WO2004099430A2 - Substrat als träger von ligaten - Google Patents
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- WO2004099430A2 WO2004099430A2 PCT/EP2004/004906 EP2004004906W WO2004099430A2 WO 2004099430 A2 WO2004099430 A2 WO 2004099430A2 EP 2004004906 W EP2004004906 W EP 2004004906W WO 2004099430 A2 WO2004099430 A2 WO 2004099430A2
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- substrate
- ligates
- ligands
- test sites
- substrate according
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
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- B01J2219/0061—The surface being organic
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- B01J2219/00614—Delimitation of the attachment areas
- B01J2219/00617—Delimitation of the attachment areas by chemical means
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- B01J2219/0063—Other, e.g. van der Waals forces, hydrogen bonding
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- B01J2219/00722—Nucleotides
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
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- C12Q1/6825—Nucleic acid detection involving sensors
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
Definitions
- Substrate as a carrier for ligates
- the invention relates to a substrate for use as a carrier for ligates.
- dynamic ranks of a sensor means the area in which the sensor reproducibly and specifically reacts to changes in the concentration of a particular analyte.
- the “dynamic ranks” of a sensor is generally about a factor of 10 to 100 in Analyte concentration and is limited to small concentrations by the sensitivity of the detection method. For high concentrations, the sensor becomes saturated from a certain range, so that a further increase in the analyte concentration does not cause a signal change.
- the object of the invention is to provide a sensor which makes it possible to detect the concentration fluctuations of constituents of an analyte liquid in parallel, which constituents can be present in the test substance in concentrations that differ by orders of magnitude.
- characteristic parameters of the active areas of the sensor surface such as occupancy parameters e.g. the geometric area of the test sites or their coverage with ligates.
- the characteristic occupancy parameter defines the number of the respective ligates on the sensor surface and thus also the number of association events for a given analyte concentration via the association constant.
- Fluorophore chemical compound that is able to emit a longer-wave (red-shifted) fluorescent light when excited with light.
- Fluorophores fluorescent dyes
- UV ultraviolet
- VIS visible
- IR infrared
- the absorption and emission maxima are typically shifted from each other by 15 to 40 nm (Stokes shift).
- Ligand Term for molecules that are specifically bound by the ligate examples are substrates, cofactors or coenzymes of a protein (enzyme), antibodies (as ligand an antigen), antigens (as ligand of an antibody), receptors (as ligand of a hormone), hormones (as ligand of a receptor) or nucleic acid oligomers (as ligand of the complementary nucleic acid oligomers).
- Ligate Term for (macro) molecule with specific recognition and binding sites for the formation of a complex with a ligand examples include substrates, cofactors or coenzymes of a protein (enzyme), antibodies (as ligate of an antigen), antigens (as ligate of an antibody), receptors (as ligate of a hormone), hormones (as ligate of a receptor) ) or nucleic acid oligomers (as a ligate of the complementary nucleic acid oligomers).
- Probe Biomolecules applied to the sensor surface that can specifically bind one or more molecules from the test substance (targets).
- the chains are alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl.
- Preferred spacers are those of chain length 1-20, in particular chain length 1-14, the chain length being the shortest continuous connection between the structures to be connected.
- the test site each carry one or more types of probe molecules, each of which can specifically bind one or more molecules of a test substance.
- the size of these areas and their surface coverage with probes can be optimized to the order of magnitude of the target concentration.
- Target molecules in the test substance that can bind specifically to one or more biomolecules on the sensor surface (probes).
- These spacers can in turn be bound to various reactive groups which are naturally present on the nucleic acid oligomer or are attached to it by modification.
- "N" is any integer, in particular a number between 1 and 20.
- the spacer for connecting the disulfide function to the nucleic acid oligomer can each have a different chain length (shortest continuous connection between disulfide function and nucleic acid oligomer), in particular any chain length between 1 and 14. These spacers can in turn be connected to different ones naturally existing on the nucleic acid oligomer or attached to it by modification attached reactive groups.
- the placeholder "n” is any integer, in particular a number between 1 and 20.
