ ) Alkoxy radicals, (C ₁ -C ₆ ) thioether radicals, hydroxy, trifluoromethyl, bromine, chlorine, fluorine, phenyl and / or un-, mono- or disubstituted amino groups, the amine substituents consisting of (C ₁ -C ₆ ) alkyl radicals or phenyl are selected, are substituted.

The organic is more preferred Semiconductor material made from conjugated oligomers with recurring Units of pyrrole or thiophene, e.g. α-sexithiophene, or their derivatives, or from ortho- or ortho- and peri-fused polycyclic aromatic hydrocarbons with 4 to 20 fused rings selected. As such ortho or ortho and peri-annellated polycyclic aromatic hydrocarbons can Quaterphenyl, tetracene, pentacene, chrysene, pyrene, perylene and coronene as well as derivatives thereof become. Pentacen is the material of the organic semiconductor layer within the scope of the field effect transistor according to the invention particularly preferred. Pentacen forms after thermal deposition films arranged in a vacuum. Pentacen also has a relatively high melting point of 271 ° C and on and is up to about 300 ° C not decompose.

According to the invention, capture molecules selected from the group consisting of nucleic acids including DNA, RNA, LNA ("locked nucleic acid") are involved in this organic semiconductor layer via physical or chemical interactions - these are artificial RNA derivatives in which the ribosering between the 2'-oxygen and the 4'-carbon is "pinched"; see Brasch, Corey, Chem. Biol. 2001, Vol. 8 (1), pages 1 to 7) and PNA, proteins, enzymes, peptides, low molecular weight organic Ligands and supramolecular systems, coupled. In the context of the present invention, low molecular weight organic ligands are understood to mean those which come from compound libraries which are based on methods of combinatorial chemistry. Dendrimers, for example, can be mentioned as supramolecular systems which can be used as capture molecules in the context of the present invention. Particularly preferred capture molecules are oligonucleotides. Coupling the catcher molecules to the organic semiconductor layer via physical or chemical interactions means covalent, ionic or electrostatic bonds of the catcher molecules to the organic semiconductor material in the context of the present invention. For example, the organic semiconductor material, such as polythiophene, α-sexithiophene or pentacene, can first be derivatized using Friedel-Crafts acylation and then finally covalently bound with, for example, oligonucleotides as capture molecules via acid amide bonds. Alternatively, the organic semiconductor layer can first be applied, for example by thermal deposition in a vacuum in the case of polycyclic aromatic hydrocarbons such as, in particular, pentacene or conjugated oligomers such as α-sexithiophene, and then coating the organic semiconductor layer with a layer composed of corresponding capture molecules by immersion be carried out in a solution containing these capture molecules, the coupling of the capture molecules to the organic semiconductor layer then being achieved via electrostatic interactions. Furthermore, the organic semiconductor material, ie its molecules, and the capture molecules can be applied simultaneously.

In one embodiment of the present invention, a layer composed of linker molecules can be applied to a silicon dioxide layer, which functions as a separating layer of the gate region, to which in turn the capture molecules, selected from the group consisting of nucleic acids, proteins, enzymes, via covalent bonds. Peptides, low molecular weight organic ligands and supramolecular systems. Such linker molecules are not subject to any specific limitation, as long as they are capable of covalently binding to the OH groups present on the surface of the SiO ₂ separating layer and furthermore have a functional group which can be used for covalent binding with those used as probes in biochemical reactions Capture molecules is capable. Such linker molecules are usually based on an organic silicon compound. Such bifunctional silicon-organic compounds can be, for example, alkoxysilane compounds with one or more terminal functional groups selected from epoxy, glycidyl, chlorine, mercapto or amino. Preferably the alkoxysilane compound is a glycidoxyalkylalkoxysilane, such as 3-glycidoxypropyltrimethoxysilane, a mercaptoalkylalkoxysilane, such as γ-mercaptopropyltrimethoxysilane, or an aminoalkylalkoxysilane, such as N-β- (aminoethyl) -γaminopropyltrimethoxysilane. The length of the alkylene radicals which act as a spacer between the functional group, such as epoxy or glycidoxy, which binds to the capture molecule or the probe, and the trialkoxysilane group is not subject to any restriction. Such spacers can also be polyethylene glycol residues.

