Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/28—Electrolytic cell components
  • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
  • 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
  • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/403—Cells and electrode assemblies
  • G01N27/404—Cells with anode, cathode and cell electrolyte on the same side of a permeable membrane which separates them from the sample fluid, e.g. Clark-type oxygen sensors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • 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
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2458/00—Labels used in chemical analysis of biological material
  • G01N2458/30—Electrochemically active labels

Definitions

• the present invention relates to a method for detecting one or more chemical or biological species that can either undergo a redox reaction or directly or indirectly release a molecule capable of undergoing a redox reaction, wherein current generated by said redox reaction is detected at least one electrode position.
• Electrodes With one or more measuring positions, consisting of an electrode, takes place at the only oxidation or reduction of a mediator.
• the fabrication of the electrodes for this process is simple (e.g., gold wire, circuit boards, metal evaporated plastic), but the current signals obtained are often not sensitive enough.
• EP 886 773 B1 discloses a method for detecting molecules or molecular complexes in a diluent or solvent, wherein a measurement sample is brought into contact with an interdigital ultra-microelectrode assembly.
• a measurement sample is brought into contact with an interdigital ultra-microelectrode assembly.
• an electric potential to the electrode structures, an alternating electric field is generated, and the current or potential changes caused by species present or produced in the measurement sample are measured.
• the resulting current flow is mainly due to the detected molecules and molecular complexes in the influenced by the electrode near the room.
• the influence can be effected by diffusion, by attachment or by binding of the species to be measured.
• the measurements are carried out in particular by means of impedance spectroscopy.
• the electric field used for detection can be generated by AC voltage with very small amplitudes between about 10 mV and 50 mV; the frequencies can be between 1 mHz and 10 MHz.
• the molecules to be measured can be bound on the microelectrode surfaces themselves. This can be done by physical (adsorption) or chemical bonding, e.g. Thiol compounds are applied to gold electrodes and measured. However, it is also possible to apply to the electrodes antigens or the like which react with an antibody in the measurement sample or to monitor hybridization reactions in the nucleic acid chemistry.
• the abovementioned publication shows the detection of the binding of ⁇ -galactosidase-streptavidin to an S-biotinylated electrode surface made of gold.
• the change in impedance is measured by a so-called Nyquist plot, which shows the disruption of the dielectric between the electrodes by the complexed molecule and thus the binding between the biotin and the streptavidin-enzyme complex represents.
• the ultra microelectrode arrangement can also measure the binding of the ⁇ -galactosidase streptavidin to the biotin amperometrically on the basis of the detection of p-aminophenol.
• the alternating electric field is superimposed with a constant potential by which one electrode is brought to a potential of +250 mV with respect to an Ag / AgCl reference electrode, with the aid of which the aminophenol is oxidized to quinone imine.
• the electrodes themselves are not chemically altered by the binding of the species to be detected to the capture molecules.
• the smallest possible AC voltage (usually at or below 50 mV) is used, which generally does not have to be based on a reference. Partially the frequency is varied over very wide ranges, in order to read from it further dependencies.
• FIG. 2a A diagram in which this measurement is plotted against the time [s] on the basis of the coordinates Potential [V] is shown in FIG. 2a.
• Impedance spectroscopy may additionally utilize oxidation and / or reduction effects, e.g. mentioned in the above document.
• an AC resistance is measured by means of a low AC voltage in the range of usually about 10-50 mV.
• a defined potential is applied with DC voltage to at least one of the two electrodes (typically by reference electrode reference), which specifically causes a reduction or oxidation of an existing oxidizable, reducible or redox reaction accessible mediator, which in turn causes DC currents, but not necessarily evaluated become.
• reference electrode reference typically by reference electrode reference
• cyclic voltammetry can also be used as the measuring principle in these arrangements.
• the electrodes are very slowly - usually over several seconds or even minutes away - from a predetermined positive voltage to a predetermined negative voltage and switched back again.