- oligo-spacer-SS- Two identical or different nucleic acid oligomers, spacer oligo, which are connected to one another via a disulfide bridge, the disulfide bridge being connected to the nucleic acid oligomers via any two spacers and the two spacers having a different chain length (shortest continuous connection between disulfide bridge and the respective nucleic acid oligomer), in particular any chain length between 1 and 14.
- These spacers can in turn be bound to various reactive groups that are naturally present on the nucleic acid oligomer or are attached to them by modification.
- PNA Peptide nucleic acid synthetic DNA or RNA in which the sugar-phosphate unit is replaced by an amino acid.
- synthetic DNA or RNA synthetic DNA or RNA in which the sugar-phosphate unit is replaced by an amino acid.
- the sugar-phosphate unit is replaced by the -NH- (CH 2 ) 2 -N (COCH 2 base) -CH 2 CO unit hybridizes PNA with DNA).
- Nucleic acid At least two covalently linked nucleotides or at least two covalently linked pyrimidine (e.g. cytosine, thymine or uracil) or purine bases (e.g. adenine or guanine).
- the term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as e.g. on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous structures (e.g. phosphoramide, thio-phosphate or dithio-phosphate backbone).
- An essential feature of a nucleic acid in the sense of the present invention is the sequence-specific binding of naturally occurring cDNA or RNA.
- Nucleic acid - nucleic acid of unspecified base length e.g. oligomeric nucleic acid octamer: a nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bound to one another.
- Oligomer equivalent to nucleic acid oligomer Oligomer equivalent to nucleic acid oligomer.
- Oligonucleotide equivalent to oligomer or nucleic acid oligomer e.g. a DNA, PNA or RNA fragment of unspecified base length.
- Oligo Abbreviation for oligonucleotide Oligo Abbreviation for oligonucleotide.
- the present invention relates to a substrate for use as a carrier for ligates in a method for the detection of ligate-ligand association events.
- Test sites which have ligates bound to the surface are arranged on the substrate. At least two types of test sites are provided, the individual test sites being occupied with different types of ligates. These different types of ligates each detect complementary types of ligands which are present in an analyte solution in different concentration ranges.
- the test sites have a characteristic occupancy parameter, so that the ligands can be detected in the concentration range in which the respective ligand is present in the analyte solution.
- a sensor with a given number of specific coupling points reaches a saturation value from a certain analyte concentration in the test substance, so that a further increase in the concentration can no longer be detected.
- This can theoretically be described with first-order binding kinetics for the binding of a probe S on the sensor surface and a target T in the test substance to form a surface complex ST with an association constant K:
- the present invention provides a sensor with a "dynamic ranks" optimized with regard to the analysis of analyte liquids which contain analytes in very different concentrations.
- the present invention is based on the idea that by optimizing the "dynamic range" of a sensor, changes in the concentrations of constituents of a liquid test substance can also be detected in parallel if the initial concentrations of these constituents vary by many orders of magnitude.
- the substrate of the invention is preferably used as a carrier of biomolecules in a method for the electrochemical or fluorescence spectroscopic detection of components of an electrolyte solution.
- the substrate according to the invention can also be used in an electrochemical or fluorescence spectroscopic method for the detection of biomolecules.
- the present invention describes a sensor with spatially limited areas of different probe molecules (spots), each of which can specifically bind one or more target molecules from a test substance.
- the size and / or surface coverage of the probes (characteristic coverage parameters of the substrate) of the spots of the invention are optimized for the concentration ranges of the corresponding targets to be detected in such a way that the proportion of binding events of all spots for, for example, the non-pathogenic state is independent of the actual concentration of the spots Targets is roughly the same. This leaves the specific "dynamic ranks" of a sensor normalize to this "initial state".
- the advantage of this method lies in the adjusted sensitivity of all spots with regard to the changes in concentration of the corresponding targets to be detected, regardless of their initial concentration.
- the sensor is thus optimized for the “tolerable” concentration range between the “permitted”, non-pathogenic value and the “critical”, pathogenic value of each analyte.
- the composition of the analyte pool of a healthy organism is generally the rule sufficiently known that the present method can be used to provide evidence of a disease (exceeding the "critical" concentration value of an analyte) on the basis of changes in individual analytes.
