DE102006057975B4 - Method and device for detecting microorganisms and / or their activity - Google Patents

Abstract

Method for the detection of microorganisms and / or their activity with biosensors, characterized in that on a surface of a substrate partially at least one ligand for binding a receptor or at least one receptor for a ligand or at least one ligand for binding a receptor and at least one receptor for either the or another ligand is immobilized chemically, physically or biologically, wherein the ligands and the receptors are components of the quorum sensing system of microorganisms, being used as ligands signal molecules and receptor receptors corresponding to the signal molecules and wherein one of the ligands the signaling molecule is autoinducer-2 (AI-2) or another molecule that binds to an autoinducer-2 binding receptor, and wherein physical or physiological responses to receptors are caused by binding of ligands emitted by microorganisms in the process of quorum sensing Sch chemical changes are recorded.

Description

The The invention relates to a method for the detection of microorganisms and / or their current state of activity using quorum sensing components based biosensors and devices as biosensors to carry out the method of use z. B. in biotechnology and environmental technology as well as medical technology.

bacterial Contaminations in drinking water or in biotechnological production processes, such as in yeast fermentation or in cell cultures, are a serious one and often a health-threatening problem. To take appropriate countermeasures is the most possible early Detection of bacteria necessary.

The Control of activity of microorganisms in biochemical processes to achieve optimal Yield requires methods for detecting the current cell state.

Microbiological Analyzes, that is Sampling and determination of the germs by cultivation on selective nutrient media a number of disadvantages: on the one hand sampling is a potential source of contamination, on the other hand, the cultivation and analysis of the bacteria is time- and costly. About that In addition, the microbiological analysis allows the detection of biofilm-forming bacteria only very limited. By molecular biological Procedures, such as PCR analyzes, can be reduce the detection time, the need for sampling and DNA isolation however, this process is also sluggish. By the selection of primers for the PCR reactions or probes are used in hybridizations a strong limitation of the spectrum of bacteria to be detected in purchase. In addition is With these procedures no distinction between living and dead microorganisms possible.

WO 01/94543 A2 discloses a so-called hapten inhibition assay for the quantitation of homoserine lactone-type autoinducers and analog compounds in which the concentration of an autoinducer in solution is determined by competition with a known amount of immobilized autoinducer for the binding sites on anti-autoinducer antibodies. Furthermore, it discloses a method based on the principle of affinity chromatography for the purification of receptors capable of binding to autoinducer. These are isolated by binding to immobilized autoinducer from a complex protein mixture.

Honestly et al. deal with the possibility the development of intelligent implants and propose a model of a sensor that predicts the emergence of so-called biofilms on implants early by the detection of quorum-sensing molecules (Ehrlich et al. Engineering approaches for the detection and control of orthopedic biofilm infections. Clin Orthop Relat Res. 2005 (437) 59-66). Of the Detection of the molecule RAP (ribonucleic acid (RNA) III activating protein), one of the Bacterial species Staphylococcus aureus and Staphylococcus epidermidis in the context of the species-specific quorum sensing secreted peptide, should with the help of the corresponding receptor TRAP (Target of RNAIII-activating protein).

task The invention is methods and means for the detection of microorganisms and / or their activity indicate the rapid and early detection of microorganisms enable.

The Task is solved by a method having the features of claim 1 and by a device with the features of claim 19.

The Methods and devices are characterized in particular by that a faster and earlier Proof of both a wide range as well as a very specific bacterial species and / or their activity is possible.

To becomes on a surface a substrate partially at least one ligand for binding a receptor or at least one receptor for a ligand or at least a ligand for binding a receptor and at least one receptor for either the or another ligand chemical, physical or biological immobilized by ligands bound by microorganisms emitted in the process of quorum sensing, at receptors physical or physical-chemical changes are measurable.

To is on a surface a substrate partially at least one ligand for binding a receptor or at least one receptor for a ligand or at least one Ligand for binding a receptor and at least one receptor for either the or another ligand chemical, physical or biological immobilized, whereby by bonds between ligands, by microorganisms in the process of quorum sensing, and receptors caused physical or physical-chemical changes are measurable.

basis The invention is the communication between microorganisms for physical or to use physical-technical processes.

quorum Sensing is a name for the communication between microorganisms by means of small hormone-like Signaling molecules, so-called autoinducer, as ligands and receptors. This process plays a crucial role in the regulation of cell density, which allows bacterial populations to be similar to multicellular organisms to behave and thereby benefit from advantages. To the through Quorum sensing regulated behaviors include production of antibiotics, symbiosis, conjugation, virulence, formation of biofilms as well as the bioluminescence of some Vibrio species.