• the resulting diagram of the measured amount of current versus voltage allows conclusions about the presence of molecules to be detected between the interdigital electrodes.
• Cyclic voltammetry is, in principle, a very slow electrochemical redox process with corresponding mass conversion, in which oxidation and / or reduction maxima of the respective substance are determined. 4.
• redox recycling also called redox recycling, differs.
• the two electrodes are each constantly subjected to an oxidation and a reduction potential (with reference electrode reference), which should be at or above the oxidation and at or below the reduction potential of the redox molecule to be detected. Due to the extreme proximity of the two electrodes, the oxidized molecule migrates to the oppositely charged electrode, where it is reduced and migrates back. Measured are the two streams that are generated directly by the oxidation or reduction of the redox molecules at the electrodes.
• a further disadvantage is that the close proximity of the electrodes to one another involves the risk of short-circuits: if the anode and cathode touch at a distance of often only about 400 nm, the respective measuring position is altogether useless. If a plurality of species in a liquid are to be detected with the electrode arrangement, with several measurement positions occupied by different capture molecules being exposed to the same liquid to be examined, then the entire test must be repeated with a new electrode arrangement.
• the electrode structures are often in the
• the object is achieved by providing a method for detecting one or more (chemical or biological) species, which can either undergo a redox reaction or directly or indirectly release a molecule that can undergo a redox reaction, said by the Redox reaction generated current is detected at at least one electrode position, wherein the at least one electrode position on or near which the species to be detected is repeatedly switched between two different potentials such that it occupies potentials with respect to a reference electrode which are close to or lower than the redox potential of said species or said molecule, such that said species / said molecule (the latter after release) alternately reduces at or near said electrode position in the toggle and is oxidized.
• the current formed over time by the repeated reduction and oxidation of the species / molecule is detected and optionally compared to an electrode position at or near which said species or molecule is absent.
• the detection and evaluation of the amounts of electricity and the conclusion that the species sought - qualitatively or quantitatively determinable - is present or not, is generally carried out according to the features of claim 1, preferably according to claim 2 and optionally also according to claim 3. Ideally, positioning a time-varying amount of the species", it should be understood that the amount of species on, at or near the electrodes changes and preferably increases during the period ti-.2.
• the method according to the invention therefore differs in that no interdigital electrodes must be used with a fixed potential difference, but that the voltage is cyclically varied at only one electrode so that these alternately as a cathode and a reference electrode Anode acts.
• FIG. 2d This is shown schematically in FIG. 2d.
• switching back and forth between two different potentials on this electrode usually with, for example, 0.1 to 10 Hz
• switching back and forth between two different potentials on this electrode becomes possible when there is a species or molecule on or near it that is accessible to a redox reaction , this one oxidized by one potential and reduced by the other.
• the generated currents - a positive oxidation and a negative reduction current - are measured in their sum (see FIG. 1, where 1 is a measuring position consisting of one electrode, 2 the oxidized redox mediator, 3 the reduced redox mediator, 4) the electrode abandoned low frequency alternating voltage and 5 schematically represent the readout of the generated redox current over time).
• the temporal change of the total current signal added up is summed up (sum of the amounts of oxidation and reduction current, resulting in an increase in current over time), which corresponds to a change in the concentration of the redox mediator.
• the occurring capacitive Umladestrom is not considered, since it is independent of the redox currents of the mediator. Although it relaxes quickly after each reload, it is almost identical for each new measurement cycle and therefore does not change over time.
• the sensitivity of this arrangement corresponds approximately to that of the "conventional" redox cycling on interdigital anode and cathode.
• the complicated manufacturing process of interdigital electrode arrays by e.g. Semiconductor technology is omitted here, however.
• One or more electrodes with cyclically changing potentials can be used as a measuring position or as a complete array with different measuring positions.
• the method according to the invention makes it possible to detect (directly) molecules which are present in a detection fluid, provided that these molecules can undergo a redox reaction.
• the method according to the invention can be used for the detection of molecules which are present in a measuring liquid or another detection fluid and are accessible to a redox reaction.