- ligands are preferably detected which are present in the analyte solution in concentration ranges whose mean values differ by at least a factor of 10.
- the mean values of the concentration ranges in which they are present in the analyte solution differ by at least a factor of 100, particularly preferably by at least a factor of 1000, very particularly preferably by at least a factor of 10,000.
- the present invention also includes the use of the substrates in methods for the detection of ligate-ligand association events.
- the substrates can be used in particular in fluorescence spectroscopic and electrochemical detection methods.
- Chronoamperometry (CA), chronocoulometry (CC), linear sweep voltammetry (LSV), cyclic voltammetry (CSV),retemating current voltammetry (ACV), voltammetry techniques with different pulse shapes, in particular square wave voltammetry (SWV), differential pulse voltammetry are used as electrochemical detection methods (DPV) or Normal Pulse Voltammetry (NPV), AC Impedance Spectroscopy, Chronopotentiometry and Cyclic Chronopotentiometry in question.
- CA Chronoamperometry
- CC chronocoulometry
- LSV linear sweep voltammetry
- CSV cyclic voltammetry
- ACCV alternating current voltammetry
- SWV square wave voltammetry
- DUV electrochemical detection methods
- NPV Normal Pulse Voltammetry
- AC Impedance Spectroscopy
- the characteristic occupancy parameter of the substrate is the size of the area of the individual test sites.
- the area of the test sites preferably differs by at least a factor of 10, particularly preferably by at least a factor of 100, particularly preferably by at least a factor of 1000 and very particularly preferably by at least a factor of 10000.
- substrates which have test site areas between 1 ⁇ m 2 and 1 mm 2 Preference is given to substrates which have test site areas between 1 ⁇ m 2 and 1 mm 2 . Substrates which have test site areas between 10 ⁇ m 2 and 100000 ⁇ m 2 are particularly preferred.
- solid bodies with a freely accessible surface that can be functionalized with biomolecules and wetted with a liquid test substance are suitable as sensor substrates.
- Solid substrates include plastics as well as metals, semiconductors, glasses, composites or porous materials.
- the term surface is independent of the spatial dimensions of the surface. The surface of the sensor must be divisible into separate areas. This can be achieved by structuring the solid-state substrate into active and inactive areas or by partially functionalizing its homogeneous surface.
- the structuring of the solid state substrates into active and inactive areas can be e.g. by lithography, vacuum deposition, electrochemical deposition, doping or laser treatment.
- the structuring on homogeneous substrates can be achieved by applying and structuring passivation layers.
- any material that forms a closed layer on a surface and thus separates the substrate surface from the environment is suitable as a passivation layer. At a later time the material can e.g. can be removed in its entire thickness without residue by laser ablation at the desired locations.
- micro-contact printing ⁇ CP mico-contact printing
- Whitesides 1994 A. Kumar, G.M. Whitesides, Science, 1994, 263, 60
- a microstructured stamp is wetted with a liquid, then brought into direct contact with the substrate to be processed, and a lateral chemical structure is thus imprinted on the surface.
- electrically conductive materials such as platinum, palladium, gold, cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead, aluminum, manganese, any doped or undoped semiconductors and binary or ternary connections are used as surfaces of the sensor substrates.
- electrically conductive materials such as platinum, palladium, gold, cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead, aluminum, manganese, any doped or undoped semiconductors and binary or ternary connections are used as surfaces of the sensor substrates.
- homogeneous electrically conductive surfaces can be structured or conductive materials can be applied to spatially separated areas of a non-conductive substrate, such as glass or plastic, in any thickness.
- insulating carrier plates are used as sensor substrates, which are expediently rigid carrier plates on one side, rigid carrier plates on both sides or rigid multilayer carrier plates.