The signal molecules called autoinducers are made by certain bacteria produced with the help of certain genes and into the surrounding culture medium issued. Bacteria have a suitable receptor system for them Ligands. After reaching a certain concentration of signal molecules in the medium it comes to the binding of the autoinducer signaling molecule to the receptor. The receptor autoinducer complex directs the information into the cell interior and activates the Transcription of certain genes.

It are different classes of autoinducers. Most of these are species-specific, d. H. Different types of bacteria produce different signal molecules for communication within one's own bacterial species. The Autoinducer-2 (below also called AI-2) for example, the communication between different bacterial species (Interspecies communication) and thus represents a universal signaling molecule in communication between microorganisms.

More than 30 different, mostly gram-negative, sorts of bacteria, including Escherichia coli, Salmonella typhimurium, Salmonella paratyphi, Helicobacter pylori, Vibrio cholerae, Shigella flexneri, Staphylococcus aureus, produce the signaling molecule Autoinducer-2 with the help of a specific gene (LuxS or homologue), secrete it into the culture medium and have a suitable intracellular receptor system (Schauder, S., Shokat, K., Surette, M.G. and Bassler, B.L. (2001) The LuxS family of bacterial autoinducers: Biosynthesis of a novel quorum sensing signal molecule. Mol. Microbiol. 41: 463-476; Schauder, S. and Bassler, B.L. (2001) The languages of bacteria. Genes Dev. 15: 1468-1480; Sperandio, V., Torres, A.G. and Kaper, J.B. (2002) Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol. Microbiol. 43: 809-821.)

These special form of quorum sensing allows the bacteria to communicate between different species. Unlike the conventional one Quorum Sensing produces different types of bacteria a similar Signal. The receptors are proteins of the LuxP type. LuxP is as soluble Protein in the periplasmic space and binds AI-2. The complex from LuxP and AI-2 mediated over a two-component system sends a signal to the cell interior.

According to the invention for the detection of microorganisms the ligands produced by microorganisms be secreted in the process of quorum sensing, by physical or physical-chemical methods detected.

To become the components (receptor or ligand) of the bacterial quorum Sensing Systems used for the functionalization of surfaces of substrates. Such surfaces can also the surfaces at the same time be of sensors, so that significantly advantageous embodiments result. Quorum sensing is also for eukaryotes, such as Yeasts are described, with some of the basic mechanisms still unclear. But here is a corresponding use receptor and ligand of the quorum sensing system possible.

to Functionalization of the surface of the substrate are ligands signal molecules such as species-specific autoinducer, for example, autoinducer-1, or autoinducer of interspecies communication and corresponding receptors used. It is one of the ligands the signaling molecule autoinducer-2 (AI-2) or another autoinducer-2 (AI-2) receptor binding molecule.

at the signaling molecules On the one hand, it can be about the authentic, of the particular organism delivered molecules which are either formed directly by the microorganism itself or heterologously expressed by a host organism or chemically synthesized. It can also be about any other type of biologically and / or chemically synthesized molecule, which with a corresponding quorum sensing receptor or a derivative a quorum sensing receptor can interact.

Examples of species-specific autoinducers and their respective receptors are based on Gram-negative bacteria on the LuxI / LuxR system (Swift, S., Karlyshev, AV, Fish, L., Durant, EL, Winson, MK, Chhabra, SR, Williams , P., Macintyre, S. and Stewart, GSAB (1997) Quorum Sensing in Aeromonas hydrophila and Aeromonas salmonicida: Identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules J. Bacteriol 179: 5271- 5281). LuxI refers to autoinducer synthase and LuxR refers to the autoinducer receptor. The autoinducer-1 are formed from acylated homoserine lactones (AHL). LuxI is crucial for education. The autoinducer formed differ mainly in the length of their acyl chains and / or their degree of saturation. The highly specific recognition of the respective autoinducer-1 is ensured by a corresponding receptor. So far, quorum sensing systems have been identified in over 50 species (DeLisa, MP and Bentley, EB (2002) Bacterial autoinduction: looking outside the cell for new metabolic engineering targets, Microb Cell Fact 1: 5). Any of the corresponding receptors or autoinducer-1 or corresponding derivatives of receptor or autoinducer are suitable for the method of the invention.