• detection fluid is meant liquids, gels or other higher-viscosity materials and gases. It is favorable if the detection fluid is guided in a fluid channel via the one or more measuring electrodes. If necessary, several detection fluids can be measured simultaneously, wherein each of the fluid channels is guided over one of the measuring electrodes. It may be favorable to use one of the measuring electrodes to carry a reference fluid whose content or absence of molecule which is accessible to a redox reaction is known. This facilitates semi-quantitative or quantitative statements.
• 4-aminophenol can be detected directly in a measuring liquid by allowing this liquid to flow in the vicinity of measuring electrodes and linger there.
• Each electrode is continuously switched back and forth between the redox potentials of p-aminophenol +200 mV and -350 mV with 1.0 Hz.
• an iridium / iridium oxide reference electrode can serve.
• a counter electrode mounted at any position, dissipates differential currents.
• a plurality of electrodes may be provided, each located in separate fluid channels.
• the species to be detected binds to the invention
• the redox mediator also referred to as redox mediator
• Mediator can be generated chemically, physically or enzymatically.
• gold electrodes may be provided on which a protein is bound by adsorption.
• any redox molecules can be used for the present invention; however, those which can be cleaved by enzymes are particularly suitable for the detection of enzymes or enzyme-containing complexes.
• redox molecules are ferrocene derivatives, potassium hexacyanoferrate (II), (III) and organic ruthenium and osmium complexes such as ruthenium hexamine, osmium bispyridyl dichloride.
• organic and possibly also inorganic redox molecules are preferably present in encapsulated form, the capsules, eg liposomes, for example, being able to carry a group which reacts only with a combination of catcher molecule and species to be detected, but not with the catcher molecule alone.
• Such binding possibilities offer, for example, so-called intercalation compounds, which are known from DNA technology and react only with double-stranded nucleic acid, but not with single-stranded nucleic acid.
• intercalation compounds which are known from DNA technology and react only with double-stranded nucleic acid, but not with single-stranded nucleic acid.
• the expert knows a number of Ways to realize these techniques, see for example WO 2002/081739 or WO 2002/082078.
• the species-containing detection fluid is allowed to flow over the one or more electrodes already occupied by the capture molecules. If multiple electrodes are used, the electrodes can be occupied with different capture molecules that can bind / complex different species. With this method, therefore, several different species can be detected in a detection fluid, which is particularly useful in the DNA analysis of
• the plurality of electrodes may in this case be arranged in a common fluid channel or in different fluid channels.
• One or more of the electrodes serving as measuring positions can remain without catcher molecules in this embodiment. These are then suitable as comparison electrodes in order to be able to compare or calibrate the respective measured values against a suitable zero value, which enables quantitative or semi-quantitative detection results.
• the electrode / measuring positions may be made of any conductive material, e.g. consist of a precious metal such as gold or platinum, but also of carbon compounds such as graphite or nanotubes; gold is favorable because many biologically relevant species can be attached to gold via a thiol bridge. If the measuring positions are occupied with biomolecules, they can serve as a platform for a biological test.
• the redox mediator is hereby e.g. generated by an enzyme label position-specific or converted into its electrochemically active form.
• the direct environment of the electrodes / measuring positions can, as also already mentioned, be designed in such a way that they are able to bind catcher molecules.
• materials such as oxides with hydroxide surfaces or modified silanes / siloxanes or inorganic / organic hybrid materials such as the silicon-containing Ormocere® are suitable for this purpose.
• These can easily be designed or modified on their surface with suitable groups for the present invention, for example with amines, hydroxyl groups, carboxyl groups. In the abovementioned cases, one will usually foresee a connection of catcher molecules on the / the electrodes, but this is not necessary.
• the direct environment of the electrodes / measuring positions may alternatively or additionally also be made hydrophobic in order to keep the contact angle of droplets applied to electrode material as large as possible (in particular for the connection of catcher molecules).