- the insulating carrier plate can be a one-sided or double-sided flexible carrier plate, in particular made of a polyimide film, or a rigid-flexible carrier plate. It advantageously consists of a base material that is selected from the group BT (bismaleimide triazine resin with quartz glass), CE (cyanate ester with quartz glass), CEM1 (hard paper core with FR4 outer layers), CEM3 (glass fleece core with FR4 outer layers), FR2 ( Phenolic resin paper), FR3 (hard paper), FR4 (epoxy glass hard fabric), FR5 (epoxy glass hard fabric with cross-linked resin system), PD (polyimide resin with aramid reinforcement), PTFE (polytetrafluoroethylene with glass or ceramic), CHn (highly cross-linked hydrocarbons with ceramic) and glass ,
- BT bismaleimide triazine resin with quartz glass
- CE cyanate ester with quartz glass
- CEM1 hard paper core with FR4 outer layers
- CEM3
- carrier plates have a certain number of conductor tracks with a gold surface, which are coated with a solder resist layer as a passivation.
- laser gold ablation is used to burn free gold spots into the lacquer for later functionalization.
- spots of any size and geometry can be written in the lacquer, whereby only the width of the conductor tracks represents a limit.
- laser ablation not only removes the lacquer layer at desired points, but also ensures that the surface of the gold is temporarily melted and the pores are reduced by melting it briefly. By melting the substrates, a few gold layers are additionally ablated from the surface, thus removing impurities.
- the conductor track substrates just described are suitable both for electrochemical measurement methods and for fluorescence spectroscopy. Functionalization of the active areas with ligates
- the active areas of the sensor are functionalized with ligates, which act as probes for the ligands present in the test substance.
- ligates which act as probes for the ligands present in the test substance.
- all types of ligates are suitable for examining analyte liquids for the presence of their specific ligands. Molecules that specifically interact with a ligand to form a complex are referred to as ligates.
- ligates in the sense of the present document are substrates, cofactors or coenzymes as complex binding partners of a protein (enzyme), antibodies (as complex binding partners of an antigen), antigens (as complex binding partners of an antibody), receptors (as complex binding partners of a hormone), hormones (as complex binding partners of a receptor), nucleic acid oligomers (as complex binding partner of the complementary nucleic acid oligomer) or metal complexes.
- the number of probes on the sensor surface scales linearly with the surface, for example the application of probe monolayers under conditions which enable an occupancy of less than the densest packing.
- volume methods for immobilizing the probes, for example via functionalized polymers, are also conceivable as long as the number of probes continues to scale with the area.
- the free substrate sites are preferably wetted with modified nucleic acid oligomers in aqueous solution.
- the nucleic acid oligomer which is to be applied to the free surface is modified via a covalently attached spacer of any composition and chain length with one or more reactive groups, these reactive groups preferably being located near one end of the nucleic acid oligomer.
- the reactive groups are preferably groups that can react directly with the unmodified surface.
- nucleic acid oligomers of the general formula (nx HS spacer) oligo, (nx RSS spacer) oligo or oligo spacer SS -Spacer-oligo, which react with a gold surface to form gold-sulfur bonds, (ii) nucleic acid oligomers with amines that attach to platinum or silicon surfaces by chemical or physical sorption and (iii) nucleic acid oligomers with silanes that form a covalent bond with oxidic surfaces. With these types of attachment of nucleic acid oligomers, deposits of less than the densest packing are generally realized, so that there is sufficient space on the surface for later hybridization.
- HS spacer thiol
- SS disulfide
- the molecule can also be modified with an electrochemical label if necessary via a further spacer of any composition and chain length, if the functionalization of the free substrate sites and the subsequent hybridization using electrochemical
- Oligonucleotides with a redox label can be used electrochemical methods to investigate the hybridization events, if the
- Target molecules are provided with a redox label.
- Another electrochemical detection variant is a displacement assay, in which short-chain signal oligomers are also bound to the unlabeled probe oligomers
- Redox labels of unlabeled target oligomers of the complementary sequence are displaced.
- Transition metal complexes in particular those of copper, iron, ruthenium, osmium or titanium with ligands such as pyridine, 4,7-dimethylphenanthroline, 9,10-phenanthrenequinone diimine, can be used as redox labels of the ligates or ligands, Porphyrins and substituted porphyrin derivatives can be used.
