DE10330717A1 - Apparatus and method for detection of analyte - Google Patents

Abstract

The invention relates to an apparatus and a method for the detection of analyte. DOLLAR A The object of the invention to provide an apparatus and method for the detection of analyte, which overcome the disadvantages of the prior art and in particular neither two specific binding partner (= sandwich) for the analyte to be detected nor require a complex prior labeling of the analyte of a particular Class of molecules is achieved by providing the device for detection of analyte in a sample of a support substrate provided with at least two electrodes surrounding a gap having immobilized specific binding partners capable of complementary complementary analyte directly or indirectly to couple, wherein the gap is dimensioned in its width and depth so that binding events of the immobilized specific binding partner with the complementarily associated analyte are possible, wherein after successful binding events, the complementarily associated analyte targeted with electr Isch conductive detection substrate is loaded and allows an electrical current flow over the space in the voltage applied to the electrodes voltage, wherein the electrodes are in communication with a measuring unit, so that the current flow is permanently detectable exists.

Description

The The invention relates to an apparatus and method for detecting Analyte.

A Device for the detection of analytes by means of analyte-specific Reaction after solid phase binding is in the form of a biochip eg. known from the document WO 01/14425 A1.

at this is a protein chip for mass diagnostics or analysis of test samples consisting of a solid substrate, on a variety of spots from test proteins in a defined Are fixed, whereby these proteins belong to the group of antigens, Receptors and enzymes are assigned and the sample proteins on the surface over her Amino groups on the functional groups of the applied on the surface Chemicals are bound.

Of the Disadvantage of this protein chip is that in the application of the respective Spots crosstalk The individual protein samples can be made, resulting in uselessness of the chip or leads to non-usable test results.

From the Scriptures US 5,077,210 is a method for the production of biochips is known in which are provided on solid surfaces silanes with terminal thiol groups, which in turn can react via corresponding cross-linker with amino groups of bioactive molecules.

The disadvantage of this protein chip is that only a coupling of the biomolecules on their amino group can be done, resulting in the coupling of antibodies to it; that the antigen binding, NH ₂ -bearing region is blocked, whereby no antigen-antibody reaction can take place.

Farther it is known that one Antibodies oxidized on carbohydrate residues are directed via their aldehyde residues on surfaces (Qian, W.P., Yao, D.F., Xu, B., Yu, F., Lu, Z.H., and Knoll, W., 1999: "Atomic force microscopic studies of site-directed immobilization of antibodies using their carbohydrate residues. "Chemistry of Materials, 11 (6), 1399-1401). The disadvantage of these surfaces is that no Separation of surface areas is achieved, so that a Spotability of different samples without sample crosstalk is not given is.

Of the Disadvantage of all these known biochips is that they are not electric, but optically read out.

So is for example for The optical readout known that automation of Image analysis based on the chip technology by reading the different microarray structures can be made. Most available technologies based on the detection of fluorescently labeled binding pairs, held in a specific way on the chip surface are, wherein the fluorescence detection by optical readout of the reactive centers of the microarray is executed. The use of fluorescent or chemiluminescent samples is thereby as in the classical methods described above and becomes with CCD imaging combined (Eggers, M. et al., 1996: Professional Program Proceedings. Electro '96. IEEE, New York, NY, USA, 364 pp.).

From the Scriptures DE 198 60 547.1-52 is an affinity sensor for detecting specific binding events consisting of a support substrate provided with at least two electrodes surrounding a space having immobilized specific binding partners capable of coupling complementarily associated binding partners directly or through other specific binding molecules, the complementarily associated binding partners are loaded with electrically conductive particles directly or via connecting molecules and the gap in its width and depth is dimensioned so that binding events of the immobilized specific binding partner with the complementarily associated binding partners are possible and the coupling of the binding partners an electrical current flow over in the Interposed positioned conductive allows at the voltage applied to the electrodes, the electrodes are in communication with a measuring unit, so that the current flow permanently de is detectable.

