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

Abstract

A method for automated in situ determining an analyte content of a liquid sample by means of a bioanalyzer, wherein a measurement duct has at least one substrate, comprising a repeatedly performable sequence of steps as follows: (i) preparing a sensor matrix, which has a plurality of receptors, which bind the analyte and/or a further target molecule, or bring about a chemical conversion of the analyte or of the further target molecule, leading through the measurement duct a preparation solution of at least a first chemical species, 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, through the measurement duct, wherein analyte contained in the liquid sample or in the liquid to be measured, and/or other target molecules contained in the liquid sample or the liquid to be measured, bind, preferably selectively and specifically, on the receptors or are chemically converted by the receptors, and determining a measured variable correlated with the amount of the target molecules bound or converted by the receptors, and deriving therefrom the analyte content of the liquid sample; and (iii) regenerating, especially clearing, the at least one substrate, wherein the sensor matrix and, in given cases, molecules bound thereto, especially analyte molecules, other target molecules or other molecules, are released from the substrate and/or at least partially decomposed.

1 - 24 . (canceled) 
     
     
         25 . 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, comprising a repeatedly performable sequence of steps as follows for measuring at least within a measuring region:
 (i) 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;   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, and 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/or other target molecules contained in the liquid sample or the liquid to be measured, bind, preferably selectively and specifically, on the receptors or are chemically converted by the receptors, and determining a measured variable correlated with the amount of the target molecules bound or converted by the receptors or with the amount of the analyte bound or converted by the receptors and deriving therefrom the analyte content of the liquid sample; and   (iii) regenerating, especially clearing, the at least one substrate, wherein the sensor matrix and, in given cases, molecules bound thereto, especially analyte molecules, other target molecules or other molecules, are released from the substrate and/or at least partially decomposed.   
     
     
         26 . The method as claimed in  claim 25 , wherein:
 the plurality of first chemical species bound on the substrate forms a first binding layer, and the other functional groups of the first chemical species provide bonding locations for subsequent bonding of receptors.   
     
     
         27 . The method as claimed in  claim 25 , wherein:
 the plurality of first chemical species bound on the substrate forms a first binding layer, and the other functional groups of the first chemical species form binding locations for subsequent binding of one or more further binding layers, wherein an uppermost binding layer provides a plurality of binding locations for subsequent binding of receptors.   
     
     
         28 . The method as claimed in  claim 26 , wherein:
 the binding locations provided for subsequent binding of receptors are selected from a group comprising a few binding locations suitable, in each case, for binding a plurality of different receptors.   
     
     
         29 . The method as claimed in  claim 26 , wherein:
 the provided binding locations comprise binding locations for binding biotin.   
     
     
         30 . The method as claimed in  claim 26 , wherein:
 the provided binding locations comprise immunoglobulin binding locations.   
     
     
         31 . The method as claimed in  claim 30 , wherein:
 the immunoglobulin binding locations are provided by binding a biotinylated, immunoglobulin binding molecule on a binding location for binding biotin.   
     
     
         32 . The method as claimed in  claim 31 , wherein:
 the binding location for binding biotin is a binding location where the plurality of first chemical species bound on the substrate forms a first binding layer, and the other functional groups of the first chemical species provide bonding locations for subsequent bonding of receptors.   
     
     
         33 . The method as claimed in  claim 25 , wherein:
 the 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 is an optical, measured variable, which is ascertained especially based on a luminescence measurement, a reflection measurement or an absorption measurement.   
     
     
         34 . The method as claimed in  claim 25 , wherein:
 the analyte or the other target molecule is marked directly or indirectly with a marker, which serves for introducing a property influencing the 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.   
     
     
         35 . The method as claimed in  claim 34 , wherein:
 the marker is selected from a group comprising enzymes, fluorophores and metal or semiconducting, nano particles.   
     
     
         36 . The method as claimed in  claim 35 , wherein:
 the marker is bound via at least one affine interaction, preferably by means of an antibody and/or a protein tag, directly or indirectly on the target molecule (especially a competitor) to be marked or on the target molecule after binding of the target molecule to be marked on the sensor matrix.   
     