- riboflavin of quinones such as pyrrolloquinoline quinone, ubiquinone, anthraquinone, naphthoquinone or menaquinone or derivatives thereof, of metallocenes and metallocene derivatives such as ferrocenes and ferrocene derivatives, cobaltocenes and cobaltocene derivatives, of porphyrins, methylene blue, hydroquinone derivatives, daaminomycin derivatives Derivatives (para or ortho dihydroxy benzene derivatives, para or ortho dihydroxy anthraquinone derivatives, para or ortho dihydroxy naphthoquinone derivatives) and similar compounds are possible.
- quinones such as pyrrolloquinoline quinone, ubiquinone, anthraquinone, naphthoquinone or menaquinone or derivatives thereof
- metallocenes and metallocene derivatives such as ferrocenes and ferrocene derivative
- ligates or ligands can be given a fluorophore as a second functionalization via a further spacer of any composition and chain length if the functionalization of the free substrate sites and the subsequent hybridization are to be examined with the aid of optical methods.
- fluorescence spectroscopy can also be performed with a fluorophore on the target molecules and unlabeled probes.
- fluorescent dyes such as e.g. Texas Red®, rhodamine dyes, Cy3 TM, Cy5 TM, fluorescein etc. (cf. Fluka, Amersham and Molecular Probes catalog) can be used.
- Two techniques are particularly suitable for the functionalization of the exposed substrate sites.
- small volumes are selectively applied to the spots of the substrate using a commercially available spotter, each spot being able to be functionalized with different molecules.
- all exposed spots can be functionalized with the same probe molecules, for example by immersing the substrate in the probe liquid or by wetting the entire substrate. Varying the surface concentration of the ligates
- the number of probes on the sensor surface can also be set without varying the active spot size. This means that different amounts of probe molecules can be realized on spots of the same size with just one sensor design.
- a particularly preferred embodiment of the present invention is therefore substrates whose characteristic occupancy parameter is the occupancy density of the test sites with ligates.
- the occupancy densities of the test sites with ligates particularly preferably differ by at least a factor of 10, particularly preferably by at least a factor of 100 and very particularly preferably by at least a factor of 1000.
- the assignment can e.g. the incubation time, the number of coupling groups per molecule, the molarity of the loading buffer or the concentration of the molecules in the incubation solution.
- the surface coverage is set using a coadsorbate.
- a suitable coadsorbate is added to the incubation solution of the probe molecules in a certain concentration and brought into contact with the sensor surface, or the coadsorbate is applied in a second coating step after the functionalization with the probes.
- the coadsorbate preferably has the same coupling group as the probe molecule, thus occupies part of the active surface and ensures a reduced surface coverage of the probe.
- the surface coverage can be adjusted via the concentration of the coadsorbate in the respective incubation solution.
- short-chain thiols of the general structure SH- (CH 2 ) n -X are particularly preferred, where X can be any head group.
- Fig. 1 Theoretical curves of the relative proportion of binding events ([TS] / S 0 ) for different target concentrations.
- concentration of the probes on the sensor surface is normalized to the association constant of the binding.
- Fig. 2 Schematic image of a section of the sensor substrates based on circuit board technology, a) top view of the conductor track substrate with free substrate locations of different active area and geometry, b) cross section through a substrate with 3 identical electrode spots.
- the gold sites of the substrate exposed by laser ablation are functionalized with double-modified nucleic acid oligomers, which have a thioi group at one end for binding to the gold surface and at the other end have an electrochemical label (e.g. osmium complexes).
- the desired number of probes on the sensor surface is set either via the electrode size or by using a short-chain thiol of a certain concentration as a coadsorbate.
- the nucleic acid oligomers of the test liquid also have an electrochemical label (eg ferrocene derivatives), so that both the assignment with the nucleic acid oligomers and the hybridization efficiency can be determined using electrochemical methods.
- a preferred measurement method for analyzing occupancy and hybridization efficiency is AC (alternating current) voltammetry. From the ACV current at the redox potential of the label, according to O'Connor et al. (J. Electroanal. Chem., 466, 1999, 197-202) calculate the number of labels involved. The experiments can thus be evaluated quantitatively.