Of the Disadvantage of this electrically readable chips is that the mark generally either to all molecules of the substance class of interest must be attached or a sandwich approach is used in which another molecule, the one Wearing marker, specifically to the detected and already bound on the surface Analyte binds. This sandwich process is through the necessary Double specific binding is complicated by both the additional steps as well as due to the need for a second molecule which binds to the one you are looking for.

In the other method, the labeling of all molecules of a given ensemble (such as cDNA in expression studies), methods such as the polymerase chain reaction (PCR) are used by which a label can be inserted into these molecules. However, these methods can only be used for very specific classes of molecules, and also usually expensive and expensive. Especially in terms of new labeling methods, such as the marking with colloidal metal particles for electrical detection, this method is difficult or even not applicable.

in addition comes that both methods have the distinct disadvantage that the binding reaction by the modification of a binding partner influenced or changed will and not be possible is, with unmodified molecules to work out those influences would not have.

One faster and more efficient detection of biomolecules is just through the swift increasing number of known sequences from genome and proteome become central problem of modern life sciences.

A important method for the detection of biologically interesting molecules on the specific binding of these analyte molecules to a binding partner. If this analyte-specific binder to simplify the reading previously attached to a solid substrate, can after the binding is removed by washing all other molecules become. In a subsequent step the presence of the desired analyte on the substrate is now detected.

These can be labeled free by determining the change of certain physical Properties of the substrate through the connection, such. Mass increase (quartz crystal balance or cantilever-based measurements) or change optical (dielectric) properties (surface plasmon resonance) or change electrochemical properties.

These Label-free methods, however, generally require a complicated one Evaluation based on a complex measurement signal, that's what they are expensive and expensive.

A easier evaluation and also often an increase in sensitivity to achieve through the use of markers (label), of which radioactive and fluorescent labels are the best known.

Besides But there are also dyes or colloidal metal particles. The Markers are generally either attached to all of the molecules of interest Attached substance class or it will be a sandwich approach using another molecule, bearing a mark, specifically to the detected and already bound on the surface Analyte binds.

This Sandwich process is complicated by the necessary duplicate specific binding, both through the additional steps as well due to the need for a second molecule which binds to the one sought.

at the other method of marking all the molecules of a given ensemble (such as cDNA in expression studies), methods such as PCR used to insert a marker into these molecules.

These Procedures are only for very special classes can be used, and also usually elaborate and expensive. Especially with regard to new labeling methods, e.g. the use of colloidal metal particles for electrical detection, Such methods are difficult or impossible to apply.

Of the Invention is based on the object, a device and method to provide for the detection of analyte, the disadvantages of the prior art overcome the technology and in particular neither two specific binding partners (= sandwich) for the need to be detected analyte still a complex prior labeling of the analyte of a particular molecules class require.

According to the invention This object is achieved by the characterizing features of the first or third claim solved. Advantageous embodiments are characterized by the subordinate claims 2 and 4 to 6 indicated.

The Essence of the invention is that by the electrical detection the detection is simplified, since instead of an optical design for Figure and an image processing algorithm for conversion the analog optical signals into a digital result and a lot direct conversion of the electrical signal into digital information can be done so that the use of more robust readout devices is made possible, which in turn also outside the laboratory and, for example, directly on site (e.g., point of care diagnostics).

The inventive combination a specific binding between different classes of molecules of analyte and binding molecules with a subsequent class-specific marking step of Analyts allows label-based affinity assays without second analyte-specific Binding partner or previous marking routines.

If such reactions are used for the specific binding of the desired analyte to the electrically readable chip, surprisingly completely new possibilities arise for the subsequent marking step. This does not have to be molecule-specific, as is customary in the prior art, but can be carried out in a substance-specific (molecular-class) manner without the specificity of the signal suffering.

by virtue of of the invention are those known, class of molecule-specific labeling methods, such as for example for the evaluation of gel-electrophoretic methods in proteins as silver staining are used, also for the chip technology applicable.