In Gram-positive bacteria are processed post-translationally instead of AHL Peptide signaling molecules as autoinducer used (de Kievit, T.R. and Iglewski, B.H. (2000) Bacterial quorum sensing in pathogenic relationships. Infect. Immune. 68: 4839-4849; Winans, S.C. and Bassler, B.L. (2002) Mob Psychology. J. Bacteriol. 184: 873-883). The receptor is a membrane-bound histidine kinase of a Two-component signaling system.

Also Components of this system can be used according to the invention become. As an example, the signal transduction in Staphylococcus aureus (de Kievit, T.R. and Iglewski, B.H. (2000) Bacterial quorum sensing in pathogenic relationships. Infect. Immune. 68: 4839-4849; Winans, S.C. and Bassler, B.L. (2002) Mob Psychology. J. Bacteriol. 184: 873-883). The signal peptide becomes the precursor peptide of the agrD gene product processed. The receptor is the membrane-bound AgrC protein.

According to the invention Use of AI-2 or another binding to an AI-2 receptor molecule or the use of the LuxP receptor or its homologs, such as for example, the LuxP receptor from Vibrio harveyi, beneficial. By using the quorum sensing components of the interspecies communication of Microorganisms is a simultaneous detection of many different Bacterial species possible.

Become additionally to the signal molecule AI-2 or the molecule binding to an AI-2 binding receptor species-specific Ligands or the corresponding receptors used is suitable the detection system, for example, also for selective monitoring certain pathogenic microorganisms and their activity in the medical, pharmaceutical and food-chemical sector.

The for the functionalization of the sensor surface used receptors of the quorum sensing system are preferably recombinant Protein synthesis in microorganisms such as Escherichia coli. Therefor are the corresponding genes of z. Genomic DNA or c-DNA PCR amplified, prepared as synthetic genes or equivalent Fusion genes produced by suitable recombinant DNA techniques and into suitable expression vectors, for example pET-23, Novagen, cloned and optionally with a protein tag for purification or optionally provided connection. After expression of the proteins the purification of the proteins takes place, for example by means of affinity chromatography on Ni-NTA columns (Qiagen). Alternatively, too other expression systems or host organisms such as Yeasts are used.

to Immobilization is the receptor via a suitable linker bound to the substrate surface. The immobilization on the substrate surface is done so that the functionality of the receptor not impaired becomes. The binding of the receptor proteins preferably takes place via the Known methods for binding proteins to substrate surfaces such as z. In Cao, L. Curr Opin Chem Biol. 2005 Apr; 9 (2): 217-26.

suitable connectivity offer, for example, the following reaction pathways: At a surface of the gold substrate, the linker is attached to the gold surface via a thiol, for example, 11-mercaptoundecanoic acid, and goes over one by N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimides (NHS) activated carboxyl group has an amide bond with primary amines of the protein. For substrates with metal oxide surfaces, the Protein over an epoxy group-presenting silane, z. B. 3-glycidyloxypropyl-trimethoxysilane, bound to the substrate surface, that with the primary Amines of the protein secondary Forms amine bonds.

The be used for functionalization of the substrate surface signal molecules chemically synthesized, preferably according to Semmelhack MF, Campagna SR, Federle MJ, Bassler BL. "At expeditious synthesis of DPD and boron binding studies ", Org. Lett. 2005 Feb 17; 7 (4): 569-72.

To immobilize the chemically synthesized signal molecules, the autoinducer molecule is, for example, via a homobifunctional crosslinker, z. B. 1,4-butanediol diglycidyl ether, bound to the substrate which undergoes ether bonding with hydroxyl groups of the autoinducer. The bond to the surface of the substrate is carried out by the binding of the crosslinker to amino groups on the substrates, cystamine monolayer on gold surfaces or aminosilanes or amino polymers on oxide surfaces. The autoinducer molecule can also be bound to the gold surface of the substrates with EDC / NHS-activated 11-mercaptoundecanoic acid.

Around to prevent non-specific binding of molecules to the substrate surface, becomes an antifouling molecule simultaneously with the protein or autoinducer, for example Hexaethylene glycol, bound to the substrate surface. The antifouling molecule blocks the surface areas not occupied by the protein or autoinducer his special free end groups so that there are no further ones molecules can connect.

advantageous Embodiments of the invention are in the claims 2 to 18 and 20-26.

At a surface a substrate is according to the embodiment of the claim 2 partially bound at least one ligand become receptors bound to ligands and bind receptors of microorganisms in the Process of quorum sensing issued and / or already in the medium existing ligands. These are receptors with ligands of the medium replaced. This replacement is metrologically by the changes caused thereby of charge, mass, optical properties, magnetic Characteristics, conformation or enzymatic activity.

