US20130217003A1 - Method for determining an analyte content of a liquid sample by means of a bioanalyzer - Google Patents

Method for determining an analyte content of a liquid sample by means of a bioanalyzer Download PDF

Info

Publication number
US20130217003A1
US20130217003A1 US13/881,907 US201113881907A US2013217003A1 US 20130217003 A1 US20130217003 A1 US 20130217003A1 US 201113881907 A US201113881907 A US 201113881907A US 2013217003 A1 US2013217003 A1 US 2013217003A1
Authority
US
United States
Prior art keywords
substrate
binding
receptors
analyte
bound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/881,907
Other languages
English (en)
Inventor
Michael Hanko
Angela Eubisch
Axel Fikus
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Endress and Hauser Conducta GmbH and Co KG
Original Assignee
Endress and Hauser Conducta Gesellschaft fuer Mess und Regeltechnik mbH and Co KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Endress and Hauser Conducta Gesellschaft fuer Mess und Regeltechnik mbH and Co KG filed Critical Endress and Hauser Conducta Gesellschaft fuer Mess und Regeltechnik mbH and Co KG
Assigned to ENDRESS + HAUSER CONDUCTA GESELLSCHAFT FUR MESS- UND REGELTECHNIK MBH + CO. KG reassignment ENDRESS + HAUSER CONDUCTA GESELLSCHAFT FUR MESS- UND REGELTECHNIK MBH + CO. KG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FIKUS, AXEL, EUBISCH, ANGELA, HANKO, MICHAEL
Publication of US20130217003A1 publication Critical patent/US20130217003A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
    • C25F—PROCESSES FOR THE ELECTROLYTIC REMOVAL OF MATERIALS FROM OBJECTS; APPARATUS THEREFOR
    • C25F1/00—Electrolytic cleaning, degreasing, pickling or descaling
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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