The The invention will be described below with reference to the drawings and exemplary embodiments explained in more detail. It shows:

1a FIG. 1 shows a schematic representation of an embodiment of the arrangement according to the invention, FIG.

1b FIG. 2 shows a schematic representation of a further embodiment of the arrangement according to the invention, FIG.

2a to c: a schematic representation of the analyte-specific detection steps,

3a to c: a schematic representation of the analyte-specific labeling reactions.

As in 1 As shown, the device consists of a support substrate provided with at least two electrodes surrounding a gap having immobilized specific binding partner capable of directly or indirectly coupling complementary analyte, the gap being in width and depth so is dimensioned that binding events of the immobilized specific binding partner are possible with the complementarily associated analyte, after binding events of the complementary associated analyte is specifically loadable with electrically conductive detection substrate and allows an electric current flow over the space in the voltage applied to the electrodes voltage, wherein the Electrodes with one in the 1 Measuring unit, not shown, are in communication, so that the current flow is permanently detectable.

The detection of analyte 4 in a test sample takes place on the planar substrate 1 by using analyte-specific binding partners 2 in one or more detection fields 3 be immobilized, then the test sample in contact with the detection fields 3 is brought and then after a washing process to remove unspecifically bound analyte 4 the presence and / or amount of the analyte to be detected 4 by electrical evaluation of the detection fields 3 is determined, wherein the analyte 4 and the analyte 4 specific binding partner 2 belong to different classes of compounds distinguishable by labeling reactions ( 2a to c).

The immobilized analyte 4 specific binding partner 2 bound analyte 4 is doing in a single or multi-level class of compounds-specific reaction with labeling substrate 5 marked and this marker substrate 5 then subjected to electric current, so that an electrical readout of the analyte 4-binding partner 5 bonds is possible.

In a preferred embodiment, the analyte 4 containing test sample in contact with detection fields 3 and then after a washing process to remove nonspecifically bound analyte 4 the presence and / or amount of analytes to be detected 4 determined by electrical evaluation of the detection fields, wherein the analyte 4 and the analyte-specific binding molecules 2 belong to different compound classes distinguishable by labeling reactions.

These are the analyte-specific binding molecules 2 DNA, RNA, PNA or their derivatives and the analyte 4 to proteins, hormones, vitamins, sugars or their derivatives or to therapeutics.

The analyte-specific labeling reactions according to the invention are particularly preferably carried out by an addition of metal atoms to the analyte 4 , wherein after this addition reaction, which is directly or indirectly possible, a reinforcing reaction of the type that the metal atoms serve as nuclei for further material deposition of the same or other metals, so that an optimal electrical detection can take place. As metals, for example, gold or silver are used ( 3a to c).

Particularly advantageous are the individual detection fields 3 with pre-structured metallic electrodes 6 or electrodes attached during the reading process 6 provided electrically read, so that the presence of the analyte 4 on the basis of an analyte 4 -specific deposition of conductive material on the respective detection fields 3 by a measurable, electrical conductivity of the surface of these detection fields 3 is detectable.

1st embodiment:

DNA for binding of proteins using the example of thrombin

to Cleaning becomes chip substrates with microstructured electrode arrangements every 10 minutes cleaned in acetone, ethanol and water. The activation the surfaces done for one hour at 70 ° C in a solution from conc. Ammonia solution: hydrogen peroxide: Water in proportion 1: 1: 1 (v / v / v). Are not gold or other metallic structures on the chip surface the conc. Ammonia solution against conc. Hydrochloric acid replaced become. the activation then takes place at room temperature for 10 min. Subsequently be the chips thoroughly with dist. Washed water to remove any remains of the activating solution remove, and for 10 min at 80 ° C dried. For further processing, the chips should be absolutely dry be.