     
         37 . The method as claimed in  claim 36 , wherein:
 the marker is bound via the at least one affine interaction by means of a protein-tag directly or indirectly on the target molecule to be marked or on the target molecule after binding of the target molecule on the sensor matrix; and   the protein tag is selected from a group comprising a few tags markable, directly or indirectly, via, in each case, the same affine interaction (i.e. via the same interface).   
     
     
         38 . The method as claimed in  claim 37 , wherein:
 the tag is selected from a group comprising highly affine protein tags, especially the HA-tag, the His-tag, and fluorescing proteins, especially GFP.   
     
     
         39 . The method as claimed in  claim 37 , wherein:
 the tag and/or the target molecule to be marked is a recombinantly manufactured, protein or peptide.   
     
     
         40 . The method as claimed in  claim 35 , wherein:
 the at least one substrate is an electrically conductive substrate.   
     
     
         41 . The method as claimed in  claim 40 , wherein:
 the regenerating, especially the clearing, of the substrate occurs through 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.   
     
     
         42 . The method as claimed in  claim 41 , wherein:
 the counter electrode is embodied as another substrate, on which binds the functional group of the first chemical species binding on the substrate, and is arranged in the measurement channel, especially lying opposite the substrate or coaxially with the substrate.   
     
     
         43 . The method as claimed in  claim 25 , wherein:
 the at least one substrate comprises a thiophilic, electrically conductive substrate and the functional group of the first chemical species binding on the substrate comprises a thiol, or disulfide, group, wherein a layer of the first chemical species is formed by binding of the sulfur atoms of the thiol, or disulfide, groups on the substrate.   
     
     
         44 . A bioanalyzer for performing a method comprising the steps of: (i) 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; 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, and 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/or other target molecules contained in the liquid sample or the liquid to be measured, bind, preferably selectively and specifically, on the receptors or are chemically converted by the receptors, and determining a measured variable correlated with the amount of the target molecules bound or converted by the receptors or with the amount of the analyte bound or converted by the receptors and deriving therefrom the analyte content of the liquid sample; and (iii) regenerating, especially clearing, the at least one substrate, wherein the sensor matrix and, in given cases, molecules bound thereto, especially analyte molecules, other target molecules or other molecules, are released from the substrate and/or at least partially decomposed, the bioanalyzer comprising:
 a microfluidic system having at least one measurement duct; and   means for leading liquid through the measurement duct;   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 an amount of target molecules bound on the receptors or on an amount of converted target molecules, wherein:   said 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.   
     
     
         45 . The bioanalyzer as claimed in  claim 44 , wherein:
 said substrate is electrically conductive and a counter electrode is arranged within the microfluidic system in conducting contact with the substrate, at least during the regeneration, especially clearing, standing; and   the bioanalyzer further includes means for producing an electrical current flow between the substrate and the counter electrode.   
     
     
         46 . The bioanalyzer as claimed in  claim 45 , wherein:
 said counter electrode is arranged within the measurement duct lying opposite the first substrate or extending coaxially with the substrate, and preferably serves as another substrate.   
     
     
         47 . A method for adapting a single-use laboratory method for determining an analyte content of a liquid sample by means of an affinity bioassay for application, especially multiply repeated application, in a bioanalyzer comprising: a microfluidic system having at least one measurement duct; and means for leading liquid through the measurement duct; 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 an amount of target molecules bound on the receptors or on an amount of converted target molecules, wherein: said 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, the method comprising steps as follows:
 (a) selecting a suitable binding structure for constructing one or more binding layers for binding a receptor of the affinity bioassay, and setting a density of the binding structure; and   (b) optionally selecting an alternative determination method and/or providing an alternative additional target molecule and/or a method for direct or indirect introduction of a marking;   (c) reviewing all components of the preferably multiply usable affinity bioassay for mutual cross reactivity;   (d) ascertaining suitable method parameters of the bioanalyzer, especially volume flows, concentrations and lengths of time for leading liquids for performing a measurement with the preferably multiply usable affinity bioassay;   (e) checking and, in given cases, adjusting method parameters of the bioanalyzer for performing regeneration, respectively clearing, of the substrate;   (f) optionally reviewing matrix effects of the liquid sample or the measured liquid; and   (g) creating a calibration curve and validating the method.   
     
     
         48 . The method as claimed in  claim 47 , wherein:
 the affinity bioassay does not serve for determining deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).