Definitions

  • the invention relates to a method for determining an analyte content of a liquid sample by means of a bioanalyzer.
  • Biosensors which can be subdivided into affinity based and metabolic biosensors, serve for the preferably selective and specific detection, respectively for concentration measurement, of a predetermined analyte contained in a liquid sample to be examined.
  • the large group of heterogeneous biosensors includes, in such case: a receptor layer, which can have a large number of receptors immobilized on a surface, e.g. receptors such as antibodies, haptens, DNA, cells or enzymes; a signal transducer, which outputs a measurement signal correlated with the amount of the analyte bound on the receptor layer; and a signal processing system for amplification and/or additional processing of the measurement signal output by the signal transducer.
  • the analyte can be a substance to be detected in a liquid sample, for example, a substance such as a protein, a peptide, an antibody, an enzyme or some other substance to be detected in biochemical, biological or medicinal systems.
  • the analyte can, however, also be cells, cell components or DNA or, for example, active ingredients, which are to be detected or determined in the context of environmental monitoring or in the monitoring of water quality.
  • a substance, which preferably binds with high specificity and selectivity to a receptor is here generally referred to as the target molecule or also as a ligand for such receptor.
  • the analyte represents a target molecule for the receptors.
  • Other target molecules can, however, also be molecules different from the actual analyte, and these likewise attach to the receptors of the receptor layer, and compete, for example, with the analyte, for binding locations of the receptor layer.
  • metabolic biosensors there is catalytic conversion of the analyte and/or other target molecules after their temporary binding to the receptor layer, which, in the case of metabolic biosensors, contains at least one enzyme, a cell, a cell component or other catalytically effective biomolecule, which can catalytically convert the analyte, in given cases, with additionally present substances.
  • the liquid sample is brought in contact with the receptor layer, so that analyte molecules and, in given cases, other target molecules present in the liquid sample attach to the receptor layer or are converted by the receptor layer.
  • physical and/or chemical changes are effected, for example, a change of coating thickness, index of refraction, light absorption or electrical charge.
  • Suitable signal transducers for registering said changes include, for example, optoelectric sensors, and amperometric or potentiometric sensors.
  • the target molecules bound on the receptor layer can also be marked directly or indirectly with a marker, which has, for example, luminescent or magnetic properties, or be marked subsequently directly or indirectly after the binding of the target molecules on the receptor layer.
  • a marker which has, for example, luminescent or magnetic properties
  • an optical sensor is suitable as signal transducer for registering the luminescence, or a magnetic sensor for registering magnetic properties.
  • markers are also enzymes, which catalyze a following chemical reaction, wherein the course of the reaction can be registered with correspondingly suitable signal transducers.
  • the receptors are immobilized on solid carrier materials, i.e. bound to these. This can occur, for example, by unspecific adsorption of the receptors on the surface of the carrier material, the so called substrate.
  • covalent bonding of the receptors or bonding via a number of other bonding layers are established methods for preparation of receptor layers.
  • streptavidin can be bonded covalently on a solid carrier surface and, for preparing the receptor layer, biotin-conjugated antibodies connected on the streptavidin binding layer using the affinity interaction between biotin and streptavidin.
  • the total layer system constructed on the surface of a solid carrier material as the ‘sensor matrix’ of the biosensor, wherein the last layer is the receptor layer.
  • the sensor matrix with the receptor layer is, in many applications, applied on a biochip integrated in a microfluidics system. These biochips are, as a rule, single-use products, which are discarded, respectively replaced by a new biochip, after a single performed determination or concentration measurement of the analyte. Also, in the case of metabolic biosensors, the chip, or the cartridge, which contains the sensor matrix, must be replaced at regular intervals. The reason for this is, generally, the low stability and robustness of biological receptors as well as the often high affinity, with which target molecules are bound on the receptors. Thus, coupled with conditions, under which target molecules bound on the receptors are shed from the receptors is, as a rule, also a more or less large destruction of the receptor layer.
  • an apparatus for electrochemical detection of a nucleotide sequence.
  • This apparatus has a replaceable analytical cassette, preferably embodied as a single use-component, for introducing the liquid sample.
  • the cassette has a measuring electrode with a receptor layer, on which the nucleotide sequence selectively and specifically binds.
  • the analytical cassette is connectable with a calculation system via an analog interface, wherein an electrochemical detection of the nucleotide sequence is performable by means of the calculation system.
  • biosensors especially of affinity based biosensors
  • process measurements technology for example, for automated monitoring of a biotechnological process in an industrial plant or for automated monitoring of water, for example, for residues of medicines or for the content of endocrinally active substances
  • biosensors for such measuring tasks can be a bioanalyzer, which is fed, especially online, liquid samples of a process medium to be monitored.
  • the liquid samples can be pretreated, preferably automatically, with reagents, for example, with other target molecules, markers, etc.
  • the bioanalyzer includes furthermore: a receptor layer, to which the liquid sample, or the liquid to be measured, as obtained by pretreating, is fed; a signal transducer, which outputs a measurement signal correlated with the number of analyte, or target, molecules bound on the receptor layer; and a signal processing system, which further processes the measurement signal and outputs a measured value, especially one derived from the measurement signal.
  • the supplying of the samples and, in given cases, reagents to the receptor layer and the draining of the liquid sample, or liquid to be measured, after transpired measuring can be performed in an automated or semi-automated analyzer by means of pump apparatuses, pneumatic systems or other liquid transport systems.
  • small, supply and drain lines for the sample and, in given cases, reagents should have cross sections, which are as small as possible, e.g. in the sub-mm range.
  • Such liquid lines with cross sections in the sub-mm range are referred to in the following also as microfluidic ducts.
  • a module of a bioanalyzer, which has such microfluidic ducts, is also referred to as a microfluidic unit.
  • biosensors suitable for performing a plurality of measurements one after the other, wherein the sensor matrix, respectively its receptor layer, is essentially completely regeneratable, or renewable, in situ, with high reproducibility.
  • EP 2 189 793 A1 Known from EP 2 189 793 A1 is that, for removing the target molecules from the receptors of the respective receptor layer, enzymes can be applied, which selectively decompose the target molecules connected to the receptors. In such case, it must be heeded, however, that the used enzymes do not equally attack the receptors, and this strongly constrains the ability to apply this technique. Due to the chemical nature of such enzymatic decomposition of the bound target molecules, maintaining the same receptor density following multiple performances of the method appears extremely improbable.
  • a further approach for regeneration of the receptor layer of a biosensor involves binding the receptors via oligonucleotides, thus short DNA, or RNA, molecules.
  • oligonucleotides are covalently bonded to a surface and the receptors or molecules, which form a corresponding binding layer for binding receptors, are conjugated with the complementary oligonucleotide.
  • Specific affine interaction between the complementary oligonucleotides leads to an immobilizing of the receptors on the surface and therewith to the forming of a receptor layer.
  • the affine interaction between the complementary oligonucleotides can be removed chemically or by increasing the temperature, so that the receptors are released from the surface.
  • this method in the presence of proteins, e.g.
  • Seokheun Choi Junseok Chae “Reusable biosensors via in situ electrochemical surface regeneration in microfluidic applications”
  • Biosensors and Bioelectronics 25 (2009) 527-531
  • Seokheun Choi Junseok Chae, “A regenerative biosensing surface in microfluidics using electrochemical desorption of short-chain self-assembled monolayer”
  • Microfluidics and Nanofluidics 7 (2009) 819-827 or Christie A
  • a system which delivers even after a plurality of regenerations, or renewals, of the receptor layer, for example, after 50, 100 or more regeneration- or renewal cycles, for equal target molecule concentrations, essentially equal measurement signals, i.e. measurement signals of comparable intensity, especially in the case of reference of the measurement signals to the reference measurement.
  • an automated or semi-automatically working bioanalyzer that a plurality of different receptors can in simple manner be immobilized in a measuring region accessible for the signal transducer of the bioanalyzer, in order to detect the analyte in a direct method or another target molecule in a competitive method or in a binding inhibition test.
  • a plurality of different receptors can in simple manner be immobilized in a measuring region accessible for the signal transducer of the bioanalyzer, in order to detect the analyte in a direct method or another target molecule in a competitive method or in a binding inhibition test.
  • an automated performing of a plurality of different assays respectively analytical methods, to provide an essentially universal basic structure (e.g. a microfluidic unit with a certain liquid duct structure for passing the liquid sample and reagents through the measuring region), in which the plurality of different methods can be performed.
  • a method for automated in situ determining of an analyte content of a liquid sample by means of a bioanalyzer which includes a microfluidic system having at least one measurement duct and means for leading one or more liquids through the measurement duct, wherein the measurement duct has at least one substrate,
  • preparing a sensor matrix which has a plurality of receptors, which bind the analyte and/or a further target molecule, preferably selectively and specifically, especially due to an affine interaction, or bring about a chemical conversion of the analyte or of the further target molecule, comprising a step of leading through the measurement duct a preparation solution of at least a first chemical species, which includes at least one functional group binding on the substrate and at least one other functional group, wherein a plurality of the first chemical species are bound on the substrate via the functional group binding on the substrate, wherein the other functional group of the plurality of the first chemical species bound on the substrate serves as a receptor or for subsequent binding of a receptor; (ii) leading the liquid sample, or a liquid to be measured obtained by treating the liquid sample with at least one reagent, through the measurement duct, wherein analyte contained in the liquid sample or in the liquid to be measured, and/
  • the at least one substrate can especially be regenerated, in that the sensor matrix and, in given cases, molecules bound thereto, especially analyte molecules, other target molecules or other substances bound on the sensor matrix, are essentially completely released from the substrate and/or at least partially decomposed.
  • the at least one substrate can be completely, or essentially completely, cleared, i.e. at least sufficiently cleared that there remain on the substrate residual components of the sensor matrix and possibly substances bound on the sensor matrix only in so little measure that these, in the case of repeating the steps (i)-(iii), i.e. in the case of a new preparing of the sensor matrix, a new leading of a new liquid sample and, in the case of the new regenerating of the substrate, no noticeable influence, especially no noticeable influence on the accuracy of measurement, is exerted.
  • the sequence of steps (i)-(iii) is referred to in the following for short also as the “measuring cycle”. Since the substrate is regularly regenerated, respectively recleared, by removal of the sensor matrix and molecules possibly bound thereto, and, thereafter, the sensor matrix prepared anew, a replacement of the substrate or other mechanical reaching into a bioanalyzer, especially into its measurement duct, in which the method described is performed, for maintenance purposes, is not necessary.
  • the analysis of the liquid sample can, consequently, be performed completely automatically with a large number of measuring cycles following one after the other. Since the substrate is completely or essentially completely cleared, it is assured that in sequential measuring cycles the sensor matrix has an essentially comparable character, which assures very good comparability of the measuring, respectively analytical, results won in the different measuring cycles.
  • a measurement which includes especially the leading of the liquid sample or the liquid to be measured through the measurement duct and the determining of a measured variable correlated with the amount of target molecules bound or converted by the receptors or the amount of analyte bound or converted by the receptors and deriving therefrom the analyte content of the liquid sample according to step (ii), can at least be performed within a predetermined measuring region.
  • the measuring region is a part of the surface of the measurement duct.
  • the substrate is arranged at least partially in the measuring region, it can, however, also extend beyond the measuring region within the measurement duct or within other liquid ducts of the microfluidics system.
  • the receptors, on which the target molecules, especially the analyte, are bound are bound on the substrate via the first chemical species.
  • the receptor can be formed by the other functional group of the first chemical species.
  • the receptor layer is formed in a single preparation step by immobilizing the first chemical species on the substrate.
  • the sensor matrix is composed then only of the receptor layer. If the receptor in the form of a functional group is not a direct component of the first chemical species, the other functional group can serve for binding the receptor.
  • the binding of the first chemical species on the substrate forms a first binding layer, on which, in turn, for example, by specific binding on the other functional group, the receptor can be directly bound or bound via one or more other binding layers.
  • the one or more additional binding layers can be formed sequentially by passing through, in each case, an additional preparation solution, which contains other chemical species binding on the, in each case, uppermost binding layer for forming an additional binding layer.
  • a sensor matrix is formed of one or more binding layers and a terminating, receptor layer bound on the uppermost binding layer.
  • the lowest binding layer formed by the first chemical species and each other of the binding layers are bound on binding locations of the chemical species forming the, in each case, layer lying therebeneath, preferably through specific affine interaction, e.g. via a biotin-streptavidin binding or the binding between an antibody and its antigen.
  • the receptor is preferably bound via a specific affine interaction on the uppermost binding layer.
  • additional binding layers i.e. the indirect binding of the receptor on the first chemical species via second and possibly other chemical species offers the advantage that these can be so embodied that, on a given binding layer system, numerous different receptors can be prepared as receptor layer, wherein, however, all receptors use the same functionality relative to their binding, so that there is no need of matching binding layers and receptors.
  • Step (ii), which includes the leading and analyzing of the liquid sample, or of the liquid to be measured produced by treating, e.g. mixing, the liquid sample with a reagent, can be repeated one or more times, before, in step (iii), a regeneration of the substrate occurs.
  • the receptor layer can always be reconstructed anew on the same substrate in situ, i.e. without mechanical reaching into the measurement duct, and so, repeatedly, the analyte content of liquid samples supplied the bioanalyzer one after the other can be ascertained in automated fashion.
  • the method steps (i) to (iii) can be performed at least 50 times, preferably at least 150 times, further preferably at least 300 times.
  • the terminology, functional groups means, generally, atom groups, which decisively determine the material-properties and the reaction behavior of the compounds carrying them.
  • the concept of the functional group includes here and in the following also: atom groups or molecule regions, which can form binding, preferably specific, interactions with other chemical species; as well as also markers bound on a chemical species, especially a molecule, for example, markers formed of molecules, peptides, proteins or protein tags.
  • a biotin molecule bound on an alkyl thiol and serving as binding partner for a receptor-target molecule binding forms a first functional group of the alkyl thiol.
  • the thiol group of the alkyl thiol forms a second functional group.
  • the functional group of the first chemical species that binds on the substrate is preferably matched to the substrate material.
  • the substrate can be, for example, of metal or can comprise at least one metal coating.