For the completion of the field effect transistor according to the invention can bind or couple capture molecules such as oligonucleotides or DNA molecules via the linker molecules according to the usual in the prior art Processes are carried out, for example when using epoxysilanes as linker molecules by subsequent Reaction of the terminal epoxy groups with terminal primary amino groups or thiol groups of oligonucleotides or DNA molecules, which in corresponding Analysis methods as immobilized or fixed capture molecules for the in target molecules present to be analyzed act. You can for example those that can be used as capture molecules Oligonucleotides using the synthesis strategy as described in Tet. Let. 22, 1981, pages 1859 to 1862. The oligonucleotides can doing while of the manufacturing process at either the 5 or 3 end position can be derivatized with terminal amino groups.

In another embodiment The present invention can be applied as a separating layer of the gate region provided silicon dioxide layer a composed of linker molecules Layer can be arranged, to which in turn via both covalent bonds the molecules that make up the organic semiconductor layer, e.g. α-sexithiophene or pentacene, as well as capture molecules, selected from the group consisting of nucleic acids, proteins, enzymes, peptides, low molecular weight organic ligands and supramolecular systems, be bound. This can be achieved, for example, that in a first step is either the molecules that make up the organic semiconductor layer build up, or the capture molecules in deficit with the functional groups of the linker molecules, e.g. terminal epoxy groups when using epoxysilanes as linker molecules and subsequently in a second step the remaining, unreacted functional Groups of linker molecules with either the molecules which build up the organic semiconductor layer, or the capture molecules, each after which were used in the first step in the deficit, implemented become.

The substrate of the field effect transistor according to the invention is not subject to any specific limitation. It can be both a solid Substrate selected made of semiconductor materials, in particular silicon, or glass, as also a flexible plastic film, especially based on PET or PEN, his. PEN is elastic and transparent and is particularly suitable advantageously for the manufacture of integrated circuits Basis of the field effect transistor according to the invention.

In the context of a method for the detection of biochemical (binding) reactions using the field effect transistor according to the invention, the actual detection reaction of the target molecules to be analyzed takes place within the "active" organic semiconductor layer of the field effect transistor. The capture molecules, such as oligonucleotides, which act as "probes" directly on or are coupled to the “active” organic semiconductor layer of the field effect transistor according to the invention, that is, on the one hand into the layer of the organic semiconductor can be introduced or are in direct contact with it, for example selectively bind to the complementary nucleic acid molecules to be detected (hybridization). The hybridization event is then the event to be detected.

The (binding) reactions of the as Probe molecules acting with the Target molecules of the Analytes to be examined are thus found virtually within the "active" organic semiconductor layer (within of the semiconducting channel) of the field effect transistor according to the invention instead and therefore have a direct change in electrical conductivity or impedance in the active organic semiconductor layer of the field effect transistor according to the invention result.

Another object of the present invention thus relates to the use of the field effect transistor according to the invention in Methods for the detection of biochemical reactions and / or bonds as well as for this especially for the investigation of enzymatic reactions, nucleic acid hybridizations, Protein-protein interactions and protein-ligand interactions.

According to another aspect of Invention includes a method for the detection of chemical or biochemical reactions and / or ties the steps:

• (a) providing at least one field effect transistor according to the invention;
• (b) bringing an analyte to be examined into contact with the at least one field effect transistor;
• (c) Measure the change at least one electrical parameter of the field effect transistor in dependence of the event of a (binding) reaction of those contained in the analyte targets with the capture molecules arranged on the field effect transistor.