At a surface of a substrate are according to the embodiment of the claim 3 partially bound at least receptors and of microorganisms in the process of quorum sensing and / or already existing in the medium signaling molecules of the medium bound to receptors, with a physicochemical change done so that metrologically the changes caused thereby the charge, the mass, the conformation, the optical properties, the magnetic properties, the charge distribution or the enzymatic activity be recorded.

at the change of the optical properties Also dyes are coupled.

Of the Biosensor is advantageously according to the embodiment of claim 4 by addition of particles, for. B. of particles of defined mass, calibrated to the ligands or receptors. Preferably, the particles defined mass metal clusters, especially gold clusters. A Calibration can also by predetermined particle concentrations of the same or different types of particles, for. B. defined by adding Quantities of AI-2.

The surface of the substrate with signal molecules and thus bound receptors according to the embodiment of the claim 2 is advantageously according to the embodiment of claim 5 regenerated by a repeated coating with the receptor.

to increase the sensitivity are according to the embodiment of claim 6 for generation of fusion proteins by recombinant techniques amino acid sequences of Receptors so changed that a change of charge, mass, light, conformation or enzymatic reaction takes place, and / or a change of charge, mass, light, conformation or enzymatic reaction done chemically or physically. This is how fusion proteins become Base generated by quorum sensing receptors, their properties by means of Recombinant techniques changed so are that detected by the type of sensor used signals as charge, mass, conformation, light, magnetic field strength, enzymatic Reaction changed become. The merger motive leads to a measurable change the signals to be detected specific to the respective physicochemical Sensor.

To the development of claim 7 receptors with the Fusion of charged peptides to fusion proteins with altered Net charge changed. This training is particularly suitable for sensor types used on the charge change based. The fusion preferably takes place at the DNA level by means of suitable recombinant DNA techniques. A charge change can also be done chemically. After the development of the claim 8 preferably contain the peptides at positive net charge Arginine, histidine or lysine and at negative net charge aspartic acid or Glutamic acid.

To The development of claim 9 are receptors with the Fusion of proteins or peptides altered so that the mass of the fusion protein significantly increased becomes. This development is particularly suitable for sensor types, based on the detection of mass changes. The fusion takes place for example with the β-galactosidase from E. coli. The fusion is preferably carried out at the DNA level by means of suitable recombinant DNA techniques. The mass change can also be different, z. B. done chemically. For example, a mass change may be via biotinylation.

According to the embodiment of claim 10, receptors are associated with the fusion of proteins or peptides that have intrinsic fluorescence or which enzymatically cause a light reaction by addition of suitable substrate molecules. This development is particularly suitable for sensor types whose measuring principle is based on the detection of qualitative or quantitative changes in the emission of incident light. The fusion occurs z. Example, with proteins or peptides which have an intrinsic fluorescence, for example, green fluorescent protein or its derivatives, or which are capable of causing a light reaction enzymatically by the addition of suitable substrate molecules, for. B. luciferase. Furthermore, all enzyme reactions can be used, which are to be monitored metrologically. The fusion is preferably carried out at the DNA level by means of suitable recombinant DNA techniques. The mergers can also be chemical.

While the mentioned modifications specific to the respective sensor type are, a general signal amplification after the training of claim 11 achieved by adding receptors in their amino acid sequences to be changed that affinity between signal molecule and Receptor enlarged or is reduced. These are for binding the autoinducer important amino acids specifically by suitable recombinant DNA techniques (eg, direct mutagenesis by PCR).

there can Receptors in their amino acid sequences so changed be that binding affinity between signaling molecule and receptor for the respective sensor types is optimized. This can be done through change individual amino acids of the receptor, which has an increased or decreased affinity of binding of the signaling molecule require. Likewise, combinations of several are possibly changed amino acids apply.

It can also changes be made, indicating the steric orientation of the receptor influence.

To the Detection of a small number of signaling molecules by microorganisms will be sent out in the process of quorum sensing, after the training of claim 12 by recombinant techniques an additional Added protein to receptors, so that fusion proteins are generated. Either the fusion proteins or the signaling molecules be over sensitive to the enzymatic activity the fusion protein acting amino acid or nucleic acid sequences or Lipids to the surface bound the substrate, so that upon release of a receptor fusion molecule, the corresponding activity effective and self-enhancing detachment of the fusion proteins or the signal molecules initiated by the substrate.