• the electrodes / measuring positions may also be surrounded by rings or webs, which also help to prevent the catcher material droplets from running. Such a configuration is described for example in DE 199 16 867 A1.
• the rings or webs may be permanent or intermediate.
• punches for the application of
• the electrodes / measuring positions can be arranged on any carrier material, for example on an organic substrate such as plastic, but also on printed circuit boards or the like.
• a silicon wafer or the like can serve as a carrier, even if this is not usually necessary becomes.
• the combination of gold electrodes and a plastic substrate can be applied in a simple manner to the substrate, likewise the conductor structures.
• the conductor patterns may be made of gold, but - e.g. to save costs - also made of copper or aluminum or another common material.
• the shape of the electrodes / measuring positions is not critical, which is why they can have any shape.
• they may be flat and round, oval, rectangular, oblong, square, or otherwise polygonal depending on the requirements of the test and other conditions such as placement of one or more fluid channels.
• You may be continuous or have recesses whose surface is suitable, for example, as described above for the connection of catcher materials. If required, they can be embedded in the surrounding substrate, for example to improve the flow conditions in the fluid channel in which they are located. Alternatively, they may be applied to the substrate, for example vapor-deposited, printed, plated, soldered or applied in any other way. These variants are significantly cheaper compared to the embedding.
• the electrodes / measuring positions can be of any size.
• the surface of the electrodes In order to be able to drive and measure a large number of electrodes simultaneously, for example on a chip, it is advantageous to design the surface of the electrodes to be relatively small, for example with one Surface of approx. 0.05 to 0.5 mm 2 . If necessary, the electrodes can be designed as microelectrodes or ultramicroelectrodes (with length and / or width dimensions in the ⁇ m or sub- ⁇ m range). Finally, it should be noted that the electrodes / measuring positions may also have a three-dimensional structure, for example as drops, solder bump, wire or plate.
• the method typically requires a reference electrode, which need not be on the same substrate as the measuring electrode (s). It is sufficient if the reference electrode is in conductive communication with the measuring electrodes via the detection fluid (e.g., buffer with or without redox species).
• the reference electrode is not energized. It consists of a material suitable for the respective process, for example iridium / iridium oxide, calomel, Ag / AgCl.
• a counter electrode is further required, which is charged to apply the desired current to the measuring electrodes to a corresponding equivalent value. Also, this electrode does not necessarily have to be on the same substrate as the measuring electrodes. It is typically - but not necessarily - made of gold.
• the advantage of the method according to the invention is that an electrical readout can take place on electrodes of arbitrarily shaped shape by means of redox cycling, the sensitivity of which is comparable to a readout on interdigital electrode structures.
• the production of the electrodes can be done much easier and cheaper avoiding digital structures, as expensive semiconductor and coating techniques with masks, polymer coating,
• Exposures and washouts for the required submicron electrode structures for conventional redox cycling can be dispensed with.
• the production yield of these electrodes increases, as no rejects can be caused by electrical short circuits, which in the conventional sub-micron I nterdal ig. caused by the accidental accumulation of particles from the cleanroom processes.
• the inventive method is preferably used for the so-called chip technology.
• a measuring position in the form of electrodes on a chip, for example, has the size of about 50 to 200 mm 2 .
• Each measuring position can be acted upon by a fluid channel with a detection fluid or a comparison fluid, for example a pure buffer.
• the reference electrode and the counter electrode may need but not, also be arranged on the chip. All electrodes on the chip are connected to electrical conductor structures for applying the potentials and reading the current changes.
• the electrical control can be done via a separate potentiostat, but it can also be arranged as an electronic component on a separate chip (2-chip solution).
• This chip is used multiple times, while the chip with the measuring positions is usually used only once and then discarded, especially when the electrodes or their surroundings have been covered with catcher material.
• the electronics it is also possible to arrange the electronics directly in the chip, which also carries the measuring positions. All of these variants are known in the art, see, for example, E. Nebling et al., "Electrical Detection of Viral DNA Using Ultramicroelectrode Arrays", Anal. Chem. (2004), 76 (3): 689-696, J.