  • receptor means here a functional group as above defined, on which a complementary molecule, also referred to as target molecule, binds, preferably selectively and specifically, especially due to an affine interaction, or by which the conversion of other chemical species is enzymatically catalyzed. In such case, it need not necessarily concern a receptor in the biological sense, such as, for example, an antibody.
  • the analyte can be bound as target molecule directly on the receptor layer (direct method). It is also possible to bind on the receptor layer the analyte and a target molecule different from the analyte, in order to register a measured variable correlated with the number of target molecules bound on the receptor layer and to ascertain therefrom the analyte concentration (competitive method). It is furthermore possible to mix the liquid to be measured with a pretreatment solution before the leading through the measurement duct, wherein the pretreatment solution contains a reaction partner of the analyte in a known concentration, and then to lead the mixture through the measurement duct. In this case, the remaining reaction partner not bound to analyte molecules serves as target molecule, wherein, from the number of target molecules bound on the receptor surface, the concentration of the analyte in the measured solution can be deduced (binding inhibition test).
  • determining an analyte content means a purely qualitative detection of an analyte, a semi-quantitative determining of the analyte fraction in a certain sample volume of the liquid to be measured or even a quantitative concentration ascertainment or activity determination for the analyte in the liquid to be measured.
  • the determining of the analyte content according to the method described here and in the following is not used for determining deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • chemical species means here and in the following especially a molecule, a complex, a biomolecule aggregate or a microorganism.
  • the molecules can include, for example, enzymes, peptides, proteins or DNA.
  • complexes are protein complexes, which are also referred to as protein aggregates.
  • Microorganisms can include, especially, cells, such as e.g. yeast cells.
  • the substrate is electrically conductive. It can, in such case, be, for example, an electrically conductive surface region of the measurement duct, which is embodied e.g. as a metal film on a wall of the measurement duct.
  • the substrate can comprise, for example, a chemically stable metal with high thiophilic properties, especially a metal such as gold or platinum.
  • the functional group binding on the substrate can comprise a sulfur atom bonding, especially covalently, to the substrate. Especially suitable for this are thiol, or disulfide, groups.
  • Examples of the first chemical species include thiols, especially alkyl thiols and polyethelene glycol thiols.
  • Such molecules are known to those skilled in the art as binding molecules, so-called linkers, for biochemical molecules, especially for receptors of a biosensor (compare the above cited article).
  • alkyl thiols form covalent bonds between the substrate, for example, a gold, or platinum, substrate, and the sulfur atom of the thiol group and form on the surface frequently self organized monolayers, also referred to as self-assembled monolayers, or SAMs, for short.
  • SAMs self-assembled monolayers
  • the forming of such SAMs is, however, not absolutely required for the construction of a sensor matrix, respectively, receptor layer.
  • a control unit which includes an electronic data processing unit, e.g. a microcomputer, controls the leading of the liquids through the measurement duct and the registering of measured values.
  • the means for leading the liquids can comprise especially pumps, e.g. syringe pumps, or pneumatic means.
  • the measurement signals output from a signal transducer of the bioanalyzer suitable for registering the measured variable can be converted by the control unit into a digital signal and a measured value of the measured variable can be formed therefrom.
  • the regenerating of the substrate can occur using purely chemical means, e.g. by leading sodium hydroxide solution containing peroxide through the measurement duct for dissolving the sensor matrix.
  • the regenerating, respectively the clearing, of the substrate can also occur by producing an electrical current flow between the substrate and a counter electrode in electrically conductive contact with the substrate via an electrolyte, in order to clear the substrate, especially essentially independently of the present occupation of the substrate by the sensor matrix and, in given cases, other substances bound thereto.
  • an electrical current flow is produced between the substrate and a counter electrode, in order to effect a clearing of the substrate, which means a complete or almost complete removal and/or an at least partial decomposition of the sensor matrix and substances possibly bound thereto.
  • one of the species applied for preparing the layers contains more than one binding location for a species of the layer lying therebeneath or for a species, which forms the following layer, then, in the case of a high degree of coverage of the individual layers, covalently or non-covalently bonded junctions can occur at the molecular level, so that a cross linked network is formed.
  • an electrochemical dissolving of the layer structure by producing an electrical current flow between the substrate, for example a substrate in the form of an electrically conductive, surface region of the measuring duct, and, a counter electrode in electrically conductive contact with the substrate via an electrolyte does lead to an as complete as possible release of the total layer structure with clearing of the substrate.
  • an as complete as possible clearing of the conductive surface can be achieved, when the electrical current flow produced on the surface is sufficient for electrolytic production of oxygen or hydrogen or alternately of oxygen and hydrogen (electrochemical cleaning).
  • an optimizing of the electrochemical cleaning is such that the release of the sensor matrix and molecules bound thereto or other chemical species occurs with at least partial decomposition of the sensor matrix.
  • the step of producing an electrical current flow between the substrate and the counter electrode can especially be so performed that hydrogen and/or oxygen is formed on the substrate and/or the counter electrode by electrolysis.
  • the cleaning action of the oxidation, respectively reduction, of the receptor layer, respectively the sensor matrix, effected by the electrical current flow and the resulting hydrogen, or oxygen, is amplified still more.
  • alternate oxidizing and reducing conditions can be established on the substrate.
  • the electrically conductive contact between the substrate and the counter electrode is brought about by an electrolyte, which contains cyanide- and/or halogen ions in concentrations of less than 150 mmol/l, preferred less than 15 mmol/l, further preferably less than 1.5 mmol/l, especially when the substrate involves a gold, or platinum, film.
  • the counter electrode can be arranged lying within the measurement duct, especially opposite the substrate.
  • the counter electrode can be, for example, of a chemically very inert material and is preferably applied in the form of a film.
  • Such an inert electrode material is formed, for example, by a layer of boron-doped, diamond-like carbon.
  • Such electrodes are used, for example, in water treatment, and are applied there for electrolytically generating hydroxyl radicals for decomposing organic residues.
  • the counter electrode can likewise be embodied in the measurement duct, especially lying opposite the substrate, as a further substrate, on which the functional group of the first chemical species binding on the substrate binds.
  • both the substrate as well as also the counter electrode can comprise an electrically conductive material binding thiol groups or disulfide groups.
  • the substrate and the counter electrode serving as further substrate can be formed, for example, as gold or platinum films on oppositely lying walls of the measurement duct. In performing step (i), there is, in this way, formed both on the substrate as well as also on the counter electrode a sensor matrix having a large number of receptors.
  • analyte and/or other target molecules contained in the liquid sample or in the liquid to be measured are preferably selectively and specifically bound on the receptors of the sensor matrix formed on the substrate and on those of the additional substrate, or chemically converted by these.
  • a measured variable correlated with the amount of the analyte bound or converted by the receptors of the two sensor matrices or with the amount of the target molecules bound by the receptors of the two sensor matrices can be determined. This is especially advantageous, since the effectively available substrate surface is enlarged thereby, which, because of the correspondingly greater measurement signal, affects the accuracy of measurement positively.
  • the regeneration according to step (iii) serves for clearing both substrates.
  • the electrical potential of the substrate can be set, especially controlled, during the producing of an electrical current flow between the substrate and the counter electrode, in reference to an electrical reference potential of a reference electrode in electrolytic contact with the substrate via the electrolyte, especially by means of an electrolyte bridge.
  • the control can be done by the control unit of the bioanalyzer.
  • electrolytic contact means an ion conducting contact via an electrolyte, especially an ion containing, aqueous solution.
  • a diaphragmless, electrolyte bridge is involved.
  • electrolytic contact means an ion conducting contact via an electrolyte, especially an ion containing, aqueous solution.
  • a diaphragmless, electrolyte bridge is involved.
  • a reductive removal of a receptor layer by means of a two electrode circuit. Since a reference electrode is absent, there can be no checking of the potentials actually resulting on both electrodes. In this way, it can, depending on the conditions reigning in the microfluidic system and on the electrolyte concentration and -composition, come to too large positive or negative potentials on the substrate or on the counter electrode. These large potentials, when they occur repeatedly, can lead to the destruction of the electrode functionality. This defeats, however, the object of the invention disclosed here.
  • the reference electrode can be embodied as an electrically conductive coating of an additional liquid duct connected with the measurement duct. It is also possible to use, as reference electrode, a currentlessly operated, pseudo reference electrode in the form of a metal wire, especially a platinum- or gold wire, which can be arranged in the measurement duct or in an additional liquid duct connected with the measurement duct. Should in the course of a measurement cycle it come to an occupation of the pseudo reference electrode by the used reagents, then the pseudo reference electrode is likewise regenerated, or cleared.
  • the potential of the substrate serving as working electrode can continuously or discontinuously, especially linearly, be varied between a minimum- and a maximum value, in order to reclear the substrate by electrochemical cleaning, especially with oxygen-, respectively hydrogen, evolution.
  • the potential is moved back and forth, especially linearly, multiple times, especially between 2 and 1000 times, between the maximum- and the minimum values.
  • the process duration as well as also the cleaning duration can be variable within broad limits and changed, or set, depending on the cleaning result.
  • the control unit of the bioanalyzer can provide a corresponding input facility for a service person or means for loading predetermined process parameters.
  • the electrical current level occurring on the substrate can be registered, in order so to obtain one or more cyclic voltammograms, from which the state of the electrically conductive surface section can be deduced.
  • alternative analytical methods for example, optical, voltammetric or other electrochemical analytical methods, can be applied for evaluating the state of the substrate.
  • the electrical current level of the electrical current flow produced between the substrate and the counter electrode can be varied continuously or discontinuously between a minimum- and a maximum value, in order to reclear the substrate by electrochemical cleaning.
  • the electrical potential difference established between the substrate and the reference electrode must not subceed a minimum value, nor exceed a maximum value, in order to avoid damaging the substrate.
  • the counter electrode likewise serves as a substrate, then all data and conditions given here for the substrate connected as working electrode hold equally for the counter electrode.
  • one or more cleaning- and/or auxiliary liquids can be led through the measurement duct.
  • a cleaning liquid led through before or after producing an electrical current flow between the substrate and the counter electrode serves mainly for chemical cleaning of the substrate.
  • Suitable as cleaning liquids are, for example, acids or alkaline solutions or solvents.
  • Serving as auxiliary liquids are, for example, electrolyte solutions, which support the electrochemical processes on the electrically conductive substrate advantageously (e.g. Na 2 SO 4 solution or addition of H 2 O 2 ).
  • Auxiliary- or cleaning liquids led through the measurement duct during the electrical current flow serve for supporting the electrochemical cleaning.
  • the electrolyte and/or the auxiliary liquid are/is led during the electrical current flow continuously or discontinuously through the microfluidic measurement duct.
  • a depletion of needed auxiliary reagents, or an enrichment of reactive, electrolytically produced substances can be prevented, which has a positive effect on the lifetime of the substrate surface.
  • decomposition products of the sensor matrix and also the gas bubbles formed by electrolysis are removed from the region of the substrate surface, which contributes to a more rapid and safe cleaning.
  • an acidic phosphate buffer solution can be led as auxiliary liquid through the measurement duct.
  • Such an auxiliary liquid is advantageous for supporting an electrochemical cleaning in the case of variation of the potential of a platinum-substrate between negative values in the range between ⁇ 0.5 V and ⁇ 1.25 V (reduction, in given cases, with associated forming of hydrogen) and positive values in the range between +1.5 V and +2.25 V (oxidation, in given cases, with, associated forming of oxygen) in reference to the potential of a silver/silver chloride reference.
  • the substrate can be checked for residual components of the sensor matrix, especially by means of an electrochemical measuring method, e.g. cyclic voltammetry or impedance spectroscopy, an optical measuring method, e.g. ellipsometry, or an adsorption test.
  • an electrochemical measuring method e.g. cyclic voltammetry or impedance spectroscopy
  • an optical measuring method e.g. ellipsometry
  • an adsorption test e.g. ellipsometry
  • the electrical current level during the regeneration can be taken into consideration for evaluating and/or for control of the regeneration, respectively regeneration efficiency.
  • an adsorption test a substance, which binds on residual components of the sensor matrix on the substrate, is led through the measurement duct and then the amount of the adsorbed substance is ascertained.
  • the checking can also be applied for control of the regeneration.
  • the auxiliary liquid can include, for example, also photochemically or electrochemically produced ozone, which, because of its strongly oxidizing effect, cares for an effective decomposition of the sensor matrix. Also, other chemical substances or physical methods effective for decomposing the sensor matrix can be used. In such case, can provision of an electrical current flow is not needed, nor then need substrates be electrically conductive.
  • the substrate can be glass, on which, with the assistance of a first chemical species, which includes a (trialkoxy)silyl group as first functional group for binding on the glass substrate, the first binding layer for the construction of the sensor matrix is prepared.
  • a first chemical species which includes a (trialkoxy)silyl group as first functional group for binding on the glass substrate
  • the first binding layer for the construction of the sensor matrix is prepared.
  • step i all steps needed for a measurement procedure, including the construction of the sensor matrix (step i), determining the analyte content (step ii) and regeneration, respectively removal, of the sensor matrix (step iii) are performed in situ, i.e. by successive leading of corresponding solutions through the measurement duct in a predetermined sequence, without other handling of the measurement duct.
  • the plurality of first chemical species bound on the substrate forms a first binding layer, and additional functional groups of the first chemical species provide bonding locations for subsequent bonding of receptors.
  • the other functional group of the first chemical species can comprise, for example, biotin.
  • the receptor can, in this case, be bound directly or indirectly via a biotin-binding second chemical species bound on the other functional group of the first chemical species and having at least one biotin-binding location on the other functional group of the first chemical species.
  • the biotin-binding second chemical species can comprise a biotin-molecule binding, e.g. avidin, an avidin derivative or an avidin-similar molecule, especially streptavidin or NeutrAvidin.
  • a biotin-molecule binding e.g. avidin, an avidin derivative or an avidin-similar molecule, especially streptavidin or NeutrAvidin.
  • biotin-molecule binding e.g. streptavidin, NeutrAvidin or avidin, or an avidin derivative
  • streptavidin e.g. streptavidin
  • NeutrAvidin e.g. avidin
  • avidin derivative e.g. avidin, or an avidin derivative
  • the receptor bound on the other functional group of the first chemical species can be bound directly or indirectly on the first chemical species via at least one non-covalent bonding.