The electrical parameters of the field effect transistor measured in step (c) can be the electrical conductivity or the electrical resistance of the channel region, the charge carrier density of the channel region ( 70 ) and / or the capacitance between the gate electrode ( 50 ) and the channel area ( 70 ) his.

In step (a), for example, a large number of field effect transistors according to the invention be arranged in an array. For such array arrangements a flexible transparent PEN film is particularly suitable as Substrate material. You can based on the device according to the invention available in the prior art Microtechnologies are parallelized.

The use of the field effect transistor according to the invention in such analysis methods offers the following advantages:
direct electrical reading is possible without a previous labeling reaction,
the field effect transistor system according to the invention is mechanically deformable when using plastic foils such as PEN foils,
high sensitivity, possibly a quantification of the binding events, at best even without amplification of the signals,
- Analysis methods can be carried out both in a batch process and in online screening.

The particular advantage of an analysis procedure using the field effect transistor according to the invention in the direct detection of the target molecules present in an analyte to be examined the change electrical parameters such as electrical channel conductivity or - resistance or gate channel capacity. That means a labeling reaction (Labeling) of the target molecules to be examined in the analyte is not necessary is.

The present invention is described below Reference to the accompanying drawings of preferred embodiments described as an example. It shows:

• 1 is a schematic cross-sectional view of a preferred embodiment of the field effect transistor according to the invention;
• 2 a schematic representation of the sensitive interface of a preferred embodiment of the field effect transistor according to the invention, the sensitivity being realized by DNA single strands as capture molecules which are covalently fixed on a monomolecular silane layer as a linker molecular layer via amide bonds to SiO ₂ as a separating layer of the gate region, and a schematic View regarding the change in channel resistance as an event to be measured.

1 shows a field effect transistor 10 according to an embodiment of the invention. The field effect transistor has a substrate 20 which can be, for example, silicon, glass or in particular also a flexible plastic film, in particular based on PET or PEN.

A first metal electrode is on the substrate 30 and a second metal electrode 40 for example applied by means of a vapor deposition process or a sputtering process. The first metal electrode 30 serves as the drain region of the field effect transistor 10 while the second metal electrode 40 as the source region of the field effect transistor 10 serves. The first 30 and second 40 metal electrodes can be made independently of one another from, for example, platinum, palladium, gold or silver. Alternatively, any metallic alloys can be used to form the first and second metal electrodes.

The gate region of the field effect transistor is also on the substrate 10 forming gate electrode 50 applied. On the gate electrode 50 For example, made of nickel or A gate dielectric or separating layer is usually produced for the metals or metal alloys mentioned above for the first and second metal electrodes 60 arranged. The gate area is separated from the drain area by the separation layer 30 , the source area 40 and the channel area 70 electrically separated such that the charge carrier density and thus the conductivity or the impedance of the channel region 70 is controllable via the gate area. The interface 60 can be an electrically insulating separating layer and / or a layer with a large dielectric constant. The separation layer is preferably 60 made of silicon dioxide or silicon nitride, in particular silicon dioxide.

Between the first metal electrode 30 and the second metal electrode 40 is an organic semiconductor layer 80 arranged according to the embodiment 1 in some areas also on the surface of the first and second metal electrodes 30 . 40 is arranged. According to the invention, capture molecules (not shown), such as oligonucleotides, are coupled directly to or with the “active” organic semiconductor layer of the field effect transistor according to the invention. Between the organic semiconductor layer 80 , which includes the capture molecules and the separation layer 60 is a linker layer as described above for binding the capture molecules 90 arranged.

2 shows a schematic representation of the sensitive interface of a preferred embodiment of the field effect transistor according to the invention 10 , the sensitivity being realized by DNA single strands as capture molecules, which are covalently fixed on SiO ₂ as a separating layer of the gate region on a monomolecular silane layer as a linker molecular layer via amide bonds. When complementary bases are added as the target molecules to be investigated in the analyte (illustration on the right), the channel resistance of the active organic semiconductor layer based on pentacene changes, which can be measured directly as a sensor event.