• a support material such as passivated silicon
• gold electrodes diameter 0.5 mm
• a counter electrode an iridium / iridium oxide reference electrode
• iridium / iridium oxide reference electrode On a support material, such as passivated silicon, several gold electrodes (diameter 0.5 mm), a counter electrode and an iridium / iridium oxide reference electrode are vapor-deposited. Each gold electrode corresponds to a measuring position.
• a non-specific protein (BSA) is bound by adsorption.
• the enzyme ⁇ -galactosidase is bound by adsorption.
• the gold electrodes can be flooded with a variety of liquid media by means of a microfluidic flow cell connected to a micropump, hoses and a distributor valve.
• the 12 gold electrodes are supplied with the required potentials using a 16-channel multipotentiostat and the resulting currents are read out in the nano-ampere range.
• This potentiostat consists of 2 8-way multiplexers, which read the electrodes sequentially in a short time after the respective potentials have been applied.
• At each electrode is between the potentials of +200 mV and -350 mV with 1, 0 Hertz continuously switched back and forth.
• the reference is the iridium / iridium oxide reference electrode.
• the counterelectrode dissipates differential currents.
• the redox potentials of the mediator p-aminophenol are within the range of +200 mV and -350 mV.
• an enzyme substrate dissolved in buffer (1.0 mg / ml in PBS pH: 7.0) is passed over the electrodes.
• This substrate is p-aminophenyl- ⁇ -D-galactopyranoside (pAP- ⁇ -gal) and is converted by the enzyme ⁇ -galactosidase from an electrochemically inactive form to the electrochemically active redox mediator p-aminophenol.
• pAP- ⁇ -gal p-aminophenyl- ⁇ -D-galactopyranoside
• Figure 3a shows the parallel current measurement over time at each one of 12 gold electrodes with redox cycling on each electrode based on the resulting superimposed measurements of the current signals for all electrodes.
• the conversion of p-aminophenol is measured individually on each electrode (+200 mV, -350 mV with 1, 0 Hz).
• the liquid flow is stopped (second 28, see arrow), but the measurement of the current continues.
• an oxidation current peak positive
• a reduction current peak negative
• FIG. 3b shows the sum of the current signal amounts from the oxidation and reduction current for each individual electrode (arrow: stopping the liquid flow).
• the positions 1 - 8 show a largely horizontal course of the current signals.
• the positions 9-12 show an increase in the current over time after switching off the liquid flow (arrow). Since the enzyme 1 - 8 is missing in the electrodes, no active redox mediator is formed here and thus no redox currents result. On the electrodes 9 - 12, the enzyme generates the active redox mediator, and thereby the current at these positions increases over the Time continuously.
• the electrodes 1-8 show a different high, almost constant current.
• the different amount of this charge current from electrode to electrode results from the fact that the potentials at each electrode are switched simultaneously, but the readings by the multiplexers are slightly delayed in time. At the electrodes that are measured shortly after switching (1, 2). This transshipment current is still high because its relaxation is not yet complete.
• Figure 3c shows the plot of change in current values over time (current slope) for each position individually as a bar graph.
• the horizontally extending currents of the "negative" electrodes 1 - 8 show here, regardless of their height ( Figure 3b) almost no change.
• the current at the "positive" positions 9 - 12 increases significantly over time. This clearly shows the presence of the enzyme.
• the mean value of the current slope of positions 9-12 is about 209 nA / min.
• the invention described above is thus suitable for the sensitive electrical reading of redox mediators on non-interdigital electrodes and is significantly more sensitive than just the oxidation on such electrodes, while the sensitivity of the reading of those on interdigital electrodes is comparable.
• the gold electrodes are first adsorbed with suitable capture molecules.
• a target molecule specifically bound to these capture molecules is labeled by an enzyme (e.g., ⁇ -galactosidase).
• the supplied substrate is converted by this enzyme into the active redox mediator. This can be measured electrode-specifically with the method described above sensitive.

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