  • the binding locations of the uppermost binding layer provided for binding of the receptors can be selected from a group comprising a few binding locations suitable, in each case, for binding a plurality of different receptors. These binding locations are, consequently, applicable as universally as possible.
  • the binding locations can be such for binding biotin and, in this case, comprise streptavidin or NeutrAvidin or an avidin-like molecule.
  • the biotin-streptavidin binding can serve as a universal interface for binding a plurality of different receptors, since biotinylation, respectively the binding of streptavidin on the binding molecules, is simply and universally possible with established techniques of biotechnology.
  • biotin functionalized antibodies are widely distributed for performing immunoassays.
  • binding locations for constructing an as universal as possible binding layer can be immunoglobulin binding locations.
  • these can be provided by binding a biotinylated, immunoglobulin binding molecule on a binding location for binding biotin, as such was described above.
  • the layer formed by the first chemical species bound on the substrate is simultaneously a binding, and receptor, layer.
  • the layer formed by the first chemical species forms a binding layer, on which optionally one or more other binding layers can be formed, wherein a receptor layer is provided as terminating layer, which is formed by receptors bound on the therebeneath lying binding locations of the binding layer.
  • the chemical species forming the individual layers can be bound with the chemical species forming the, in each case, thereover or thereunder lying layers by an affine interaction, especially a non-covalent bonding.
  • each layer of the sensor matrix has, relative to the, in each case, layer lying therebeneath, a, with reference to the particular conditions, complete degree of coverage, at least, however, a degree of coverage of more than 80%.
  • complete degree of coverage at least, however, a degree of coverage of more than 80%.
  • layer of a sensor matrix is not necessarily to be understood as a morphological description. Rather, the term “layer” refers to the plurality of chemical species bound on chemical species of the layer lying therebeneath or on the substrate in the same binding step and bound in the same way, preferably non-covalently, further preferably via affine interactions.
  • a layer need not only be formed of a plurality of equal chemical species, e.g. equal molecules, especially equal proteins, but, instead, can also comprise a number of different chemical species. Decisive is the binding, via essentially the same physical interactive forces, in the same binding step, e.g. the sending through of a preparation solution comprising the chemical species forming the layer.
  • the average surface density and advantageously also the average orientation of the receptors of the receptor layer, in the case of repeated or multiple preparing of the sensor matrix remain as constant as possible.
  • degree of coverage of more than 80% means that degree of coverage of a surface, in the case of which the coverage of the surface is greater than 80% of the coverage in the equilibrium state in the case of the conditions used, in each case, for binding, these essentially being the concentration, the flow rate, the binding rate and the geometry of the fluid measurement duct, for the species to be bound from the preparation solution.
  • the flow rate and the duration of the leading of the respective solution through the measurement duct can be selected in such a manner and matched to one another and to the concentration of the solution that the degree of coverage of more than 80% is reached.
  • the said parameters can be ascertained in preliminary experiments, and be then provided for performing the described method for ascertaining an analyte content.
  • the degree of coverage can be ascertained by measurements, wherein the degree of coverage of the respective species is measured either directly, for example, by ellipsometry or by means of a quartz microscale, or indirectly by feeding to the respective layer a molecule specifically binding with the respective species, and ascertaining the number of bound molecules, for example, by optical methods.
  • the particular species forming a binding layer in the case of each layer of the sensor matrix, can bind with another chemical species, which binds on the same binding locations of the layer lying therebeneath as the binding species, but which, however, has no other functional group, on which chemical species led subsequently through the measurement duct can bind.
  • the particular surface density of the binding species results from the concentration of the binding species, the concentration ratio and the size ratio of the binding species and the one or more other chemical species as well as the respective equilibrium ratios of binding and release. Analogously, so also can the receptor density of the receptor layer be set.
  • the layer sequence gold substrate/alkyl thiol-PEG-biotin/NeutrAvidin/biotin conjugated antibody mixing in of biotin-polyethelene glycol to the biotin-conjugated antibody can lessen the surface density of the receptors of the receptor layer.
  • the layer of receptors and polyethelene glycol is likewise referred to as the receptor layer.
  • the density of the binding species in the binding layers can also be set in the same way.
  • the measured variable correlated with the amount of the target molecules bound by the receptors or target molecules converted by the receptors or correlating with the amount of analyte bound or converted by the receptors can be an optical, measured variable, which is ascertained especially based on a luminescence measurement, a reflection measurement or an absorption measurement.
  • SPR measurements are less suitable, because of large disturbing influences brought about, above all, by temperature, and electrolyte, fluctuations.
  • a functional group which has luminescent properties, i.e. fluorescent, or phosphorescent, properties, or which converts a substrate solution supplied in a following step to produce light (chemiluminescence) or a color change.
  • the substrate when the measured variable is a chemical luminescence brought about directly or indirectly by the target molecule or the luminescence of a luminescent marker of the target molecule, it is advantageous, when the substrate, respectively the measuring region containing the substrate, transmits light in the wavelength range between 300 nm and 900 nm at at least 10%, preferably at least 50%, or even at least 70%.
  • the substrate can be applied on a wall of the measurement duct as a metal film having a lattice structure. A transmission can, in this case, occur through intermediate spaces of the lattice structure.
  • the analyte can be a peptide or a protein, wherein the other target molecule besides the analyte is another molecule different from the analyte, especially a peptide or protein derived from the analyte and having a tag, preferably a protein tag, which serves for direct or indirect production of the measured variable, or which influences the measured variable.
  • the target molecule different from the analyte can be, for example, a competitor, which, due to an affinity interaction, binds, same as the analyte, on the receptor (compare competitive method).
  • the target molecule different from the analyte can be a molecule preferably selectively and specifically binding on the receptor layer, especially a peptide or protein, which forms a complex with the analyte molecules, so that the number of target molecules bound on the receptor layer is a measure for the concentration of the analyte (compare binding inhibition test).
  • Such a protein or peptide having a protein tag and forming the other target molecule can be produced as a recombinant protein or peptide including the protein tag.
  • a marker can be bound directly or indirectly on the analyte bound on the sensor matrix or on another target molecule.
  • the marker serves directly or indirectly for introducing a property influencing the measured variable. It can especially be bound via the protein tag on a target molecule having a protein tag. This can be effected, for example, by leading a solution of the marker, or a chemical species carrying the marker as a functional group, through the measurement duct, wherein the marker, or the chemical species carrying the marker, has a functional group binding on the analyte, on the other target molecule or on the protein tag of a target molecule.
  • the marker binds in the latter case specifically on the target molecules bound on the receptor layer and having a protein tag, and not on analyte molecules bound likewise on the receptor layer.
  • the application of protein-tagged, target molecules, preferably as competitors, offers, compared to known methods of protein analysis, the great advantage that, for the determining of different analytes, only a marker, which directly or indirectly binds on the protein tag of the target molecule, is necessary, or the protein tag itself represents the marker.
  • GFP green fluorescing protein
  • directly bound markers are, besides GFP, also other fluorescing substances, such as e.g. fluorescing nanoparticles.
  • Indirectly bound markers can be bound advantageously on a target molecule, or the analyte, via affine interaction.
  • the described protein tags are especially suitable as universal binding structures for subsequent direct or indirect binding of the marker.
  • Suitable as markers are, besides chemical species, which are detectable, for example, due to certain fluorescent or absorption characteristics, also chemical species, which, in the presence of other chemical species, influence a property of the measuring system detectable, especially optically, by the signal transducer.
  • markers which enter chemical reactions, from which a detectable optical signal arises, especially due to chemiluminescence or a color change.
  • An example of such a marker is peroxidase, which, in the presence of a solution of luminol/H2O2, effects a chemiluminescence catalyzed through the peroxidase.
  • a bioanalyzer for performing the method of one the above described embodiments includes a microfluidic system having at least one measurement duct and means for leading liquid through the measurement duct, wherein the measurement duct includes at least one substrate, on which, at least within a measuring region, at least at times, a sensor matrix having a plurality of receptors is arranged, which, preferably selectively and specifically, bind or chemically convert target molecules or analyte present in a liquid sample or a liquid to be measured obtained by treating the liquid sample with at least one reagent, wherein the bioanalyzer includes at least one signal transducer, which outputs a measurement signal dependent on an amount of analyte bound on the receptors or converted by these or dependent on target molecules bound on the receptors or an amount of converted target molecules.
  • the amount of analyte bound, or target molecules bound corresponds to the number of analyte molecules, or target molecules, bound on the receptors.
  • a counter electrode Arranged within the microfluidic system can be a counter electrode, which is arranged especially within the measurement duct lying opposite the first substrate and preferably serves as another substrate. If the counter electrode serves as another substrate, then there is arranged also on this other substrate, at least at times, a sensor matrix having a plurality of receptors. The preparing of the sensor matrix and the regeneration of the substrate and the counter electrode serving as another substrate can occur, as above described in connection with the method for automated in situ determining of an analyte content of a liquid sample.
  • the substrate and the counter electrode can be arranged relative to the flow direction of the electrolyte flowing through the measurement duct in such a manner that gas bubbles occurring in the electrolyte due to an electrical current flow between the substrate and the counter electrode are so led that they essentially do not influence the electrical current flow.
  • the counter electrode can be embodied as an electrically conductive coating of a duct of the microfluidics system, respectively as a film, which especially can be different from the measurement duct, but bonded therewith. It can preferably be arranged within the measurement duct, for example, lying opposite to the substrate or coaxially therewith.
  • the bioanalyzer can furthermore comprise, formed within the microfluidics system, a reference electrode, which is in electrically conductive connection with at least one substrate via an electrolyte bridge, especially a diaphragm-less electrolyte bridge.
  • the substrate, the counter electrode and, in given cases, the reference electrode can be embodied as electrically conductive, connectable films, especially as metal coatings on the walls of the ducts of the microfluidics system, in which they are arranged.
  • the microfluidic system can comprise a first component, which has a surface, in which the measurement duct and, in given cases, other ducts, especially ducts connected with the measurement duct for liquid transport, are embodied as depressions, wherein at least a second component is connected with the first component and covers the depressions and liquid tightly seals them relative to the environment.
  • the so formed liquid lines extend between one or more liquid reservoirs, from which liquids can be introduced into the microfluidics system, and waste containers, into the liquids can be transferred from the microfluidic unit.
  • the first and second components can be composed especially of an electrically insulating, preferably transparent, material, e.g. glass or a synthetic material, e.g. a plastic.
  • the substrate, the counter electrode and, in given cases, the reference electrode can be formed on the second component as electrically conductive, connectable films, especially as metal coatings.
  • the microfluidic system can, in an alternative embodiment, comprise a first component, especially a planparallel first plate, and a second component, especially a planparallel second plate, arranged spaced from the first component, wherein the first and second components are connected with one another via one or more spacing elements arranged between the first and second components, especially one or more intermediate plates, wherein the one or more spacing elements have intermediate spaces and/or perforations, which form a duct structure extending between the first and second components, including the measurement duct and, in given cases, additional ducts connected with the measurement duct for liquid transport.
  • the first and second components are especially of an electrically insulating, preferably a transparent material, e.g. glass or a plastic.
  • the one or more intermediate plates can be flexible, for example.
  • they can be embodied, for example, in the form of silicone mats.
  • the first and second components can be embodied, for example, in the form of glass plates.
  • Advantageous here is the application of microscope slides.
  • the substrate, the counter electrode and, in given cases, the reference electrode can in this embodiment be in the form of electrically conductive, connectable films, especially metal coatings, on the first and/or the second component.
  • electrically conductive, microfluidic small tubes can be used as substrate, as counter electrode and, in given cases, as reference electrode.
  • the substrate can be a small gold tube
  • the counter electrode a small platinum tube
  • the reference electrode a small silver tube lined internally with silver chloride.
  • Advantages in the case of this type of embodiment lie in the simple electrical and microfluidic connectability of these small tubes.
  • An advantageous variant, in such case, is composed of a measurement duct, whose measuring region is formed of a small tube, which is at least internally electrically conductive, for example, a small tube of platinum, in whose interior is placed, coaxially and without direct contact with the small tube, a wire, which is at least electrically conductive on its surface, e.g.
  • the small tube can serve, in such case, as substrate and the wire as counter electrode and, in given cases, as another substrate. Also, the reversed functionality represents another option.
  • the coaxial arrangement of substrate and counter electrode permits, because of the small separation between substrate and counter electrode, high current densities, which enable a long service life, even in the case of slight removal of the surface of the substrate and/or the counter electrode during the electrical regeneration, respectively cleaning.
  • the measurement duct is preferably so embodied in the region of the measuring region that it transmits light in the wavelength range between 300 nm and 900 nm at at least 10%.
  • the substrate as a thin platinum layer, whose thickness amounts to between 2 and 30 nm, or in the case of a structured, for example, lined or lattice structured, opaque substrate applied on a transparent material.
  • the microfluidic unit is, or, in given cases, a number of microfluidic units are, arranged together with containers for provisioning reagents, respectively reagent solutions, and for accommodating liquid wastes, in a replaceable unit of the bioanalyzer, a so-called replaceable cassette.
  • the containers are connected within the replaceable cassette by means of microfluidic connecting systems with the one or more microfluidic units for supplying the reagents into the microfluidic unit or units, and for draining consumed liquids into the waste containers. Transport of the liquids through a microfluidic unit occurs preferably indirectly, e.g.
  • the replaceable cassette After consumption of the reagents, respectively reagent solutions, stored and needed for measurement operation, or after complete filling of the containers for accommodating liquid wastes, the replaceable cassette is to be replaced by a new one or a reconditioned one.
  • FIG. 1 a a first schematic representation of a receptor layer with bound analyte, and target, molecules
  • FIG. 2 a schematic representation of a microfluidic unit of a bioanalyzer in plan view
  • FIG. 3 a schematic, longitudinal section of the microfluidic unit of FIG. 2 ;