10

Field Effect Transistor

20

substratum

30

first Metal electrode (drain area)

40

second Metal electrode (source area)

50

Gate electrode

60

Interface

70

channel area

80

organic Semiconductor layer

90

Linker molecule layer

Claims (12)

Field effect transistor ( 10 ) comprising a substrate ( 20 ), a drain area ( 30 ), a source area ( 40 ), a channel area ( 70 ) and a gate area, the channel area ( 70 ) has a layer based on an organic semiconductor material and capture molecules, selected from the group consisting of nucleic acids, proteins, enzymes, peptides, low molecular weight organic ligands and supramolecular systems, are coupled to this layer via physical or chemical interactions. A field effect transistor according to claim 1, wherein the organic semiconductor material out  (i) conjugated polymeric hydrocarbons comprising Polyacetylene, polyphenylene, poly (phenylene vinylene) and their derivatives, including Oligomers of these conjugated polymeric hydrocarbons,  (Ii) conjugated heterocyclic aromatics, including polyaniline, polypyrrole, Polythiophene, polyfuran and poly (thiophene vinylene), including oligomers of it, or  (iii) from ortho- or ortho- and peri-annellated polycyclic aromatic hydrocarbons with 4 to 20 fused Wrestling selected is. A field effect transistor according to claim 2, wherein the organic semiconductor material from conjugated oligomers with repeating units of pyrrole or thiophene, especially α-sexithiophene, or their derivatives, or from ortho- or ortho- and peri-annellated polycyclic aromatic hydrocarbons with 4 to 20 fused Wrestling selected is. Field effect transistor according to claim 3, wherein the orthobzw. ortho- and peri-fused polycyclic aromatic hydrocarbons from quaterphenyl, tetracene, pentacene, chrysene, pyrene, perylene and Corones and derivatives thereof, in particular pentacene, are selected. Field effect transistor according to one of claims 1 to 4, wherein the gate region is a gate electrode ( 50 ) and a separation layer ( 60 ), so that the gate area from the drain area ( 30 ), the source area ( 40 ) and the channel area ( 70 ) is electrically insulated in such a way that the electrical conductivity of the channel area ( 70 ) about the electrical potential difference between the gate electrode ( 50 ) and the channel area ( 70 ) is controllable. Field effect transistor according to claim 5, wherein the separating layer ( 60 ) is an electrically insulating separation layer and / or a layer with a large dielectric constant, preferably made of silicon dioxide. Field effect transistor according to one of the claims 1 to 6, the substrate being a solid substrate selected from semiconductor materials, in particular silicon or glass, or a flexible plastic film, in particular based on PET or PEN. Field effect transistor according to one of claims 1 to 7, wherein the capture molecules are oligonucleotides. Field effect transistor according to one of claims 6 to 8, wherein between the organic semiconductor layer and the gate region, a linker molecular layer ( 90 ) is arranged, to which the capture molecules are bound via covalent bonds. Use of the field effect transistor according to one of claims 1 to 9 in methods for the detection of biochemical reactions and / or bonds as well therefor especially for the investigation of enzymatic reactions, nucleic acid hybridizations, Protein-protein interactions and protein-ligand interactions. Method for the detection of chemical or biochemical reactions and / or bonds, comprising the steps: (a) providing at least one field effect transistor ( 10 ) according to one of claims 1 to 9; (b) bringing an analyte to be examined into contact with the at least one field effect transistor ( 10 ); (c) measuring the change in at least one electrical parameter of the field effect transistor as a function of the event of a (binding) reaction of the target molecules contained in the analyte with those on the field effect transistor ( 10 ) arranged capture molecules. The method of claim 11, wherein the electrical characteristic of the field effect transistor (measured in step (c) 10 ) the electrical conductivity or the electrical resistance of the channel region ( 70 ), the charge carrier density of the channel area ( 70 ) and / or the capacitance between the gate electrode ( 50 ) and the channel area ( 70 ) is.