The Modification of the surface The chips are in a 10 mM Glycidoxypropyltrimethoxysilanlösung in dry toluene. The incubation should be at least 4 hours at 70 ° C. After that, the chips for each 2 × 5 minute thoroughly in toluene, ethanol and finally rinsed in water.

• – 5 min in 0,1 % Triton X-100
• – 2 × 2 min in HCl pH 4
• – 10 min in 100mM KCl
• – 1 min in dest. Wasser
• – 15 min 50mM Ethanolamin 0,1 % SDS in 0,1 M Tris, pH 9,0
• – 1 min in dest. Wasser

For immobilization on the modified chip surfaces, the concentration of the 3 'or 5'-amino modified oligonucleotides should be between 5 and 50 μM, in 0.1 M potassium hydroxide solution. The prepared oligol solutions are now applied in the desired pattern on the chips and incubated for at least 4 hours at 37 ° C in a humid chamber and then dried at 80 ° C. The chips are then washed to remove unbound oligonucleotides and the surface also blocked to prevent further binding of molecules. The washing steps and solutions are the following:

• - 5 min in 0.1% Triton X-100
• - 2 × 2 min in HCl pH 4
• - 10 min in 100 mM KCl
• - 1 min in dist. water
• - 15 min 50mM ethanolamine 0.1% SDS in 0.1M Tris, pH 9.0
• - 1 min in dist. water

After that The chips are dried under a stream of nitrogen.

The Incubation with the thrombin solution takes place at room temperature in a humid chamber for 2 hours. The previously detected thrombin concentrations range from 1 μg / ml to 0.1 ng / ml in PBS pH 7.4. The chips are then incubated for 5 min in PBS pH 7.4, 0.03% Tween 20 and 2 x 5 Washed in PBS pH 7.4 min and dried under nitrogen. Now the chips are covered with negatively charged gold sol and for at least Incubated for 2 hours. Subsequently the chips are briefly dest. Rinsed in water and dried under nitrogen.

to silver enhancement 80 mg of silver acetate in 40 ml of dist. Water and 200 mg of hydroquinone in Citrate buffer pH 3.8 dissolved. Now both solutions Mixed 1: 1 and the chips dipped in this solution and for max. 10 min in this solution left under light. The reaction is washed by washing with dist. Water stopped. The silver reinforcement step can be repeated as many times as the deposited silver layer for one electrical proof is sufficient. This takes place on chip substrates either with measuring tips or in a reader.

2nd embodiment

PNA (Peptide nucleic acid) DNA

to Cleaning the chips substrates each 10 min. in acetone, ethanol and water cleaned. The activation of the surfaces takes place for one hour at 70 ° C in a solution from conc. Ammonia solution: Hydrogen peroxide: water in the ratio 1: 1: 1 (v / v / v). Are not Gold or other metallic structures may be present on the chip surface the conc. ammonia solution against conc. hydrochloric acid be replaced. activation then takes place at room temperature for 10 min .. then be the chips thoroughly with dist. Washed water to remove any remains of the activating solution remove, and for 10 min at 80 ° C dried. For further processing, the chips should be absolutely dry be.

The Modification of the surface The chips are in a 10 mM Glycidoxypropyltrimethoxysilanlösung in dry toluene. The incubation should be at least 4 hours at 70 ° C. After that, the chips for each 2 × 5 minute thoroughly in toluene, ethanol and finally rinsed in water.

to Immobilization on the modified chip surfaces should increase the concentration the 3 'or 5'aminomodified PNA between 5 and 50μM, in 0.1M potassium hydroxide solution, lie. The prepared PNA solutions are now in the desired pattern applied to the chips and for at least 4 hours at 37 ° C incubated in a humid chamber and then dried at 80 ° C. The chips will be now washed to remove unbound PNA, and the surface also blocked to another bond of molecules to prevent. The washing steps and solutions are

• - 5 in 0.1% Triton X-100
• - 2 × 2min in HCl pH 4
• - 10 min in 100mM KCl
• - 1 min in at least. water
• - 15 min 50mM ethanolamine 0.1% SDS in 0.1M Tris, pH 9.0
• - 1 min in at least. water

After that The chips are dried under a stream of nitrogen.