  • FIG. 4 a a schematic representation of the microfluidic unit of FIG. 2 before an electrochemical cleaning, in plan view;
  • FIG. 5 a schematic representation of a second embodiment of a microfluidic unit of a bioanalyzer, composed of a composite of a first component, a perforated, intermediate plate forming the duct and a second component, wherein:
  • a is a schematic representation of the first component in plan view
  • FIG. 6 a schematic representation of a third embodiment of a microfluidic unit of a bioanalyzer, wherein:
  • a is a schematic longitudinal section taken with the cutting plane C-C of FIG. 6 b );
  • FIG. 1 a is a schematic representation of a regeneratable sensor matrix of a bioanalyzer with receptors R 350 and analyte molecules R 400 bound on these and likewise other target molecules R 450 bound on the receptors R 350 .
  • Serving as substrate 3 for the sensor matrix is a thiol groups binding, electrically conductive surface. This can be, for example, a gold, or platinum, surface, which is formed, for example, by a coating, a film or a solid metal piece, e.g. a small tube.
  • Platinum and gold have high thiophilic properties and have, furthermore, the advantage that they are chemically stable, and, in the face of the electrochemical dissolving of the receptor layer and the preparation of a new receptor layer described in detail below, are not changed chemically, especially they do not oxidize.
  • binding molecules R 200 for the receptors R 350 Arranged on the substrate 3 is a layer of a first chemical species serving as binding molecules R 200 for the receptors R 350 .
  • the binding molecules R 200 for forming a binding layer on the substrate 3 can, in the case of a thiophilic surface, such as, for example, a gold or platinum surface, be, for example, alkyl thiols having another functional group, wherein the thiol groups covalently bond with the gold or platinum surface.
  • a further example of binding molecules R 200 is ⁇ -mercapto ⁇ -biotin polyethelene glycols (referred to, for short, also as thiol PEG biotin). This can be bound from aqueous solution via a metal sulfur binding on the substrate 3 .
  • mercapto polyethelene glycol which has only one thiol group binding on the substrate 3 , not, however, another functional group for binding the receptor R 350 , it is possible, on the one hand, to set the surface density of biotin groups.
  • the introduced polyethelene glycol prevents the unspecific adsorption of subsequently added proteins on the substrate surface.
  • the binding molecules R 200 have furthermore at least one other functional group, which serves as binding location for the specific binding of a complementary molecule bound via a functional group to the receptor R 350 .
  • the other functional group of the binding molecules R 200 is formed by a biotin group of the alkyl thiols. If ⁇ -mercapto ⁇ -biotin polyethelene glycol serves as binding molecule R 200 , then its biotin group serves likewise as functional group for specific direct or indirect binding of the receptor R 350 .
  • Receptor R 350 is in the present example bound preferably selectively and specifically on a molecule R 300 binding on the binding molecules R 200 .
  • Molecule R 300 is in the present example a biotin-binding molecule, e.g.
  • binding molecules R 200 and the molecule R 300 preferably selectively and specifically binding on the other functional group of the binding molecules R 200 and via which the receptor R 350 is bound on the binding molecules R 200 , form a universal interface for binding a large selection of receptors R 350 on the same binding molecules R 200 and therewith for (indirect) binding of a plurality of different receptors on the substrate 3 .
  • the biotin streptavidin connection for binding of the receptor R 350 on the binding molecule R 200 in the example shown here is well suited as a universal interface, since the biotinylation, respectively the marking of molecules with streptavidin is an established technique in biotechnology.
  • biotin-functionalized antibodies are widely used, for example, for performing immunoassays.
  • Bound on the receptors R 350 in the example shown in FIG. 1 a ) are analyte molecules R 400 and other target molecules R 450 competing with the analyte molecules R 400 for binding on the receptors R 350 and used by the bioanalyzer for detection.
  • the other target molecules R 450 are able, like the analyte R 400 , to bind on the receptors R 350 , preferably selectively and specifically, by affine interaction.
  • the analyte R 400 can be a protein, for example.
  • the protein molecules bind correspondingly, in each case, preferably selectively and specifically, on the receptors R 350 .
  • the other target molecules R 450 are preferably likewise protein molecules, which bind likewise preferably selectively and specifically, especially by affine interaction, on the receptors R 350 .
  • the other target molecules R 450 can be marked, or subsequently markable, protein molecules.
  • the other target molecules R 450 are provided in the present example with a protein tag.
  • a protein tag is an amino acid sequence bound on the actual protein. Protein tags serve, for example, in affinity chromatography for clean up of a target protein manufactured in a biotechnological method, through specific interaction of the protein tag bound on the target protein with a binding partner used in chromatography columns to isolate the target protein from the remaining products of the method.
  • the protein tag serves in the present example to bind, directly or indirectly, a molecule R 500 carrying a marker.
  • the marker possesses physical or chemical properties, based on which the signal transducer of the bioanalyzer can produce a measurement signal, which correlates with the number of the other target molecules R 450 bound on the receptors R 350 and indirectly with the number of analyte molecules bound on the receptors R 350 .
  • the marker bearing molecules R 500 comprise for this purpose a functional group, which, due to an affine interaction, specifically binds on the target molecules R 450 , preferably on their protein tag.
  • analyte R 400 present in a liquid sample is the protein Pfs
  • a corresponding antibody can serve as receptor R 350 .
  • a competitor serving as other target molecule R 450 in the present case e.g. Pfs (HA-Pfs) recombinantly manufactured and equipped with a hemagglutinin tag (HA tag)
  • HA tag hemagglutinin tag
  • the molecule R 500 carrying the marker can be, for example, an antibody against the HA tag, which is conjugated with a peroxidase.
  • Chemiluminescence effected by means of addition of a solution of luminol/H2O2 catalyzed by the marker peroxidase is converted by an optical signal transducer, which includes especially a photodiode, into an electrical measurement signal, whose signal strength depends on the intensity of the chemiluminescence. This depends, in turn, on the number of marker bearing molecules R 500 bound on the target molecules R 450 . From the measurement signal produced by means of the signal transducer, consequently, the number of target molecules bound on the receptor layer R 450 can be deduced and, from that information, the concentration of the analyte molecules R 400 in the liquid to be measured can be learned.
  • the measurement signal can be processed by a measurement circuit and/or an electronic data processing unit of the bioanalyzer and, therefrom, the concentration of the analyte in the liquid to be measured, respectively the liquid sample, can be derived.
  • a liquid to be measured formed from a liquid sample, to which has been added a peroxidase-conjugated carbamazepine derivative is led over the so formed sensor matrix.
  • the carbamazepine originally contained in the liquid sample binds, in such case, as analyte R 400 on the receptors and competes with the peroxidase-conjugated carbamazepine molecules present in known concentration, which, as earlier manufactured conjugates, include both the functionality of the target molecules R 450 as well as also the functionality of the molecule R 500 carrying the marker.
  • FIG. 1 b Another universal layer structure for binding receptors R 350 is presented in FIG. 1 b ).
  • the layer structure shown in FIG. 1 a ) of a substrate 3 a binding layer of a large number of binding molecules R 200 and molecules R 300 binding preferably selectively and specifically on a functional group of the binding molecules R 200 is likewise used for binding the receptors R 350 .
  • This is, however, supplemented by another binding layer, on which also non-biotinylated receptors R 350 can be bound in various ways.
  • This additional binding layer is composed in the example here of a biotinylated immunoglobulin-binding molecule R 310 , e.g.
  • biotinylated antibody against immunoglobulins of the type IgG (biotin anti-IgG) or of a biotinylated protein A/G a biotinylated antibody against immunoglobulins of the type IgG (biotin anti-IgG) or of a biotinylated protein A/G.
  • FIG. 2 shows in plan view a microfluidic system 100 of a bioanalyzer for detection, or for determining a concentration, of an analyte in a liquid sample.
  • Microfluidic system 100 includes a base 1 a , for example, of a transparent synthetic material, which has on a basal plane an open duct structure formed by depressions in the base 1 a and including duct sections 10 , 15 , 20 , 30 , 40 and junctions 12 , 18 . Lying on the base 1 a and sealing the duct sections 10 , 15 , 20 , 30 , 40 relative to one another and against the environment is a terminating cover plate 1 b , which is formed, for example, likewise of a transparent synthetic material.
  • Base 1 a and cover plate 1 b are fixedly connected with one another by a shape, force or material bonded, interlocking connection 1 c , for example, an adhesive bonding or a pressed joining.
  • the duct sections 10 , 15 , 20 , 30 , 40 sealed by the cover plate 1 b form liquid lines, through which are led for performing a measurement the liquid sample to be analyzed and, in given cases, other reagents.
  • Base 1 a includes, moreover, connections 110 , 120 connectable with liquid supplies and connections 130 , 140 connectable with liquid drains, through which connections liquids can be fed to the duct structure, or drained therefrom.
  • the supply and drain lines connect the microfluidic system 100 with remotely arranged, liquid reservoirs, in which liquids for performing a measurement, respectively an analysis, are accommodated. Via the supply and drain lines, both the liquid sample to be examined as well as also, in given cases, reagents accommodated in the liquid reservoirs and to be added to the liquid sample are suppliable to the microfludic system 100 .
  • the delivery and the draining of the liquids through the duct structure of the microfluidic system 100 can occur, for example, by means of pumps or pneumatically.
  • the duct structure can, as shown in FIG. 2 , also be seen as the basic structure of a microfluidic system, which is independent of the concrete embodiment of the liquid lines.
  • the illustrated structure can also be constructed, for example, using microfluidic hoses and/or small tubes.
  • the liquid lines have preferably a cross section between 10 7 and 1 ⁇ m 2 , preferably between 10 6 and 10 2 ⁇ m 2 , further preferably between 10 5 and 10 4 ⁇ m 2 .
  • a measurement duct 30 formed by a duct section of the microfludic system 100 includes the substrate 3 already described based on FIG. 1 and embodied as an electrically conductive surface region of the measurement duct.
  • Substrate 3 can be embodied, for example, as a metal film, for example, as gold or platinum film, placed on the cover plate 1 b .
  • the region of the substrate coming in contact with liquid flowing in the measurement duct 30 serves as measuring region 7 , in which the sensor matrix is constructed and, in given cases, analyte contained in a liquid sample or liquid to be measured led through the measurement duct 30 is bound on the sensor matrix and detected by means of a signal transducer, especially an optical, signal transducer.
  • a further duct section 40 which is connected with the measurement duct 30 via a junction 18 , includes, serving as a counter electrode 4 , an additional, electrically conductive, surface region, which is formed, for example, as a metal film placed on the cover plate. Both the substrate 3 as well as also the counter electrode 4 are connected with a voltage source, a potentiostat or a galvanostat, via electrical connections (not shown). If an electrolyte flows through the duct sections 30 and 40 , the substrate 3 and the counter electrode 4 are in electrically conductive contact via the electrolyte. By applying a potential difference between substrate 3 and counter electrode 4 , an electrical current flow through the electrolyte can be effected.
  • a third electrically conductive surface region is arranged, which can serve optionally as a reference electrode 2 .
  • the reference electrode 2 is likewise embodied as a metal layer, especially a silver layer on the cover plate 1 b of the microfluidic unit 100 for forming an Ag/AgCl, reference electrode.
  • an electrical connection can be connected, for example, to a potentiostat or galvanostat.
  • a method for preparing the sensor matrix and for detection, or for determining a concentration, of an analyte in a measured solution will now be described with reference to the microfluidic system illustrated in FIG. 2 and a sensor matrix with receptors according to FIG. 1 .
  • a sensor matrix is prepared on the substrate 3 .
  • the sensor matrix includes a receptor layer with receptors R 350 , on which bind, preferably selectively and specifically, the target molecules R 450 to be determined.
  • a first preparation solution of binding molecules R 200 having a thiol group and another functional group, e.g. alkyl thiols with an additional functional group, is led through the measurement duct 30 .
  • the binding molecules R 200 bond covalently to the substrate 3 via their thiol groups and form there ideally a monolayer.
  • the preparation solution is led through the measurement duct 30 at a preferably constant flow rate for a predetermined, first length of time, until the degree of coverage of the arising binding layer, as defined above, safely amounts to more than 80%.
  • the length of time required therefor can be determined in preliminary experiments. Such depends especially on the flow rate, the type of binding molecules R 200 , the concentration of the preparation solution, the binding rate and on additional environmental variables, for example, temperature.
  • a second preparation solution is led through the measurement duct 30 .
  • This second preparation solution contains the actual receptors R 350 , which are bound on a functional group R 300 preferably selectively and specifically binding on the first binding layer.
  • the binding molecules R 200 forming the first binding layer can have a biotin group, on which a biotin-binding group R 300 , for example, a streptavidin group, bound on the receptor R 350 specifically binds.
  • the feeding of the second preparation solution is performed over a length of time, which is so selected that the degree of coverage of the receptor layer formed by receptors bound on the binding molecules R 200 amounts to at least 80%, as above defined.
  • the length of time required therefor can be determined in preliminary experiments.
  • the preparing of the sensor matrix requires in the present example only two steps, namely binding of the binding molecules R 200 on the substrate 3 and the following binding of the receptors R 350 via their functional groups R 300 on the binding molecules R 200 . It is, however, also an option that the receptor R 350 is not bound on a functional group specifically binding directly on the first binding layer, but, instead, first is bound via one or more other binding molecules, and therewith, via one or more other binding layers, on the first binding layer.
  • the one or more additional binding molecules are bound in one or more additional steps by the leading of corresponding preparation solutions, which contain, in each case, the binding molecule to be bound on functional groups of the last applied layer. In such case, there results a layer structure of the sensor matrix having one or more binding layers and a terminating receptor layer.
  • all layers of the sensor matrix have a degree of coverage at least 80%, as already explained above.
  • the preparing of the sensor matrix has ended and detection of an analyte or the determining of a concentration of the analyte in a liquid sample, or in a liquid to be measured obtained by treating the liquid sample with one or more additional reagents, can be performed.
  • the individual method steps for determining a concentration of the analyte depend on the method of detection to be executed.
  • the analyte can be bound on the receptor and the binding detected directly or through binding of an additional receptor on the analyte immobilized on a first receptor (sandwich test method).
  • a target molecule binding competitively on the receptor can be detected, as already described above.
  • the measured solution containing the analyte can also be earlier mixed with an additional solution (reagent), which contains a target molecule binding on the analyte, so that an analyte and target molecule complex is formed. The mixture is then led through the measurement duct and the remaining free target molecules bound on the receptor layer and detected (binding inhibition test).
  • a competitive test method is performed.
  • a mixture of the liquid to be measured containing the analyte R 400 with a solution, which contains, serving as competitor, other target molecules R 450 of known concentration is led through the measurement duct 30 .
  • the target molecules R 450 compete with the analyte molecules R 400 for binding on the receptors R 350 .
  • the target molecules R 450 are proteins marked with a protein tag and are, except for the tag, essentially identical with the analyte R 400 .
  • the measurement duct 30 contains marker bearing molecules R 500 , which bind specifically on a functional group of the target molecule R 450 , in the present example, on the protein tag of the target molecule R 450 .
  • the marker bearing molecule is, in the present example, an anti-HA antibody peroxidase conjugate. Alternatively, the marker bearing molecule can also be indirectly bound.
  • a biotinylated anti-HA antibody can be bound, on which, subsequently, is bound streptavidin marked with a fluorescent dye as marker bearing molecule.