at the release of a receptor fusion protein by the binding to a signal molecule secreted by microorganisms is the corresponding enzymatic activity is effective. After replacement of the surface of the substrate, the released receptor fusion protein acts enzymatically on still bound receptor molecules or signaling molecules and release these from this surface from. A single or a few signal molecules lead to the triggering of a self-reinforcing Cascade of detachments from the surface of the substrate. In this way, detection of threshold values becomes Processes through the use of quorum sensing possible. Advantageously the extra Protein according to the embodiment of claim 13 a proteolytic or nucleolytic or lipolytic function, e.g. A protease or restriction endonuclease or lipase.

to Generation of a signal cascade occurs a fusion of the receptors with proteins or peptides that have a specific nuclease or protease activity have, for. A restriction endonuclease or the TEV protease. At the same time, the binding of the receptor or the signal molecule to the surface of the substrate a linker, consisting in the first case of double-stranded DNA with the recognition sequence for the particular restriction endonuclease, or in the second case a peptide linker with the recognition sequence for the particular protease. To replacement the fusion protein consisting of receptor and endonuclease or protease, this acts on the substrate-bound linker sequences and thus amplifies the signal. The fusion is preferably on DNA level using appropriate recombinant DNA techniques.

To The development of claim 14 are advantageously on a surface a substrate partially signal molecules and it with fluorescent markers attached receptors bound.

there can according to the embodiment of claim 15, the fluorescent marker be of one species or of different species. advantageously, are also several fluorescent markers of different fluorescence on a surface detectable.

Preferably become after the development of claim 16 either fluorescent proteins or Quantum Dots.

This allows different microorganisms z. B. be detected in mixed cultures. The connection of the alternative fusion constructs to the optical sensor takes place via the respectively corresponding signal molecule. Dependent Depending on the particular sensor principle, the immobilization of the specific autoinducers or receptors is locally defined or undefined.

On This way, simultaneous detection of quorum sensing activities is more diverse Microorganisms on one and the same sensor platform possible.

To the development of claim 17 is the signal molecule an autoinducer-2 (AI-2) and / or another signal molecule or at least one combination it.

Of the Receptor is according to the embodiment of claim 18 either a LuxP molecule or a genetically engineered derivative.

In addition to changes, which are carried out at the receptor, also the respective signal molecule can be changed, to provide evidence depending on to optimize each sensor. So z. B. for mass-selective sensors the Mass of the signaling molecule preferably increased be by chemically molecules to the signaling molecule be coupled. For optical sensors can be coupled according to dyes. Charge detectors can charge created or changed become.

Also can the binding affinity be changed between receptor and signal molecule via modifications to the signaling molecule. So can modified with the receptor interacting groups. Also may be useful in the case of using AI-2 as a ligand, instead of boron to integrate another element into the signaling molecule, the binding properties or the steric orientation of the To influence the receptor.

The Substrate is advantageously according to the embodiment of claim 20 a piezoelectric transducer. In the piezoelectric transducer is the piezoelectric effect for converting force or pressure into one used electrical voltage. The occurring charges are the acting force proportional. The charges can be detected as voltage, so that the substrate itself is a mass sensor. amendments the acting forces lead directly to corresponding charge and resulting detectable voltage changes. Piezoelectric transducers are advantageously suitable for dynamic Force measurements even with very high frequencies, so that too fast Force changes over the equivalent voltage changes can be measured. With increasing resonance frequency of the piezoelectric transducer increases the sensitivity to mass changes at. This is a very sensitive biosensor for the detection of Microorganisms realized as a microbalance.

Depending on used sensor principle can the quorum sensing components used for functionalization specifically selected become. For the sensor system by means of microbalances of a piezoelectric transducer For example, it makes sense to compare the autoinducer with its immobilize to the receptor significantly lower mass on the sensor surface and the mass change that by the detachment of the receptor caused by the signal molecules immobilized on the substrate surface is going to detect.

The Substrate is according to the embodiment of claim 21 a Electrode of a controllable semiconductor component or of a capacitor, so that in each case a charge sensor is realized.

To the one is that a controllable semiconductor element advantageously in the form of a field effect transistor, wherein a charge carrier transport only by majority carrier takes place. The binding, redistribution and release of charges on the substrate surface leads to a change to be measured of the conductive Channels in the field effect transistor.

To the another is a capacitor, whereby the binding and immobilization of the receptor or the autoinducer to a change in electrical energy in the electric field between the electrodes as a two-terminal of a capacitor leads.

The Substrate is a photodetector according to the embodiment of claim 22 and / or an opto-electrical component. Occurring fluorescence changes can over one Fluorescence spectrometer can be measured directly as a photodetector. With a coupling of an opto-electric device, the effect the fluorescence is triggered or reinforced become.