- Example 1 PCB substrates.
- FIG. 2a shows a section of this conductor track picture. The detail shows 4 of the 48 working electrodes (20A to 20D) and part of the counter electrode 28.
- the entire conductor pattern is covered with a 15 ⁇ m to 20 ⁇ m thick passivation layer 22 (FIG. 2 b) made of structurable, optically curable lacquer (2-component solder resist, Elpemer GL 2467 SM-DG, from Peters).
- Recesses 24, 24A to 24D are made in the passivation layer by high-energy pulses from an excimer laser and are used to hold the biomolecules 26.
- a passivation layer with a thickness of 15 ⁇ m to 20 ⁇ m, you need to remove the lacquer and melt it for a short time
- the melting of the surface leads to the closure of surface pores of the gold layer, to a reduction in the surface roughness and, by ablation, fewer gold layers to the removal of surface impurities.
- the laser irradiation of the substrate can take place directly or via an optic or mask and enables recesses of any size and geometry.
- FIG. 2b shows a section through a conductor track substrate with 3 identical spots.
- Each of the conductor tracks 20 consists of a copper core 14 which is continuously covered by a nickel barrier layer 16 and a gold layer 18.
- the copper core has a thickness of approximately 28 ⁇ m. It represents an inexpensive and highly conductive basic component of the conductor tracks.
- the base copper core is coated with a 6 ⁇ m thick, continuous nickel layer as a diffusion barrier. A 2 ⁇ m thick gold layer is applied to this nickel layer.
- the conductor tracks of the exemplary embodiment are approximately 150 ⁇ m wide and are arranged on the carrier plate at a distance of approximately 200 ⁇ m (center-center).
- the working electrodes, the counter electrode and a reference electrode, if provided, are each connected to connection contact surfaces (not shown) of the electrical substrate for contacting.
- the conductor tracks have circular recesses with diameters of 10 ⁇ m, 30 ⁇ m, 100 ⁇ m and rectangular recesses with the dimensions 100 ⁇ m ⁇ 700 ⁇ m (cf. 24A to 24D in FIG. 2a). These recesses thus have areas of 78.5 ⁇ m 2 , 706.5 ⁇ m 2 , 7850 ⁇ m 2 or 70000 ⁇ m 2 , so that the active area of the electrodes is varied by a factor of 1000.
- Example 2 Functionalization of the spots of the substrate with nucleic acid oligomers.
- the free substrate sites of various sizes described in Example 1 are e.g. functionalized with the nucleic acid oligomers via a spotting method.
- the oligonucleotides are synthesized in an automatic oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) in accordance with the synthesis protocols recommended by the manufacturer for a 1.0 ⁇ mol synthesis.
- the oxidation steps are carried out with a 0.02 mol / l iodine solution in order to avoid oxidative cleavage of the disulfide bridge.
- Modifications to the 5'-position of the oligonucleotides are carried out with a coupling step which is extended to 5 min.
- the amino modifier C2 dT (Glen Research 10-1037) is incorporated into the sequences according to the respective standard protocol.
- the coupling efficiencies are determined online during the synthesis via the DMT cation concentration photometrically or conductometrically.
- the oligonucleotides are deprotected with concentrated ammonia (30%) at 37 ° C for 16 h.
- the oligonucleotides are purified using RP-HPL chromatography according to standard protocols (eluent: 0.1 mol / l triethylammonium acetate buffer, acetonitrile), and the characterization is carried out using MALDI-TOF MS.
- the amine-modified oligonucleotides are coupled to the activated redox labels (e.g. osmium complexes) in accordance with the conditions known to the person skilled in the art. The coupling can take place both before and after the oligonucleotides have been bound to the surface.
- the substrates from Example 1 are, for example, modified with double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification one: the phosphate group of the 3' end is with (HO- (CH 2 ) 2 -S) 2 esterified to P- O- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH
- Modification two: at the amino-modified 5 'end is the osmium complex [Os (bipy) 2 Cl imidazole acrylic acid] according to the respective standard protocol) as a 5x10 "5 molar solution in buffer (Phosphate buffer, 0.5 molar in water, pH 7 with 0.05 vol% SDS) applied using a spotter (Carthesian) and incubated for 2 - 24 h.