The Chips are now in a humid chamber at 40 ° C for at least 2 hours the DNA solution incubated and then washed with buffer. Now the chips are positively charged Gold sol covered and for incubated for at least 2 hours. Subsequently, the chips are briefly with least. Water rinsed and dried under nitrogen. The silver gain is 80 mg of silver acetate in 40 ml dist. Water and 200 mg of hydroquinone in citrate buffer pH 3.8 solved. Now bide solutions Mixed 1: 1 and the chips dipped in this solution and for max. 10 min in this solution left under light. The reaction is washed by dest. Water stopped. the silver reinforcement step can be repeated as many times as the deposited silver layer for one electrical proof is sufficient.

This takes place on chip substrates either with measuring tips or in one Reader.

1

planar substratum

2

binding partners

3

detecting patches

4

analyte

5

marking substrate

6

electrodes

Claims (8)

Apparatus for detecting analyte in a sample consisting of a support substrate provided with at least two electrodes surrounding a space having immobilized specific binding partners capable of directly or indirectly coupling complementally related analyte, the space being in its Width and depth is dimensioned such that binding events of the immobilized specific binding partner with the complementarily associated analyte are possible, characterized in that after successful binding events of the complementarily associated analyte is specifically loaded with electrically conductive detection substrate and an electric current flow through the space in the at Electrode applied voltage allows, the electrodes are in communication with a measuring unit, so that the current flow is permanently detectable. Device according to claim 1, characterized in that that analyte and binding partner belong to different substance classes. Method for the detection of analyte ( 4 ) in a test sample in which on a planar substrate ( 1 ) analyte-specific binding partners ( 2 ) in one or more detection fields ( 3 ) are immobilized, then the test sample in contact with the detection fields ( 3 ) and then after a washing process to remove nonspecifically bound analyte ( 4 ) the presence and / or amount of the analyte to be detected ( 4 ) by electrical evaluation of the detection fields ( 3 ), characterized in that analyte ( 4 ) and analyte-specific binding partners ( 2 ) belong to different classes of compounds which can be distinguished by labeling reactions such that the immobilized analyte-specific binding partner ( 2 ) bound analyte ( 4 ) in a single- or multistage class of compound-specific reaction with labeling substrate ( 5 ) and that this labeling substrate ( 5 ) an electrical readout of the analyte ( 4 ) Binding partner ( 5 ) Bindings. Method according to claim 3, characterized in that the analyte ( 4 ) in contact with the detection fields ( 3 ) and then after a washing process to remove nonspecifically bound analyte ( 4 ) the presence and / or amount of the analytes to be detected ( 4 ) is determined by electrical evaluation of the detection fields, wherein the analyte ( 4 ) and analyte-specific binding molecules ( 2 ) belong to different classes of compounds distinguishable by labeling reactions. A method according to claim 4, characterized in that as analyte-specific binding molecules ( 2 ) DNA, RNA, PNA or derivatives thereof and as analyte ( 4 ) Proteins, hormones, vitamins, sugars whose derivatives or therapeutics are used. A method according to claim 4, characterized in that in the analyte-specific labeling reactions an addition of metal atoms to the analyte ( 4 ), which are detected electrically, directly or after an amplification reaction in which the metal atoms serve as nuclei for further material deposition. Method according to Claim 3, characterized in that the individual detection fields ( 3 ) with pre-structured metallic electrodes ( 6 ) or attached electrodes during the reading process ( 6 ) can be electrically read by the presence of the analyte ( 4 ) based on an analyte ( 4 ) -specific deposition of conductive material on the respective detection fields ( 3 ) to the mess baren electrical conductivity of the surface of these detection fields ( 3 ) leads. Method according to claim 3, characterized that a device according to claim 1 is used.