The Substrate is according to the embodiment of claim 23, a surface plasmon resonance spectrometer as an electron density sensor. An advantage is the high sensitivity the plasmon resonance frequency versus changes in the integral electron density the immobilized protein coating and the good integration possibility in a microfluidic system.

On the substrate is located after the development of the claim 24 a gold electrode array as an electrochemical sensor, making changes in the protein coating over the current-voltage characteristic in a suitably selected reference medium can be detected immediately.

According to the embodiment of claim 25, the substrate is a component of a magnetic component. The substrate is here used as a switching, amplifying or storage device. It is advantageously part of a logical circuit structure. Another advantage is that such a device can also be connected via logic links from the outside. Thus, for example, the initial state can be reached again.

The Substrate is a calorimeter according to the embodiment of patent claim 26 for enzymatic reactions, that the temperature changes formed in the enzymatic reaction detected. The heat output is correlated with the number of bound enzymatically active Receptor molecules.

Based the following figures and embodiments the invention will be closer explained.

there demonstrate

1 : Immobilization of the receptor on the substrate surface

2 : Immobilization of the autoinducer on the substrate surface

1st embodiment

1 : The receptor or corresponding derivatives ( 2 ) is applied to a surface of the substrate ( 1 ) chemically immobilized. Autoinducer molecules ( 3 ) bind to the receptors or their derivatives. The resulting physical or physicochemical changes are detected by sensors.

For producing a biosensor from a LuxP protein as a receptor ( 2 ) on the surface of the substrate ( 1 ), the gene coding for the LuxP by means of appropriate primers of z. B. genomic DNA PCR amplified. The PCR fragment is purified and cloned into an expression vector, for example pET-23 or Novagen, for E. coli and the sequence of the gene is verified by sequence analysis. By cloning into the vector, the receptor is provided C-terminal with a histidine tag. After expression of the gene, the receptor protein is purified by affinity chromatography on Ni-NTA columns (Qiagen).

The LuxP protein thus obtained is applied via a suitable linker to the surface of the substrate ( 1 ), which is the inorganic surface of the substrate ( 1 ) covalently connects to the respective biomolecule.

• – bei einer Substratoberfläche aus Gold wird der Linker über ein Thiol an die Goldoberfläche gebunden, zum Beispiel 11-Mercaptoundecansäure, und geht über eine durch N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimid (EDC) und N-Hydroxysulfosuccinimide (NHS) aktivierte Carboxylgruppe eine Amidbindung mit primären Aminen des Proteins ein, oder
• – bei Substraten mit Metalloxid-Oberflächen wird das Protein über ein Epoxidgruppen präsentierendes Silan, zum Beispiel 3-Glycidyloxypropyl-Trimethoxysilan, an die Substratoberfläche gebunden, das mit den primären Aminen des Proteins sekundäre Aminbindungen ausbildet.

The binding of the receptor proteins preferably takes place via known methods for binding proteins to substrate surfaces. Suitable connection options are, for example

• With a substrate surface of gold, the linker is attached via a thiol to the gold surface, for example 11-mercaptoundecanoic acid, and passes through an N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) activated carboxyl group, an amide bond with primary amines of the protein, or
• For substrates with metal oxide surfaces, the protein is attached to the substrate surface via an epoxide group-presenting silane, for example, 3-glycidyloxypropyl-trimethoxysilane, which forms secondary amine bonds with the primary amines of the protein.

2nd embodiment

2 : Autoinducer or corresponding derivatives ( 3 ) are attached to the surface of the substrate ( 1 ) chemically immobilized. Corresponding receptor molecules or their derivatives ( 2 ) are bound to this surface via the autoinducer. Autoinducer molecules ( 4 Compete for the binding to the receptors or their derivatives and lead to their replacement. The resulting physical or physicochemical changes are detected by sensors.

For producing a biosensor from a on the surface of the substrate ( 1 ) immobilized signal molecule or its derivatives ( 3 ) and a receptor coupled thereto or its derivatives ( 2 ), the autoinducer is chemically synthesized and attached to this surface via a suitable linker. The linker covalently connects the inorganic surface to the corresponding autoinducer.

Of the Autoinducer can over a homobifunctional crosslinker, for example 1,4-butanediol diglycidyl ether, bound to the substrate with hydroxyl groups of the autoinducer Etherbindungen received. The binding to the substrate surface takes place presenting by binding of the crosslinker to amino groups Layers on the substrates, cystamine monolayer on gold surfaces or Aminosilanes or amino-polymers on oxide surfaces.