  • a luminal and H2O2 containing solution is led through the measurement duct 30 , which leads to emission of luminescent radiation.
  • the emitted radiation is registered by means of a photoelectric signal transducer (not shown), e.g. a photodiode, and converted into an electrical measurement signal.
  • the measurement signal is output by the signal transducer to a measurement circuit, which amplifies and processes the measurement signal, and the processed measurement signal output to a data processing system, for example, a microcomputer.
  • the data processing system derives from the measurement signal, using a furnished evaluation program, a concentration of the analyte in the liquid to be measured and outputs this to a display system and/or, via a data line, to a superordinated unit.
  • the data processing system can be a component of a control unit, which controls the functioning of the bioanalyzer, processes, stores and outputs measurement signals, and processes and executes input commands from a service person or from a superordinated unit for servicing and/or control of the bioanalyzer.
  • different signal transducers can be used for producing a measurement signal correlated with the number of target molecules bound on the receptor layer.
  • chemiluminescence for detecting the target molecules
  • a fluorescence can be excited.
  • radiation for exciting the fluorescence is radiated into the microfluidic unit in the region of the receptor layer and emitted fluorescent radiation registered by means of a photoelectric signal transducer, e.g. a photodiode.
  • an absorption measurement can be performed for detecting the target molecules.
  • the measured radiation i.e., for example, the luminescent or fluorescent radiation
  • the transparent base 1 a of the microfluidic unit 100 or through the cover plate 1 b of the microfluidic unit 100 it is advantageous to register the measured radiation, i.e., for example, the luminescent or fluorescent radiation, either through the transparent base 1 a of the microfluidic unit 100 or through the cover plate 1 b of the microfluidic unit 100 and, in given cases, to send the necessary excitation radiation (for fluorescent or absorption measurements) correspondingly through the transparent base or through the cover plate.
  • the substrate 3 on which the sensor matrix is applied, is transparent for radiation of the applied wavelength, which, as a rule, lies between 300 and 900 nm.
  • the conductive coating forming the substrate 3 for example, the gold, or platinum, coating, sufficiently thin.
  • a gold or platinum layer of this thickness is sufficiently transparent for optical measurements with measuring radiation in the wavelength range between 300 and 900 nm.
  • the substrate can also be applied as a structured coating, e.g. in the form of a lined or lattice structure with uncoated, and therewith transparent, intermediate spaces.
  • Another variant in the case of the application of an enzyme as marker and a chromophoric enzyme substrate is one wherein the actual measuring region is followed by a transparent region, for example, in front of the exit 130 in the region of the measurement duct 30 , so that the absorption of the liquid flowing through the duct can be measured in the transparent region.
  • a transparent region for example, in front of the exit 130 in the region of the measurement duct 30 .
  • phosphatases and other enzymes are widely applied as markers in bioanalysis. Available for these are also numerous, different, enzyme substrates.
  • a regeneration of the receptor layer is done. Since the releasing of the analyte molecules R 400 , respectively the target molecules R 450 , or the releasing of the receptors R 350 bound via the binding groups R 300 on the binding molecules R 200 is not completely possible in sufficiently reproducible manner, for this, the entire sensor matrix, including the analyte molecules R 400 bound thereon, respectively the target molecules R 450 , including all additional molecules bound thereon, is released, in order to reclear the substrate 3 .
  • the binding molecules R 200 and the receptors R 350 bound via the binding groups R 300 form an interlinked covalently or non-covalently, bonded network at the molecular level, which can be completely dissolved only with difficulty, or after a long time, even at increased temperature, by a purely chemical cleaning, in the case of which a cleaning liquid, such as, for example, an acid, an oxidizing acid, a lye solution or an organic solvent, is led through the measurement duct 30 .
  • a cleaning liquid such as, for example, an acid, an oxidizing acid, a lye solution or an organic solvent
  • an electrochemical cleaning method has been found to be effective, in the case of which a potential difference is placed between the substrate 3 and the counter electrode 4 , while an electrolyte liquid is led both through the measurement duct 30 as well as also through the additional duct section 40 .
  • An electrical current flow due to the potential difference effected between the substrate 3 and the counter electrode 4 through the electrolyte liquid leads to dissolution of the sensor matrix, in given cases, covalently or non-covalently cross linked at the molecular level, including the thereon bound, additional molecules.
  • the substrate 3 , the counter electrode 4 and the reference electrode 2 are embodied in the form of a three electrode circuit, wherein, by means of a potentiostatic circuit, a potential difference between the reference electrode 2 and the substrate 3 is set and controlled by means of the counter electrode 4 .
  • a constant potential difference can be applied, for example, for a predetermined length of time. It has found to be especially protective of the metal coating forming the substrate 3 , when the potential difference applied between the substrate 3 and the reference electrode is varied continuously between a maximum value and a minimum value, e.g. by a linear sweep of potential, wherein the maximum value and the minimum value have preferably different signs, so that alternately an oxidizing potential and a reducing potential lies on the substrate 3 .
  • The, especially linear, moving of the potential of the substrate 3 relative to the reference electrode from a starting value to the maximum value, thereafter, to the minimum value and then back to the starting value (or, the other way around) is referred to as a “cycle”.
  • a number of cycles for example, 2 to 100 or even up to 1000, are passed through.
  • the actual conditions selected for the electrochemical cleaning depend, among other things, on the respective compositions of the electrolyte and the sensor matrix, the geometry of the microfluidic system and the arrangement and choice of material of the substrate 3 , respectively the counter electrode 4 . These are, as a rule, ascertained in preliminary experiments. In such case, the cleaning method is to be optimized such that, on the one hand, a complete removal of the sensor matrix is assured, while, on the other hand, the electrodes are not attacked.
  • a purely oxidative cleaning is likewise possible; it has, however, been found that, in such case, the substrate can be damaged.
  • Especially advantageous in the stated ranges of potential is that, at the maximum value, respectively the minimum value, in the presence of an aqueous electrolyte, oxygen, respectively hydrogen, evolution occurs.
  • the formed oxygen, respectively hydrogen contributes, in such case, supportively to the dissolution and decomposition of the sensor matrix components.
  • the electrical potential of the substrate is preferably set, at least at times, at more than +0.6 V, preferably at more than +1.0 V, relative to the potential of a silver/silver chloride, reference electrode.
  • the electrical potential of the substrate is preferably, at least at times, set at less than ⁇ 0.2 V, preferably at less than ⁇ 0.4 V, relative to the potential of a silver/silver chloride, reference electrode. Due to the small overvoltage for the evolution of hydrogen on platinum under these conditions, sufficient hydrogen is produced for a reductive cleaning.
  • the alternation between oxidative and reductive cleaning occurs as a result of a cyclic or periodic movement of the potential.
  • a settable, or changeable, voltage source is used, whose outputs are not referenced to ground potential.
  • maximum electrical current density occurring during a sweep of potential, respectively during a cycle amounts to more than 2 mA/cm 2 , further preferably to more than 20 mA/cm 2 , with reference to the area of the substrate within the microfluidic ducts, especially in the measuring region of the substrate.
  • positive electrical current charge flowed during a regeneration, respectively clearing, of the substrate amounts to more than +0.2 C/cm 2 , further preferably more than +2 C/cm 2 and negative electrical current charge flowed to less than ⁇ 0.2 C/cm 2 , further preferably less than ⁇ 2 C/cm 2 , in each case, with reference to the substrate area within the microfluidic ducts, respectively the measuring region.
  • the positive charge and negative charge flowed through the substrate in the electrolytic regeneration, respectively clearing have magnitudes which are essentially equal, or differ by less than 25%.
  • the electrical current occurring in the case of a linear sweep of potential on the substrate 3 can optionally be registered and evaluated in the form of a cyclic voltammogram.
  • the so measured cyclic voltammograms can especially provide information as to whether residues of the sensor matrix are still bound on the electrically conductive substrate 3 .
  • successful termination of the cleaning phase can be ascertained based on the registered cyclic voltammograms.
  • other voltammetric analytical methods such as, for example, square wave voltammetry or differential pulse voltammetry are suitable for this as well.
  • a cleaning electrolyte for the electrochemical cleaning for example, a 100 mM Na 2 SO 4 solution
  • a cleaning electrolyte for the electrochemical cleaning for example, a 100 mM Na 2 SO 4 solution
  • a cleaning electrolyte for the electrochemical cleaning for example, a 100 mM Na 2 SO 4 solution
  • a cleaning electrolyte for the electrochemical cleaning for example, a 100 mM Na 2 SO 4 solution
  • a 100 mM Na 2 SO 4 solution for example, a 100 mM Na 2 SO 4 solution
  • the leading of the cleaning electrolyte on this flow path is effected by applying a pressure difference between the supply connection 110 and the two drain connections 130 , 140 .
  • a pressure difference between the supply connection 110 and the two drain connections 130 , 140 it is assured that there is no pressure difference between the second supply connection 120 and the drain connections 130 , 140 , so that flow of the Na 2 SO 4 solution in the duct section 20 with the reference electrode 2 is prevented.
  • a diaphragmless electrolyte bridge is formed for the reference electrode, whose reference electrolyte, the KCl solution, is, via the junction, in conductive contact with the Na 2 SO 4 solution serving as cleaning electrolyte, without requiring a porous diaphragm, a gap, a fritted glass or other means physically separating the reference electrolyte from the cleaning electrolyte.
  • a potential difference is applied between the substrate 3 and the counter electrode 4 .
  • This can occur, for example, by applying a certain potential to the substrate 3 relative to the reference electrode 2 .
  • the liquid junction between the KCl electrolyte contained in the duct section 20 and the cleaning electrolyte accommodated in the duct section 10 , 15 at the junction 12 between the duct section 10 and the duct section 20 forms a diaphragmless, electrolyte bridge, via which the reference electrode 2 is connected with the substrate 3 .
  • the electrical current between the substrate 3 and the counter electrode 4 is so set that the desired potential is achieved.
  • Gas bubbles occurring in the electrochemical cleaning of the substrate 3 or the counter electrode 4 are drained in the direction of flow (compare the arrows of FIG. 4 b )) of the electrolyte liquid from the junction 18 via the drains 130 , 140 away from the duct system of the microfluidics system.
  • Equally, also molecules, molecule parts or molecule aggregates released from the substrate 3 are drained from the measurement duct 30 in the direction of flow, so that re-adsorption is prevented.
  • FIGS. 5 a ) to c ) illustrate a preferred embodiment of a microfluidic unit. Equal components are referenced with equal reference characters as used in the previous example.
  • the microfluidic unit includes a first component 1 a ′ ( FIG. 5 a )), a second component 1 b ′ ( FIG. 5 c )) and, arranged between the first component 1 a ' and the second component 1 b ′, an intermediate plate 1 c ′ ( FIG. 5 b )), all of which are shown in FIG. 5 in plan view.
  • the first component 1 a ′ shown in FIG. 5 a ) can be of a transparent material, for example, glass or a plastic. Visible in plan view are the reference electrode 2 and the substrate 3 , which are embodied as films.
  • Serving as reference electrode 2 is e.g. a silver/silver chloride, reference electrode, which was formed of a silver film applied on the component 1 a ', in given cases, via one or more intermediate layers, especially tackifiers.
  • the surface of the silver film was chlorided, for example, by treating with an iron(III) chloride solution.
  • Substrate 3 is likewise applied as a film on the first component 1 a ′, in given cases, via one or more intermediate layers.
  • it can be, for example, a 50 to 500 nm thick platinum layer applied e.g. by means of sputtering.
  • the platinum layer serves as a thiophilic substrate for binding a sensor matrix by means of the forming of a platinum sulfur binding of a first chemical species forming a first binding layer.
  • Substrate 3 and the reference electrode 2 are electrically connectable (via connections not shown in greater detail) with e.g. a measurement circuit or a voltage source, e.g. a potentiostat or galvanostat.
  • the regions 5 and 5 ′ of the component 1 a ′ are not coated with an electrically conductive material.
  • the regions 5 and 5 ′ can be coated with a chemically stable and electrically insulating material, such as, for example, silicon nitride Si 3 N 4 , in given cases, this being done only in the regions adjoining the electrodes.
  • a coating is applied, for instance, 10 to 100 ⁇ m overlapping on the electrode surfaces and so can effectively avoid an underetching of the electrodes, thus, for example, an attacking of a bonding aid, such as titanium dioxide, during the electrochemical clearing of the substrate 3 .
  • 110 , 110 ′ and 120 are the supply connections of the microfluidic unit and with 130 and 140 the drain connections of the microfluidic unit. These are executed in the illustrated example as bores, wherein, on the surface not shown in the view, for example, conventional connectors can be mounted, e.g. by adhesion, for the connection of microfluidic hose lines.
  • FIG. 5 c shows the second component 1 b ′ of the microfluidic unit in plan view.
  • the second component 1 b ′ can, as the first component 1 a ′, be formed of a transparent material, for example, glass or plastic, wherein electrodes 2 ′, 4 and 4 ′ as well as electrically insulating regions 6 , 6 ′ are provided on the second component 1 b ′ in the form of films, in given cases, via intermediate layers, especially bonding promoting, intermediate layers.
  • the reference electrode 2 ′ is embodied equally to the reference electrode 2 applied on the first component 1 a '.
  • the counter electrode includes a first counterelectrode region 4 and a second counterelectrode region 4 ′.
  • the first counterelectrode region 4 is formed of an electrically conductive, chemically inert substance, e.g. boron-doped diamond or platinum, applied in the form of a complete film.
  • the counter electrode is embodied in an interrupted manner.
  • such is a lined structure connected laterally with the counterelectrode region 4 , e.g. constructed of lines of, for instance, 10 ⁇ m width, which are arranged in parallel at mutual separations of, for instance, 20 ⁇ m.
  • other dimensions and geometries, for example, a lattice structure would be applicable here.
  • an optical signal transducer is arranged below a region or a portion of the counter electrode 4 ′, whose registration region is formed by the measuring region 7 indicated in FIG. 5 c ) by the circular area.
  • the signal transducer can have, for example, a photodiode, which registers chemiluminescent light catalytically produced by the enzyme marker and converts such into a proportional electrical current signal.
  • the counter electrode 4 , 4 ′ and the reference electrode 2 ′ have connections, not shown in greater detail, for electrical contacting.
  • the reference electrode 2 formed on the first component 1 a ′ and the reference electrode 2 ′ formed on the second component 1 b ' can be electrically conductively connected with one another and/or contacted by a shared connection from the measurement circuit, for example, a potentiostat or galvanostat.
  • the perforated intermediate plate 1 c ′ shown in FIG. 5 b comprises, for example, an elastic, 125 ⁇ m thick mat of polydimethyl siloxane (PDMS).
  • PDMS polydimethyl siloxane
  • the base surface of the first component 1 a ′ facing the viewer in FIG. 5 a ) and the base surface of the second component 1 b ' facing the viewer in FIG. 5 c ) are arranged facing one another, spaced from one another and lying opposite one another, and the intermediate plate 1 c ′ is interposed aligned with the components 1 a ' and 1 b ′ as spacing element between the two components 1 a ' and 1 b '.
  • the intermediate plate I c′ is formed of an elastic material
  • the first 1 a ' and second components 1 b ' can be connected together liquid tightly by pressing both sides against the intermediate plate 1 e arranged therebetween and so form the microfluidic unit with a duct structure sealed relative to the environment.
  • the width of the ducts amounts in the illustrated example to 500 ⁇ m.
  • the duct forming, perforated intermediate plate is produced in a photolithographic process from a photosensitive material, for example, from a photoresist.
  • the intermediate plate 1 c ′ is fixedly connected with the first and second components 1 a ′, respectively 1 b′.