- the disulfide spacer PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH of the oligonucleotide is cleaved homolytically.
- the spacer forms a covalent Au-S bond with the Au atoms on the surface, which leads to a 1: 1 coadsorption of the ss-oligonucleotide and the split off 2-hydroxy-mercaptoethanol.
- the single-strand can also be hybridized with its complementary strand.
- Split-pin needles (Arraylt chipmarker pins from TeleChem) are used for the assignment with the spotter from Cartesian Technologies (MicroSys PA), which have a loading volume of 0.2 to 0.6 ⁇ l and deliver volumes of about 1 nl per wetting process.
- the contact area of these needles has a diameter of approximately 130 ⁇ m and is therefore significantly larger than the areas of the substrate exposed during laser ablation.
- the needle is positioned over the substrate with an accuracy of 10 ⁇ m at a humidity of around 70 - 80%. The drop is released when the tip comes into contact with the passivation layer and there is no direct contact with the substrate (“pseudo-contact printing”).
- Example 3 Variation of the surface coverage by coadsorbates.
- the occupancy density of a spot with nucleic acid oligomers can be reduced in a controlled manner by coadsorption with thiols, in order to increase the relative proportion of binding events with the target concentration and electrode size remaining the same.
- the incubation solution consists of the nucleic acid oligomers (analogous to Example 2) with an additional between about 10 "5 to 10 '1 molar propanethiol.
- This free propanethiol which is present at the same time, is co-adsorbed by forming an Au-S bond and thus takes up space on the sensor surface
- the propanethiol (10 "5 to 10 " 1 molar in 500 mmol / l phosphate buffer) is applied in a second incubation step (30 min to 12 h) after the functionalization of the sensor surface with nucleic acid oligomers.
- propanethiol as a coadsorbate can reduce the surface coverage density of the nucleic acid oligomers in both variants by up to a factor of 10.
- Example 4 Varying the surface occupancy using occupancy parameters.
- the surface coverage density of the sensor spots can also be adjusted by varying selected occupancy parameters when functionalizing with nucleic acid oligomers.
- the occupancy density increases by a factor of 5.
- concentration of the incubation buffer is increased from 10 mmol / l to 500 mmol / l at a probe concentration of 30 ⁇ mol / l.
- Example 5 Hybridization with complementary nucleic acid oligomers.
- a substrate with 48 working electrodes is produced as described in Example 1 with active areas of different sizes.
- groups of 12 electrodes each, circular holes with a diameter of 10 ⁇ m (spot group 1), 30 ⁇ m (spot group 2) or 100 ⁇ m (spot group 3) and a rectangular profile with 100 ⁇ m x 700 ⁇ m ( Spot group 4) burned.
- the individual spot groups therefore have areas of 78.5 ⁇ m 2 , 706.5 ⁇ m 2 , 7850 ⁇ m 2 or 70000 ⁇ m 2 , so that the area is varied by a factor of around 1000.
- the working electrodes of a spot group are each functionalized with double-modified nucleic acid oligomers (probes) of a certain sequence (S1 to S4) analogously to Example 2.
- the surface coverage density with nucleic acid oligomers can be calculated from the redox current at the potential of the osmium complex. In the present case, the result is 5 x 10 "1 mol / cm 2 .
- the working electrodes are brought into contact with complementary, ferrocene-modified nucleic acid oligomers in a post-loading step for 30 minutes with a 1 mmol / l solution of propanethiol before hybridization.
- the spaces between the nucleic acid oligomers are rendered hydrophobic. This shifts the redox potential of ferrocene to more positive values, resulting in better separation from the osmium potential.
- the four different target nucleic acid oligomers are synthesized analogously to Example 2 but without a thiol modification at the 3 'end.
- the target nucleic acid oligomers have a sequence (T1 to T4) which is complementary to a probe nucleic acid oligomer.
- T1 to T4 the amino-modified nucleic acid oligomers are coupled at the 5 'end with ferrocene acetic acid (FcAc) in accordance with the respective standard protocol.
