title: Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase.
abstract: Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes.
authors.affiliation: Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotny Str., Kyiv 03680, Ukraine; Taras Shevchenko National University of Kyiv, 64 Volodymyrska Str., 01003 Kyiv, Ukraine; Institute of Analytical Sciences, UMR5280 CNRS/UCBL/ENS, 5 rue de la Doua, 69100 Villeurbanne, France. Electronic address: kucherenko.i.s@gmail.com.
authors.forename: I S
authors.initials: IS
authors.lastname: Kucherenko
authors.affiliation: Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotny Str., Kyiv 03680, Ukraine; Taras Shevchenko National University of Kyiv, 64 Volodymyrska Str., 01003 Kyiv, Ukraine.
authors.forename: O O
authors.initials: OO
authors.lastname: Soldatkin
authors.affiliation: Institute of Analytical Sciences, UMR5280 CNRS/UCBL/ENS, 5 rue de la Doua, 69100 Villeurbanne, France.
authors.forename: F
authors.initials: F
authors.lastname: Lagarde
authors.affiliation: Institute of Analytical Sciences, UMR5280 CNRS/UCBL/ENS, 5 rue de la Doua, 69100 Villeurbanne, France.
authors.forename: N
authors.initials: N
authors.lastname: Jaffrezic-Renault
authors.affiliation: Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotny Str., Kyiv 03680, Ukraine; Taras Shevchenko National University of Kyiv, 64 Volodymyrska Str., 01003 Kyiv, Ukraine.
authors.forename: S V
authors.initials: SV
authors.lastname: Dzyadevych
authors.affiliation: Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotny Str., Kyiv 03680, Ukraine; Taras Shevchenko National University of Kyiv, 64 Volodymyrska Str., 01003 Kyiv, Ukraine.
authors.forename: A P
authors.initials: AP
authors.lastname: Soldatkin
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D015374:Biosensing Techniques; D003402:Creatine Kinase / Q000097:blood*; D055664:Electrochemical Techniques; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005949:Glucose Oxidase / Q000378:metabolism*; D005976:Glutaral / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism*; D010725:Phosphocreatine / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D010725:Phosphocreatine; D012710:Serum Albumin, Bovine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005949:Glucose Oxidase; D006593:Hexokinase; D003402:Creatine Kinase; D005976:Glutaral
keywords: Acute myocardial infarction; Amperometric biosensor; Creatine kinase; Enzyme activity; Glucose oxidase; Hexokinase