The autoinducer can also bind to EDC / NHS-activated 11-mercaptoundecanoic acid gold surface the substrates are bound.

The coding for the LuxP gene is determined by means of appropriate primers of z. B. genomic DNA PCR amplified. The PCR fragment is purified and cloned into an expression vector, for example pET-23 or Novagen, for E. coli and the sequence of the gene is analyzed by sequence analysis fied. By cloning into the vector, the receptor is provided C-terminal with a histidine tag. After expression of the gene, the receptor protein is purified by affinity chromatography on Ni-NTA columns (Qiagen).

The protein thus obtained ( 2 ) is via the autoinducer ( 3 ) on the surface of the substrate ( 1 ). Autoinducer molecules ( 4 Compete for the binding to the receptors and lead to its replacement, resulting in physical or physicochemical changes. These are detected by sensors.

The substrate ( 1 ) of the embodiments is advantageously simultaneously a sensor.

In a first embodiment, the substrate ( 1 ) a piezoelectric transducer. The converter itself is a transducer element made of a piezoceramic provided with electrodes. The transducer element itself constitutes a plate-shaped or foil-like body. The electrodes of the transducer element are known to be connected via an amplifier with a high-impedance input to a voltmeter, which is at the same time a component of a data processing device. Thus, the attachment and detachment of receptors on the surface of the transducer element via the associated mass change on this surface can be measured as measured values. The transducer element acts as a microbalance. Advantageously, these measured values are stored in the data processing device, so that an evaluation according to the most diverse criteria can be carried out easily.

In a second embodiment, the substrate ( 1 ) the gate as an electrode of a field effect transistor. By attaching and detaching receptors at the gate equivalent charge changes take place, so that the channel resistance between source and drain changes equivalently. This resistance change is measured as a current or voltage change and detected, stored and displayed via an associated data processing device.

In a third embodiment, the substrate ( 1 ) at least one of the electrodes of the capacitor. The culture medium simultaneously acts as a dielectric between the electrodes of the capacitor. The attachment and detachment of receptors leads to charge changes and / or charge shifts at the electrodes, which can be measured by the application of an alternating voltage. The capacitor acts as an AC resistance. The capacitor is advantageously interconnected to a data processing device. The capacitor itself can be designed as a plate, ball or cylinder capacitor.

In a fourth embodiment, the substrate ( 1 ) a photodetector and / or an opto-electrical device. The photodetector is advantageously an optoelectronic semiconductor component, wherein free charge carriers are generated by absorption of light. Such photodetectors are in particular the photoresistor, the photodiode, the phototransistor and the photothyristor. The opto-electric device is a known light-emitting diode, which may also be a laser diode. The photodetector and the optoelectronic component are interconnected with a data processing device for driving and detection. In a first variant, the photodetector and the optoelectronic component are arranged at a distance from one another, the culture medium being arranged between them. In a second variant, the photodetector and the optoelectronic component are arranged in a plane next to one another. Spaced apart is a light predominantly reflective component. The culture medium is located between on the one hand the photodetector and the opto-electrical component and on the other hand the component.

In a fifth embodiment, the substrate 1 a surface plasmon resonance spectrometer as an electron density sensor, which is characterized by a high sensitivity of the plasmon resonance frequency to changes in the integral electron density of the immobilized protein coating and the good possibility of integration into a microfluidic system.

In a sixth embodiment is located on the substrate 1 a gold electrode array as an electrochemical sensor, so that changes in the protein coating can be detected directly via the current-voltage characteristic in a suitably selected reference medium.

In a seventh embodiment, the substrate is 1 a calorimeter for enzymatic reactions, which detects the temperature changes produced during the enzymatic reaction. The calorimeter is interconnected with a data processing device.

The Data processing devices of the embodiments are advantageously known computer.

1

Surface of the substrate

2

receptor

3

signaling molecule

4

from Microorganisms released in the process of quorum sensing and thus in the medium existing signal molecule

Claims (26)