  • the so formed duct structure will now be described as follows.
  • the reagents, respectively reagent solutions, required for constructing the sensor matrix (step i), performing the measuring (step ii) and regenerating, respectively clearing, the substrate (step iii) can be fed via the connections 110 and 110 ′ from supply containers, or sample feed supplies and, in given cases, mixing systems (not shown).
  • the microfluidic unit can also have other connections (not shown).
  • the reagents, or reagent solutions, which can interact with one another are fed to the microfluidic unit via two different supply lines. In this way, it is prevented that such components interact with one another already in the supply system, e.g.
  • the duct section 10 ′ coming from the connection 110 ′ meets at the duct junction 12 the duct section 10 coming from the connection 110 .
  • the liquid can be led via the duct section 40 into a waste containment (not shown) connected to the drain connection 140 , this corresponding to a bypassing of the measuring region.
  • the external circuitry of the microfluidic unit leads the flow correspondingly via liquid drive means and valves.
  • An advantage of the bypass formed by the duct section 40 is that the reagents, respectively reagent solutions, can, in this way, first be guided past the actual measuring unit, until initially possibly contained gas bubbles are removed from the supply systems and until, in the case of replacement of a first reagent, respectively first reagent solution, by a second, the resulting reagent, respectively reagent solution, concentration gradient of transition to the second reagent, respectively reagent solution, has passed through the duct system or at least the duct junction 12 .
  • the measurement duct 30 is meander shaped in the present example, wherein preferably all ducts of the microfluidic unit have the same cross section. In this way, gas bubbles, when introduced externally into the microfluidic unit and, above all, those which arise in the case of an electrochemical regeneration, respectively clearing, of the substrate, can very simply and with decreased danger, that they cling on the duct walls in regions with greater duct cross section, be moved back out of the microfluidic unit.
  • the measurement duct 30 meets at the junction 18 an additional duct section 20 , through which via the supply connection 120 reagents for assuring a stable reference potential of the reference electrodes 2 and 2 ′, for example, a 3 -molar potassium chloride solution, can be fed.
  • the reference electrodes 2 are in electrical contact with the substrate 3 and the counter electrode 4 and 4 ′ via a diaphragmless electrolyte bridge.
  • the gas bubbles arising during the electrochemical regeneration, respectively clearing, of the substrate interrupt this electrical contact between the reference electrode 2 , 2 ′ with the substrate 3 , connected as working electrode, and the counter electrode 4 , 4 ′ only momentarily, when a gas bubble is located at the end of the measurement duct 30 , shortly before the duct junction 18 .
  • the microfluidic unit according to FIGS. 5 a ) to c ) illustrates the geometric relationships between measurement duct, measuring region, substrate and counter electrode.
  • the measurement duct 30 is the region of the microfluidic system, in which the sensor matrix of the bioanalyzer, in the case of the leading through of the reagents required for its construction, is built in always equal and reproducible manner on the therein arranged substrate. In the microfluidic unit according to FIGS. 5 a ) to c ), this is the duct region 30 up to the end of the substrate, shortly before the duct junction 18 .
  • the preliminary movement of the reagents, respectively reagent solutions, through these duct sections leads to construction of a sensor matrix. This can, however, vary in its composition and density or after the passing through of gas bubbles be even partially or completely without function.
  • the substrate and the counter electrode cover supplementally, besides the measurement duct in the example shown in FIG. 5 , also the duct regions 10 , 10 ′ and 40 as well as the duct junctions 11 and 12 . Also here, other geometries are possible. Necessary for an operation of the microfluidic unit of the invention is that the substrate be present at least partially at least in the measuring region.
  • the measurement duct containing the substrate, on which the complete sensor matrix is constructed in reproducible manner can, however, also extend supplementally outside of the region 7 registered by the signal transducer.
  • An example of this is the replacement of the photodiode in the example of the microfluidic unit illustrated in FIGS. 5 a ) to c ) for registering the chemiluminescent signal caused by the enzyme marker with an optical apparatus for measuring transmission, which can be arranged, for example, on both sides of the microfluidic unit and determine transmission at the end of the duct section 30 , shortly before the duct junction 18 .
  • the entire region of the duct section 30 covered by substrate is used and no longer only the region in the registration area of the photodiode as measuring region, since this entire region is utilized for obtaining the measurement signal in this case of a transmission change of the enzyme substrate solution led through the measurement duct 30 and checked with the assistance of a signal transducer connected after the measurement duct.
  • the substrate 3 connected as working electrode and the counter electrode 4 , 4 ′ are so arranged relative to the microfluidic ducts that the duct sections 10 , 10 ′, 30 and 40 as well as the duct junctions 11 and 12 are covered by these liquid tightly. In this way, it is assured that in these regions a complete clearing of the substrate surface by electrochemical regeneration, respectively cleaning, occurs. Through the approximately equal separation between substrate and counter electrode throughout all duct regions, the clearing of the substrate surface likewise occurs largely equally throughout all duct regions and without essential differences in the distribution of the electrical current density flowing through the substrate surface.
  • the counter electrode 4 and 4 ′ can also serve as another substrate for the construction of a sensor matrix.
  • the counter electrode is composed preferably of the same material as the substrate 3 , in the illustrated example thus of platinum. Occurring on this other substrate then during the performing of the steps i to iii are preferably essentially the same processes as on the region of the substrate 3 lying opposite in the microfluidic duct.
  • FIG. 6 shows another embodiment in the form of a microfluidic unit 1000 .
  • electrically conductive, microfluidic small tubes, respectively wires are used as substrate 3 and as counter electrode 4 .
  • the substrate 3 is composed, for example, of a small gold tube and the counter electrode 4 of a gold wire arranged coaxially with the substrate 3 .
  • Advantages of this type of embodiment lie in the simple electrical and microfluidic connectability of these small tubes by means of corresponding holders 1001 , 1001 ′.
  • the small tube 3 serves, in such case, as substrate and the wire 4 as counter electrode and likewise as another substrate. Also, the reversed functionality represents another option.
  • substrate 3 and counter electrode 4 permits through the small separation between substrate and counter electrode high current densities, which enables a long service life also coupled with slight removal of the surface of the substrate and/or the counter electrode in the case of the electrical regeneration, respectively cleaning.
  • microfluidic hoses and small tubes Through corresponding connecting by means of microfluidic hoses and small tubes, also more complex microfluidic units, which are constructed not or only partially as microfluidics chips, can be implemented.
  • the substrate 3 After some cycles, in which the potential of the substrate 3 is moved between the maximum and the minimum values, the substrate 3 is essentially completely cleared. Thereafter, a renewed preparation of a sensor matrix in the above described manner can be formed, in order then to be used for a renewed measuring. In this way, at least 50, or even 100 to 200, measurements can be performed with, in each case, a newly prepared sensor matrix.
  • the sensor matrix is regularly prepared completely anew, it is also possible to perform sequential measurements of various analytes, wherein then, in each case, a sensor matrix having certain receptors is prepared, which are suitable specially for the respectively to be determined analyte.
  • a sensor matrix having certain receptors is prepared, which are suitable specially for the respectively to be determined analyte.
  • different, preferably selectively and specifically binding receptors in each case, for an analyte currently to be detected, or for target molecules applied correspondingly for the analyte detection, can be bound on the binding molecules R 200 .
  • the counter electrode and the substrate can be arranged within one and the same duct lying opposite one another.
  • the reference electrode is arranged in a duct connected via a junction with the measurement duct section, so that it is, as shown in FIG. 4 b , connected conductively with the substrate via a diaphragmless electrolyte bridge during the electrochemical cleaning.
  • a sensor matrix is constructed both on the substrate as well as also on the counter electrode, so that the two can be utilized for generating the measurement signal.
  • the substrate can be transparent for measuring radiation, so that, for example, luminescent radiation produced on the counter electrode and the substrate is transmitted through the substrate and can be registered as measurement signal by a photodiode arranged behind the substrate.
  • bioanalyzer used for monitoring a (quantitative) analyte concentration in an industrial process is the reproducibility of the measurement signals output by the bioanalyzer in the case of sequential measurements with continually interposed, regenerated sensor matrix. Especially, it must be assured that the bioanalyzer, in the case of sequential measurements, between which, in each case, a new preparing of the sensor matrix was performed, in the case of analyte concentration remaining equal, in fact outputs a measurement signal of the same signal strength.
  • the preparing of the sensor matrix is performed such that the degree of coverage of the layer of binding molecules R 200 on the conductive surface section 3 amounts to at least 80%, as above defined.
  • the length(s) of time, during which the second and, in given cases, other preparation solutions is/are led through the measurement duct is/are so selected, that, in each case, a degree of coverage of 80%, as above defined, is achieved.
  • the layer structure of the sensor matrix and, in given cases, the direct or indirect binding of marker bearing molecules on the analyte or on target molecules competing with the analyte for binding locations of the sensor matrix permits, in simple manner, the transfer of technology of methods known from laboratory use for single-use assays to a method executable repeatedly and automatically by means of a bioanalyzer. Described as an example of this here is the transfer of an immunoassay for single-use laboratory execution to an automatically working bioanalyzer for process measurements technology: In the laboratory assay, the receptor layer is produced by adsorption of antibodies against the bovine corona virus (BCV) onto the hydrophobic substrate surface of a microtiter plate.
  • BCV bovine corona virus
  • the measurement sample which is to be examined for BCV-components, is applied and subsequently rinsed, so that BCV not bound on the receptors is removed. Thereafter, a second antibody against another epitope of the BCV is applied, rinsed anew and then a conjugate of a horseradish peroxidase (HRP)-conjugated, third antibody applied.
  • HRP horseradish peroxidase
  • step a a suitable binding structure for the construction of the binding layer, or binding layers, of the sensor matrix is to be selected. Since the antibody against BCV (anti BCV) serving as receptor R 350 concerns a non-biotinylated antibody of the class IgG, the FIG.
  • step b layer sequence thiol-PEG-biotin-Neutravidin biotin-anti-IgG anti-BCV is quite suitable, wherein anti-BCV serves as receptor R 350 .
  • step b in the case of application of an HRP molecule as marker bearing molecule R 500 , besides the chromophoric ABTS reaction with following photometric determination, also the chemiluminescent reaction between luminol and hydrogen peroxide can be used. This has, in an automated bioanalyzer, the advantage that only one photodetector, such as, for example, a photodiode, is required as signal transducer.
  • a suitable blocking reagent is selected for the materials used in the automated bioanalyzer for liquid lines or other parts coming in contact with the liquid sample or the other used reagents, in order to reduce the unspecific adsorption of the components.
  • binding layer or binding layers, has/have been defined and an alternative determination method selected, there results a binding layer sequence of the sensor matrix selected from a few, universal, standard variants and formed from:
  • the receptor layer can be successively constructed while omitting, in each case, one layer and the measurement signal determined, which, in the case of occurrence of a cross reactivity, amounts to a value markedly over zero.
  • the density of the individual layers, preferably the primary binding layer is to be set by cobinding a further component non-functionalized or non-reactive relative to the following layer.
  • the automated bioanalyzer has a microfluidic duct system for liquid transport, including a measurement duct, in which the sensor matrix is formed and the analyte detection performed, additionally, the suitable volume flows, the concentrations of preparation solutions containing the chemical species used for forming the sensor matrix, and the lengths of time, during which the individual preparation solutions and, in given cases rinse solutions, are led through the measurement duct, are to be ascertained (step d).
  • the concentrations are selected to be high, the volume flows of the preparation solutions likewise high and the lengths of length of time long.
  • each component to be examined is varied relative to its concentration in the preparation solution, the volume flow rate and the length of time, during which the preparation solution is led through the measurement duct.
  • the sensor matrix embodied under the, in each case, selected conditions is supplied with a measured solution with a known analyte concentration and, by means of the selected detection method, based on the signal transducer of the bioanalyzer, a measurement signal is registered.
  • the method parameters for performing the regeneration, respectively clearing, of the substrate are to be determined in an analogous manner (step e). For this, first of all, it is established whether a cleaning by chemical means, for example, by leading a cleaning solution through the measurement duct for essentially complete clearing of the substrate will be sufficient, or whether an electrochemical cleaning should be executed, in the case of which, besides the leading of the cleaning solution through the measurement duct also the potential of the substrate relative to a counter electrode, respectively reference electrode, is varied in such a manner that, in given cases, supported by hydrogen, respectively oxygen, formation by electrolysis of the cleaning solution, an at least partial decomposition of the sensor matrix, and, in given cases, molecules bound thereon, occurs.
  • the method parameters, concentration/composition of the cleaning solution, potential of the substrate, respectively electrical current level of the electrical current flowing between substrate and counter electrode, length of time of the leading of the cleaning solution through the measurement duct, in given cases, length of time of the leading of auxiliary liquids through the measurement duct, curve of the substrate potential, respectively of the electrical current flowing between the substrate and the counter electrode can be varied, and, based on a measurement signal, which represents the degree of clearing of the substrate, the regeneration success can be determined. From a corresponding matrix, in which the cleaning success is plotted as a function of different process parameters, optimal method parameters can be determined.
  • step f matrix effects resulting from the particular measured medium.
  • suitable countermeasures for the particular case can be ascertained, such as, for example, dilution of the sample or taking into consideration the measured value deviations in the subsequent calibration of the method.
  • a corresponding validation occurs (step g). This includes reviewing storage stability of the preparation solutions and other applied reagents.
  • affinity assays for quality control for example, in a biotechnological production plant, applied in usual laboratory practice, for example, performed as a single-use, laboratory method, can be efficiently transferred to an automated bioanalyzer.
  • a substance for example, a protein or, as in the above example, a vaccine
  • the application of one or a few standard layer systems for binding of almost any receptor and/or the application of one or a few markers or molecules offer a platform, or construction kit, solution, to which, in the described manner, known laboratory methods can be transferred with relatively little effort.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Electrochemistry (AREA)
  • Materials Engineering (AREA)
  • Metallurgy (AREA)
  • Organic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US13/881,907 2010-10-29 2011-08-25 Method for determining an analyte content of a liquid sample by means of a bioanalyzer Abandoned US20130217003A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE102010043153 2010-10-29
DE102010043153.2 2010-10-29
DE102010064392.0 2010-12-30
DE102010064392A DE102010064392A1 (de) 2010-10-29 2010-12-30 Verfahren zur Bestimmung eines Analytgehalts einer Flüssigkeitsprobe mittels eines Bioanalysators
PCT/EP2011/064686 WO2012055607A1 (fr) 2010-10-29 2011-08-26 Procédé de détermination d'une teneur en analyte dans un échantillon de liquide à l'aide d'un bioanalyseur