- FcAc ferrocene acetic acid
- the measurement data show a second redox peak, the ratio of the peak currents of the osmium label and the ferrocene label corresponding to the hybridization efficiency of the experiment.
- the measurement data of the hybridization from FIG. 3 show an electrode close to saturation with a hybridization efficiency of over 90%.
- the working electrodes with the sizes adapted to the concentrations of the respective targets all show the same hybridization efficiencies of approximately 30-40%.
- Example 6 Diagnostic chip.
- vaginal smear which is examined for HPV, E-Coli and lactobacilli, among others.
- a sample is taken with the aid of standardized swabs, which is then treated using standardized methods in order to obtain the RNA of the bacteria present and the double-stranded DNA of the viruses.
- bacteria or particle numbers up to a certain limit are classified as harmless: for HPV this is 100 particles, for E-coli 100 germs and for lactobacilli 10000 germs of all relevant lactobacilli.
- a sensor chip according to the invention for the above application has three different spot sizes, which are functionalized with probe polynucleotides specific for the respective disease targets.
- Spots with an area of 1 ⁇ m 2 are used to detect the HPV-DNA in the range of 10 2 to 10 4 molecules in the test substance, while for the E-Coli RNA in the range of 10 6 to 10 8 molecules (corresponding to 10 2 up to 10 4 germs) areas of 10 4 ⁇ m 2 and for the lactobacillus RNA in the range of 10 8 to 10 10 molecules (corresponding to 10 4 to 10 6 germs) areas of 10 6 ⁇ m 2 are used.
- the choice of the electrode sizes ensures that the sensor for the respective area allows quantitative measurements from the critical target concentrations of the different pathogens and thus a parallel diagnosis of all diseases can be made.
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Abstract
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EP04731631A EP1656556A2 (de) | 2003-05-06 | 2004-05-07 | Substrat als träger von ligaten |
US10/578,661 US20070077593A1 (en) | 2003-05-06 | 2004-05-07 | Substrate as a ligate carrier |
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DE10320312A DE10320312A1 (de) | 2003-05-06 | 2003-05-06 | Substrat als Träger von Ligaten |
DE10320312.5 | 2003-05-07 |
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EP (1) | EP1656556A2 (de) |
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EP1664296A1 (de) * | 2003-08-28 | 2006-06-07 | Canon Kabushiki Kaisha | Sondenträger und verfahren zur quantifizierung einer zielsubstanz unter verwendung des sondenträgers |
WO2006114249A1 (de) * | 2005-04-26 | 2006-11-02 | Bayer Technology Services Gmbh | Neue apparatur und verfahren zur beschichtung von trägersubstraten für einen analytnachweis mittels affinitäts-nachweisverfahren |
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WO2006113785A2 (en) * | 2005-04-18 | 2006-10-26 | Brigham Young University | Laser modification and functionalization of substrates |
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- 2004-05-07 EP EP04731631A patent/EP1656556A2/de not_active Withdrawn
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Cited By (5)
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EP1664296A1 (de) * | 2003-08-28 | 2006-06-07 | Canon Kabushiki Kaisha | Sondenträger und verfahren zur quantifizierung einer zielsubstanz unter verwendung des sondenträgers |
EP1664296A4 (de) * | 2003-08-28 | 2009-05-06 | Canon Kk | Sondenträger und verfahren zur quantifizierung einer zielsubstanz unter verwendung des sondenträgers |
WO2006114249A1 (de) * | 2005-04-26 | 2006-11-02 | Bayer Technology Services Gmbh | Neue apparatur und verfahren zur beschichtung von trägersubstraten für einen analytnachweis mittels affinitäts-nachweisverfahren |
CN101208599B (zh) * | 2005-04-26 | 2014-12-10 | 拜耳知识产权有限责任公司 | 用于涂覆通过亲和力-测定法用于被分析物检测的底物基质的新的设备和方法 |
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US20070077593A1 (en) | 2007-04-05 |
WO2004099430A3 (de) | 2005-01-20 |
EP1656556A2 (de) | 2006-05-17 |
DE10320312A1 (de) | 2004-12-02 |
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