Method for the detection of microorganisms and / or their activity with biosensors, characterized in that on a surface of a substrate partially at least one ligand for binding a receptor or at least one receptor for a ligand or at least one ligand for binding a receptor and at least one receptor for either the or another ligand is immobilized chemically, physically or biologically, wherein the ligands and the receptors are components of the quorum sensing system of microorganisms, being used as ligands signal molecules and receptor receptors corresponding to the signal molecules and wherein one of the ligands the signal molecule is autoinducer-2 (AI-2) or another molecule which binds to an autoinducer-2-binding receptor, and wherein physical or physical receptors are caused by binding of ligands emitted by microorganisms in the process of quorum sensing ischemic changes are detected. Method according to claim 1, characterized that on a surface a substrate is bound in regions at least one ligand, receptors are bound to ligands that are receptors of Microorganisms released in the process of quorum sensing and / or already bind ligands present in the medium, with receptors with ligands be replaced by the medium, and that the detachment metrologically by the resulting changes in the charge, the mass, the conformation, the optical properties, the magnetic Characteristics or enzymatic activity is detected. Method according to claim 1, characterized that on a surface a substrate partially bound at least receptors, that emitted by microorganisms in the process of quorum sensing and / or signal molecules already present in the medium at receptors be bound, whereby a physicochemical change takes place, so that metrologically the resulting changes the charge, the mass, the conformation, the optical properties, the magnetic properties, the charge distribution or the enzymatic activity be recorded. Method according to claim 1, characterized that the biosensor by attaching particles to the ligands or receptors is calibrated. Method according to claim 2, characterized that the surface of the substrate with signal molecules and associated receptors by a repeated coating is regenerated with the receptor. Method according to claim 1, characterized that for the production of fusion proteins by means of recombinant techniques or chemical amino acid sequences so changed by receptors be that change of charge, mass, light, conformation or enzymatic reaction done and / or that a change of charge, mass, light, conformation or enzymatic reaction done chemically or physically. Method according to claim 6, characterized that receptors with the fusion of charged peptides to fusion proteins with changed Net charge changed become. Method according to claim 7, characterized that the peptides are preferably arginine, histidine or lysine for a positive Net charge or that the peptides aspartic acid or glutamic acid for a negative Net charge include. Method according to claim 6, characterized that receptors are altered with the fusion of proteins or peptides, that the mass of the fusion protein is significantly increased. Method according to claim 6, characterized that receptors with the fusion of proteins or peptides that have an intrinsic fluorescence or enzymatically by addition of suitable substrate molecules cause a light reaction, be changed. Method according to claim 2, characterized that receptors in their amino acid sequences so changed be that affinity between signaling molecule and receptor enlarged or is reduced. A method according to claim 1, characterized in that by means of recombinant techniques, an additional protein is added to receptors, so that fusion proteins are generated, and that either the fusion proteins or the signal molecules via sensitive to the enzymati amino acid or nucleic acid sequences or lipids are bound to the surface of the substrate so that upon release of a receptor fusion molecule, the corresponding activity is effectively initiated and a self-enhancing detachment of the fusion proteins or signal molecules from the substrate. Method according to claim 12, characterized that extra Protein has a proteolytic, lipolytic or nucleolytic function having. Method according to claim 1, characterized that on a surface a substrate partially bound at least one signal molecule and that fluorescently tagged receptors are bound to signal molecules become. Method according to claim 14, characterized that the fluorescence markers are of one species or different Varieties are. Method according to claim 14, characterized that the fluorescent markers are either fluorescent proteins or quantum Dots are. Method according to claim 1, characterized that the signaling molecule an autoinducer-2 (AI-2), and / or another signaling molecule or at least a combination of it is. Method according to claim 1, characterized that the receptor is either a LuxP molecule or a genetically engineered one Derivative is. Device for detecting microorganisms and / or their activity as a biosensor, characterized in that on a surface of a substrate partially at least one ligand for binding a receptor or at least one receptor for a ligand or at least one ligand for binding a receptor and at least one receptor for either the or another ligand chemically, physically or is biologically immobilized, wherein the ligands and the receptors Components of the quorum sensing system of microorganisms are, wherein as ligands signal molecules and as receptors corresponding receptors for the signal molecules used and wherein one of the ligands is the signal molecule autoinducer-2 (AI-2) or another autoinducer-2 binding receptor binding molecule is, and being linked by ligands, by microorganisms in the process of quorum sensing, and receptors caused physical or physico-chemical changes are measurable. Device according to claim 19, characterized the substrate is a piezoelectric transducer. Device according to claim 19, characterized the substrate is an electrode of a controllable semiconductor component or a capacitor. Device according to claim 19, characterized that the substrate has at least one photodetector and / or at least an opto-electric device. Device according to claim 19, characterized that the substrate is a surface plasmon resonance spectrometer as Electron density sensor is. Device according to claim 19, characterized that on the substrate a gold electrode array as electrochemical Sensor is located. Device according to claim 19, characterized that the substrate is a component of a magnetic component is. Device according to claim 19, characterized that the substrate is a calorimeter for enzymatic reactions, that the temperature changes formed in the enzymatic reaction detected.