Publications (1)

Publication Number Publication Date
US20130217003A1 true US20130217003A1 (en) 2013-08-22

Family

ID=44651684

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/881,907 Abandoned US20130217003A1 (en) 2010-10-29 2011-08-25 Method for determining an analyte content of a liquid sample by means of a bioanalyzer
US13/881,658 Active 2033-08-27 US10036098B2 (en) 2010-10-29 2011-08-26 Method for determining an analyte content of a liquid sample by means of a bioanalyzer

Family Applications After (1)

Application Number Title Priority Date Filing Date
US13/881,658 Active 2033-08-27 US10036098B2 (en) 2010-10-29 2011-08-26 Method for determining an analyte content of a liquid sample by means of a bioanalyzer

Country Status (5)

Country Link
US (2) US20130217003A1 (fr)
EP (2) EP2633319B1 (fr)
CN (2) CN103189746B (fr)
DE (2) DE102010064392A1 (fr)
WO (2) WO2012055607A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130213821A1 (en) * 2010-10-29 2013-08-22 Michael Hanko Method for determining an analyte content of a liquid sample by means of a bioanalyzer
US20160143542A1 (en) * 2014-11-24 2016-05-26 Ecole polytechnique fédérale de Lausanne (EPFL) Minimally Invasive Optical Photoacoustic Endoscopy with a Single Waveguide for Light and Sound
US20170052117A1 (en) * 2015-08-18 2017-02-23 Lumasense Technologies Holdings, Inc. Optode sensor with integrated reference
US10078077B2 (en) 2013-05-28 2018-09-18 Endress+Hauser Conducta Gmbh+Co. Kg Apparatus for automated determining of at least two different process parameters
EP3653121A1 (fr) * 2018-11-14 2020-05-20 Koninklijke Philips N.V. Unité de capteur, dispositif de surveillance de fluides corporels et procédé de détection d'un analyte
WO2021054262A1 (fr) * 2019-09-20 2021-03-25 Terumo Kabushiki Kaisha Procédé d'évaluation de l'état d'enrobage de l'adsorbant d'un échantillon de protéine ou de l'état d'adsorption d'un échantillon de protéine
EP3910336A1 (fr) * 2020-05-13 2021-11-17 Koninklijke Philips N.V. Prévision/détection de durée de vie de capteur de biomarqueur

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102014106918A1 (de) 2013-05-28 2014-12-04 Endress + Hauser Conducta Gesellschaft für Mess- und Regeltechnik mbH + Co. KG Verfahren und Vorrichtung zur automatisierten Durchführung von affinitätsbasierten Assays
DE102013105491A1 (de) 2013-05-28 2014-12-04 Endress + Hauser Conducta Gesellschaft für Mess- und Regeltechnik mbH + Co. KG Verfahren und Vorrichtung zur Primärsignalaufnahme für ein Affinitäts-Assay
JP6823589B2 (ja) * 2014-09-11 2021-02-03 キュー ヘルス インコーポレイテッド 被分析物の検出および定量のためのシステム
FI128124B (en) * 2016-04-25 2019-10-15 Teknologian Tutkimuskeskus Vtt Oy Optical sensor, system and methods
BR112018074349A2 (pt) * 2016-06-10 2019-03-06 Unilever Nv máquina, método para o controle de qualidade de uma formulação de fluído e método para o controle de uma máquina
EP3541314A4 (fr) * 2016-11-16 2020-06-17 University of Washington Systèmes et procédés d'imagerie par fluorescence cyclique
DE102016123700A1 (de) * 2016-12-07 2018-06-07 Endress+Hauser Conducta Gmbh+Co. Kg Sensor zur Bestimmung einer von einer Konzentration reaktiver Sauerstoffspezies abhängigen Messgröße
AU2018353924A1 (en) 2017-12-29 2019-07-18 Clear Labs, Inc. Automated priming and library loading device
DE102018111822B4 (de) * 2018-05-16 2021-10-07 Microfluidic Chipshop Gmbh Fluidisches System zur Aufnahme, Abgabe und Bewegung von Flüssigkeiten, Verfahren zur Verarbeitung von Fluiden in einem fluidischen System
EP3889577B1 (fr) * 2018-11-26 2024-01-03 National Institute for Materials Science Procédé d'analyse d'échantillon liquide
DE102020107645A1 (de) * 2020-03-19 2021-09-23 Bruker Daltonics GmbH & Co. KG Betrieb einer Mikrofluidik-Vorrichtung bei der Analyse von Probesubstanzen
TWI765383B (zh) * 2020-10-23 2022-05-21 陳文亮 生物晶片檢測裝置及其生物傳感器平台、製法和應用
AU2022264303A1 (en) * 2021-04-26 2023-10-12 Illumina Cambridge Limited Flow cells

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040028679A1 (en) * 2000-11-30 2004-02-12 Masaya Kakuta Method of measuring binding activity of ligand-binding protein having poor chemical stability to first ligand
US20100240070A1 (en) * 2007-11-07 2010-09-23 Beckman Coulter, Inc. Nonseparation Assay Methods Using Peroxide Generating Enzymes
US20130213821A1 (en) * 2010-10-29 2013-08-22 Michael Hanko Method for determining an analyte content of a liquid sample by means of a bioanalyzer

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4271140A (en) * 1978-01-23 1981-06-02 Baxter Travenol Laboratories, Inc. Method and composition for double receptor, specific binding assays
GB9810350D0 (en) 1998-05-14 1998-07-15 Ciba Geigy Ag Organic compounds
US6942771B1 (en) * 1999-04-21 2005-09-13 Clinical Micro Sensors, Inc. Microfluidic systems in the electrochemical detection of target analytes
AU2001246943A1 (en) * 2000-03-31 2001-10-30 Nederlandse Organisatie Voor Toegepast- Natuurwetenschappelijk Onderzoek Tno Method for determination of binding with natural receptors
DE10123803C1 (de) * 2001-05-16 2002-10-31 Roche Diagnostics Gmbh Elektrochemische Messzelle
WO2003011768A2 (fr) * 2001-08-01 2003-02-13 Datascope Investment Corp. Dispositif microfluidique d'analyse moleculaire
DE10210051B4 (de) 2002-03-07 2005-10-13 Siemens Ag Vorrichtung zum elektrochemischen Nachweis einer Nukleotidsequenz, Analyse-Kassette für eine solche Vorrichtung und Verfahren zur Herstellung einer solchen Analyse-Kassette
US20040081384A1 (en) 2002-10-25 2004-04-29 Datesman Aaron M. Multiple-mode planar-waveguide sensor, fabrication materials and techniques
US20090054255A1 (en) * 2004-07-01 2009-02-26 The Regents Of The University Of California Microfluidic devices and methods
CN101454461A (zh) * 2005-11-16 2009-06-10 Ambrx公司 包括非天然氨基酸的方法和组合物
GB0704151D0 (en) * 2007-03-03 2007-04-11 Univ Cranfield Sensor
EP2189793A1 (fr) 2008-11-21 2010-05-26 Micronas GmbH Procédé destiné à la régénération d'un biocapteur
CN101446555A (zh) * 2008-12-26 2009-06-03 长沙理工大学 共价固定指示剂染料制备高性能荧光传感器敏感膜的工艺
WO2010088219A2 (fr) * 2009-01-27 2010-08-05 Arizona Board Of Regents For And On Behalf Of Arizona State University Plate-forme de biocapteur réutilisable
CN101812093B (zh) * 2009-02-25 2012-12-26 中国科学院理化技术研究所 多吡啶铂配合物的一维微米或亚微米材料及其制备方法和用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040028679A1 (en) * 2000-11-30 2004-02-12 Masaya Kakuta Method of measuring binding activity of ligand-binding protein having poor chemical stability to first ligand
US20100240070A1 (en) * 2007-11-07 2010-09-23 Beckman Coulter, Inc. Nonseparation Assay Methods Using Peroxide Generating Enzymes
US20130213821A1 (en) * 2010-10-29 2013-08-22 Michael Hanko Method for determining an analyte content of a liquid sample by means of a bioanalyzer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Balasubramanian et al., “Electrochemical Desorption of Proteins from Gold Electrode Surface,” Electroanalysis, 2006, vol. 18, issue 19-20, pp. 1885–1892 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130213821A1 (en) * 2010-10-29 2013-08-22 Michael Hanko Method for determining an analyte content of a liquid sample by means of a bioanalyzer
US10036098B2 (en) * 2010-10-29 2018-07-31 Endress+Hauser Conducta Gmbh+Co. Kg Method for determining an analyte content of a liquid sample by means of a bioanalyzer
US10078077B2 (en) 2013-05-28 2018-09-18 Endress+Hauser Conducta Gmbh+Co. Kg Apparatus for automated determining of at least two different process parameters
US20160143542A1 (en) * 2014-11-24 2016-05-26 Ecole polytechnique fédérale de Lausanne (EPFL) Minimally Invasive Optical Photoacoustic Endoscopy with a Single Waveguide for Light and Sound
US20170052117A1 (en) * 2015-08-18 2017-02-23 Lumasense Technologies Holdings, Inc. Optode sensor with integrated reference
US9696259B2 (en) * 2015-08-18 2017-07-04 Lumasense Technologies Holdings, Inc. Optode sensor with integrated reference
EP3653121A1 (fr) * 2018-11-14 2020-05-20 Koninklijke Philips N.V. Unité de capteur, dispositif de surveillance de fluides corporels et procédé de détection d'un analyte
WO2020099570A1 (fr) 2018-11-14 2020-05-22 Koninklijke Philips N.V. Unité de capteur, dispositif de surveillance de fluide corporel et procédé de détection d'un analyte
WO2021054262A1 (fr) * 2019-09-20 2021-03-25 Terumo Kabushiki Kaisha Procédé d'évaluation de l'état d'enrobage de l'adsorbant d'un échantillon de protéine ou de l'état d'adsorption d'un échantillon de protéine
EP3910336A1 (fr) * 2020-05-13 2021-11-17 Koninklijke Philips N.V. Prévision/détection de durée de vie de capteur de biomarqueur
WO2021228705A1 (fr) 2020-05-13 2021-11-18 Koninklijke Philips N.V. Prédiction/détection de durée de vie d'un capteur de biomarqueur

Also Published As

Publication number Publication date
DE102010064392A1 (de) 2012-05-03
EP2633320B1 (fr) 2018-05-02
EP2633319B1 (fr) 2018-05-23
EP2633320A1 (fr) 2013-09-04
CN103189746A (zh) 2013-07-03
EP2633319A1 (fr) 2013-09-04
CN103201630B (zh) 2016-08-10
WO2012055606A1 (fr) 2012-05-03
US10036098B2 (en) 2018-07-31
CN103189746B (zh) 2016-03-02
US20130213821A1 (en) 2013-08-22
WO2012055607A1 (fr) 2012-05-03
CN103201630A (zh) 2013-07-10
DE102010064391A1 (de) 2012-05-03

Similar Documents

Publication Publication Date Title
US10036098B2 (en) Method for determining an analyte content of a liquid sample by means of a bioanalyzer
CN109863396B (zh) 用于样品分析的装置和方法
Limbut et al. A reusable capacitive immunosensor for carcinoembryonic antigen (CEA) detection using thiourea modified gold electrode
Pearson et al. Analytical aspects of biosensors
KR100635110B1 (ko) 현장분석용 랩온어칩 및 랩온어칩용 신호탐지기
JP3764779B2 (ja) 凸状領域を用いた分析方法
Susmel et al. Demonstration of labeless detection of food pathogens using electrochemical redox probe and screen printed gold electrodes
US7547384B2 (en) Electrochemical detector systems
Rusling et al. Nanomaterials and biomaterials in electrochemical arrays for protein detection
CN104203808A (zh) 具有纳米结构电极的生物传感器
JP4972295B2 (ja) 免疫分析方法及びバイオチップ
US20130130243A1 (en) Method and device for detecting and quantifying an analyte with recycling of the reagents
US9863941B2 (en) Microchip and method for detecting molecules and molecular interactions
EP2633061B1 (fr) Procédé de détection électrique de biomolécules par dissolution d'un métal et kit d'essai associé
JP2005181350A (ja) 凸状領域を用いた分析方法
EP3950132A1 (fr) Procédé de détection et de quantification d'analytes dans un dispositif microfluidique
AU2003254122A1 (en) Photonic signal reporting of electrochemical events
de Campos et al. An integrated digital microfluidic electrochemical impedimetric lipopolysaccharide sensor based on toll-like receptor-4 protein
Dincer Electrochemical microfluidic multiplexed biosensor platform for point-of-care testing
Tapuhi, Eliana*, Venter, E. Annette** & Kfir Trends in biosensor development and some potential applications
CN105874320A (zh) 用于纳米流体生物传感器的气体排空系统
KR20230057939A (ko) 중력을 이용하는 자성입자 기반 바이오칩 및 이를 이용한 분석물질 검출 방법
JP4017420B2 (ja) 生体成分検出方法
Ruijg et al. Biosensor based on enzyme labeling and redox cycling
US20090186421A1 (en) Catalytic Biosensor

Legal Events

Date Code Title Description
AS Assignment

Owner name: ENDRESS + HAUSER CONDUCTA GESELLSCHAFT FUR MESS- U

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HANKO, MICHAEL;EUBISCH, ANGELA;FIKUS, AXEL;SIGNING DATES FROM 20130221 TO 20130301;REEL/FRAME:030297/0064

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION