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WO2012050536A1 - Méthode de détermination quantitative de l'adénosine diphosphate - Google Patents

Méthode de détermination quantitative de l'adénosine diphosphate Download PDF

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Publication number
WO2012050536A1
WO2012050536A1 PCT/SK2010/050018 SK2010050018W WO2012050536A1 WO 2012050536 A1 WO2012050536 A1 WO 2012050536A1 SK 2010050018 W SK2010050018 W SK 2010050018W WO 2012050536 A1 WO2012050536 A1 WO 2012050536A1
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adenosine
adenosine diphosphate
adp
determining
diphosphate
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PCT/SK2010/050018
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English (en)
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Miroslav Stredansky
Silvia Stredanska
Pavol Szomolanyi
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Biorealis, S.R.O.
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Priority to PCT/SK2010/050018 priority Critical patent/WO2012050536A1/fr
Publication of WO2012050536A1 publication Critical patent/WO2012050536A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP

Definitions

  • This invention relates to chemical and biochemical analyses, particularly, this invention concerns a way of a quantitative detection of adenosine diphosphate (hereinafter abbreviated as ADP) using a coupled enzymatic reactions in various formats.
  • ADP adenosine diphosphate
  • This invention is especially advantageous for the determination of ADP in the presence of adenosine triphosphate (hereinafter abbreviated as ATP), for the determination of activities of the enzymes producing and consuming ADP, such as kinases, for the determination of the reaction substrates and inhibitors of the kinases and other involved enzymes.
  • ATP adenosine triphosphate
  • Adenine nucleotides exhibit a great importance in the energy metabolism of living organisms.
  • the accurate measurement of ADP allows the quantitative determination of enzymatic activities of enzymes producing or consuming ADP by their reaction and products or substrates of these enzymes.
  • ADP is converted to ATP by pyruvate kinase in the presence of phosphoenolpyruvate (hereinafter abbreviated as PEP).
  • ATP then react with luciferine and oxygen in the presence of luciferinase to emit light measured by a luminometer.
  • this method is widely used, its suitability for the measurements of the kinase activity and of the products or substrates of these enzymes is very limited, because the added ATP is interfering measurements.
  • US Patent 5916761 describes a method for determining ADP using hexokinase, oxidized nicotinamide adenine dinucleotide (phosphate) [hereinafter abbreviated as NAD(P)], and glucose-6-phosphate dehydrogenase.
  • NAD(P) oxidized nicotinamide adenine dinucleotide
  • the amount of the reduced NAD(P) proportional to the amount of ADP is measured by UV spectrophotometry.
  • An disadvantage of this method is a necessity of the unstable NAD(P) cofactor for the assay.
  • ADP is phosphorylated with kinase enzyme (e.g. galactokinase, formate kinase, pyruvate kinase) in the presence of the corresponding phosphorylated substrate (e.g. galactose- 1 -phosphate, formyl-phosphate, PEP).
  • kinase enzyme e.g. galactokinase, formate kinase, pyruvate
  • the dephosphorylated substrate e.g. galactose, formate, pyruvate
  • the corresponding dehydrogenase e.g.
  • US Patent 7410755 describes the method for determining ATPase activity and determining ADP in the presence of ATP by fluorescent or chemiluminiscent measurements.
  • ADP reacts with PEP using pyruvate kinase to form pyruvate which is oxidised with pyruvate oxidase to form hydrogen peroxide.
  • hydrogen peroxide in the presence of peroxidase oxidises specific dyes to create fluorescent or chemiluminiscent signal proportional to the amount of ADP.
  • ADP has been used also for the determination of the reaction substrates of the kinases.
  • a typical example is acetate.
  • One design of the acetate determination is based on the reaction of acetate with ATP using acetate kinase forming ADP followed by reaction of ADP with PEP using pyruvate kinase to form pyruvate, which oxidises NADH to NAD using lactate dehydrogenase.
  • the depletion of NADH is monitored either by UV spectroscopy (Trivin et al., Clin. Chim. Acta 121, pp. 43-50, 1982) or electrochemically (Tang et al., Biotechnol. Techniques 1 1, pp. 683-687, 1997).
  • UV spectroscopy Trivin et al., Clin. Chim. Acta 121, pp. 43-50, 1982
  • electrochemically Tiang et al., Biotechnol. Techniques 1 1, pp. 683-6
  • Another method determination of acetate is based on the oxidation of pyruvate (formed from acetate as described above) with molecular oxygen using pyruvate oxidase.
  • the depletion of oxygen is monitored by oxygen electrode electrochemically (Mizutani et al., Sensors Actuators B 91, pp. 195-198, 2003).
  • a drawback of this method is a high instability of pyruvate oxidase and its activators, thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), which are also very expensive.
  • creatinine and creatine are known for many years (Fossati et al., Clin. Chem. 29, pp. 1494-1496, 1983; Lindback and Bergman, Clin. Chem. 35, pp. 835-837, 1989; Killard and Smyth, Trends Biotechnol. 18, 433-437, 2000).
  • creatinine is hydrolysed by creatininase to creatine which is further hydrolysed by creatinase to sarcosine.
  • Sarcosine is subsequently oxidised by sarcosine oxidase and this reaction is monitored by various physicochemical ways.
  • Figure 1 calibration curve for ADP determined by the chemiluminiscent method using a luminometer.
  • Figure 2 calibration curve for ADP determined amperometrically by the Clark oxygen electrode.
  • Figure 3 calibration curve for acetate determined spectrophotometrically by the absorbance measurement.
  • Figure 4 calibration curve for oleate determined by fluorescent method using a fluorometer.
  • Figure 5 calibration curve for hexoses determined amperometrically using the multienzyme biosensor.
  • Figure 6 calibration curve for caffeine determined amperometrically using the multienzyme biosensor.
  • the method of the ADP determination described by this invention overcomes many of the problems outlined above.
  • the method requires stepwise or simultaneous action of several enzymes and at least one additive reaction substrate: creatine phosphate (CP).
  • CP creatine phosphate
  • the sequence of enzymatic reactions included in this method is following:
  • the present invention includes also the reagents for the determination of ADP, which reagents comprise creatine kinase, creatine phosphate, creatinase, sarcosine oxidase, and optionally other substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, and other enzymes.
  • the reagent according the present invention can be in dry form, in the form of solution, suspension, paste, film, layer, membrane.
  • the reagent can be also impregnated on to an appropriate carrier or can be incorporated into a test-strip, on a surface electrode or optode.
  • the method of the present invention is particularly suitable for the determining ADP in liquid samples.
  • the enzymes involved in this method are stable and available and can be applied in free form or immobilised at suitable conditions.
  • the reaction mixture can contain all enzymes together with CP, or the enzymes can be added stepwise in the order: creatine kinase, creatinase, sarcosine oxidase.
  • all enzymes with CP are applied together with a measured sample containing ADP and a change of a physicochemical property of the mixture is measured at suitable conditions.
  • Said suitable conditions mean above all the suitable temperature and pH.
  • the temperature is important factor. It should be kept in the range acceptable for all present enzymes.
  • the suitable temperature range is from 5 °C to 50 °C, preferably from 15 °C to 40 °C.
  • Said suitable pH value of the reaction mixture of the present invention can be kept constant in the range acceptable for all present enzymes using buffering salts.
  • the suitable pH range is from 5 to 9.5, preferably from 6.5 to 8.
  • Various buffering salts can be used for the method of the present invention.
  • the preferred buffers include phosphate, Hepes, Tris, citrate, imidazol, MOPS, borate, succinate, acetate, pyrophosphate.
  • Said enzyme activators are selected from the compounds containing Mg 2+ , Ca 2+ , K + , Mn 2+ , Co 2+ , Cu + , Zn 2+ , acetate, tartrate, succinate, citrate, glyoxalate, dithio-threitol.
  • Said enzyme stabilisers are selected from the group of sugars, polyols, aminoacids and organic salts, such as sucrose, lactose, trehalose, sorbitol, lactitol, glycerol, diethylene glycol, low molecular weight polyethylene glycols, glutamate, lysine, glycine, succinate, maleate.
  • Said surfactants are any natural or synthetic compound having detersive properties, preferably selected from the group of biologically compatible neutral surfactants (e.g. Triton® X-100, Tween®, Tergitol®, Brij®, Span®, polyethylene glycol, polyethylene oxide, polypropylene oxide), anionic detergents (e.g. sodium dodecylsulphate, cholate, taurocholate, phospholipide) and cationic surfactants (e.g. cetyltrimethylammonium bromide).
  • biologically compatible neutral surfactants e.g. Triton® X-100, Tween®, Tergitol®, Brij®, Span®, polyethylene glycol, polyethylene oxide, polypropylene oxide
  • anionic detergents e.g. sodium dodecylsulphate, cholate, taurocholate, phospholipide
  • cationic surfactants e.g. cetyltrimethylammonium bromide
  • said electron-acceptor depends on the way of the physicochemical detection.
  • said electron-acceptor when the detection is performed by optical methods, is the compound which change optical properties (increase or decrease of light absorbance or emission) when is reduced by sarcosine oxidase.
  • the preferred electron-acceptors are selected from the group of colorants, fluorescent dyes, chemiluminiscent substrates. More preferably, said electron- acceptor is selected from the group of methylene blue, thionine, neutral red, toluidine blue, Meldola's blue, ferricyanide, ferricinium and its derivates, resorufin derivates, rhodamines, coumarin derivates.
  • said electron-acceptor is oxygen, two ways of the optical detection are possible.
  • the first one is the employment of oxygen sensitive dyes, probes and optrodes, e.g. tris(2,2'-bipyridyl dichlororuthenium), platinum octaethylporphyrin and other metaloporphyrins.
  • the second one is the detection of hydrogen peroxide formed by the reaction of sarcosine with oxygen.
  • the detection of hydrogen peroxide by optical methods is well known in the art.
  • an additive enzyme peroxidase is involved together with an electron-donor which is oxidised with hydrogen peroxide to change optical properties.
  • Said electron-donor is selected from the group of spectrophotometnc peroxidase substrates, fluorescent dyes, chemiluminiscent substrates.
  • said electron-donor is selected from the group of 4-amino-antipyrine, polyphenolic compounds (e.g. pyrogallol, catechol), phenylenediamine, diaminobenzidine, 2,2'-azino-bis(3-ethylbenzthiazoline- sulfonic acid), 4-aminophenazone, 3,5-dichloro-2-hydroxybenzenesulfonic acid, 3- hydroxy-2,4,6-triiodo benzoic acid, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt, 2,4,6-tribromo-3-hydroxybenzoic acid, ferrocyanide, ferrocene and its derivates, resorufin derivates, rhodamines, coumarin derivates, fluorescein derivates, 2,4,5- triphenylimidazoles, 9-alkylidene-N-alkylacridans,
  • said electron-acceptor when the detection is performed by electrochemical methods, said electron-acceptor is the compound having, electroactive properties, it means giving measurable electrochemical signal when is reduced by sarcosine oxidase.
  • Any electrochemical procedure known in art e.g. potentiometric, amperometric, conductometric, coulometric
  • the preferred electron-acceptors for said electrochemical detection are selected from the group consisting of cytochromes, quinones, aminophenols, electron acceptor aromatic compounds (e.g. tetrathiafulvalene and N-methylphenazinium), organic dyes (e.g.
  • toluidine blue Meldola's blue, neutral red, methylene blue and thionin
  • metallocenes organometallic complexes of Os, Ru and V
  • inorganic complexes of Fe e.g. ferricyanide
  • the electron-acceptor is oxygen
  • three methods of the electrochemical detection are possible.
  • the first one is the employment of oxygen sensitive electrode (e.g. Clark electrode, gas permselective electrodes) for the measurement of the oxygen uptake which is proportional to the amount of ADP in the mixture.
  • the second one is a direct electrochemical detection of hydrogen peroxide formed by the reaction of sarcosine with oxygen on the surface of the electrochemical working electrode.
  • This electrode can be done from metal or carbon, preferably it has the active surface from platinum or from carbon nanotubes.
  • the third one is the detection of hydrogen peroxide using an additive enzyme peroxidase together with an electron-donor which is oxidised with hydrogen peroxide to change measured electrochemical signal.
  • said electron-donor is selected from the group consisting of reduced cytochromes, phenolic compounds, aminophenols, electron-donor aromatic compounds (e.g. tetracyano-p-quinodimethane), organic dyes in reduced form, metallocenes (e.g. ferrocene and its denvates), organometallic complexes of Os, Ru and V, inorganic complexes of Fe (e.g. ferrocyanide).
  • Said electrochemical signals deriving from the reduction of said electron-acceptors or the oxidation of said electron-donors are measured by any electrochemical working electrode, preferably electrode having the active surface made from noble metals, carbon materials, conducting polymers.
  • said enzymes and said other substances can be applied in the immobilised form using immobilisation procedures known in the art.
  • Three measuring arrangements are preferred in this case. The first, when said enzymes are immobilised into the surface of inside of carrier particles (e.g. beads), the repeated batch analysis saving material costs can be performed.
  • the ⁇ flow injection analysis can be advantageously carried-out, when the material with said immobilised enzymes are packed in a column or bioreactor or said enzymes are immobilised into the inner surface of tubings or capillaries.
  • biosensors are prepared when said enzymes are immobilised in the vicinity of the surface or directly on the surface of physical tranducers, e.g. said electrochemical electrodes or optrodes.
  • Said enzymes may be also incorporated in the electrode (optrode) material, where these electrodes (optrodes) are made from composite materials. All above mentioned substances, electrodes and optrodes are applicable also for biosensors.
  • the biosensor arrangement is the most preferred embodiment of the present invention, because it allows rapid, accurate, specific, and convenient ADP determination.
  • the method of the ADP determination is suitable also for the determination of AMP (adenosine monophosphate) after its transformation to ADP by myokinase.
  • Myokinase (adenylate kinase) transforms one molecule of AMP and one molecule of ATP to two molecules of ADP which is determined as described above. So the determination of AMP according the present invention requires the presence of said myokinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes.
  • the method of the ADP determination is suitable also for the determination of cyclic AMP (cAMP) after its transformation to ADP by cyclic-3', 5 '-nucleotide phosphodiesterase and myokinase.
  • Cyclic-3 ',5 '-nucleotide phosphodiesterase hydrolyses a molecule of cAMP to AMP and subsequently myokinase transforms one molecule of AMP and one molecule of ATP to two molecules of ADP which is determined as described above.
  • AMP cyclic-3 ',5 '-nucleotide phosphodiesterase, myokinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes.
  • the method of the ADP determination is suitable also for the determination of the enzymatic activity of kinases and the enzymes producing AMP by their catalytic action, e.g. cyclic-3 ',5 '-nucleotide phosphodiesterase and acyl-CoA synthetase.
  • Kinases catalyse the phosphorylation of their specific substrates with a simultaneous transformation of ATP to ADP which is determined as described above.
  • the measurement is preferably carried-out kinetically in the presence of ATP, kinase corresponding substrate, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes.
  • the kinase activity is commonly expressed in ⁇ of ADP forming per 1 min.
  • Table 1 are given examples of kinases which can be measured by the method of the present invention and their corresponding substrates.
  • the measurement is preferably carried-out kinetically in the presence of ATP, corresponding substrate of the determining enzyme, myokinase, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally other substances.
  • the method of the ADP determination is suitable also for the determination of substrates of kinases.
  • Kinases catalyse the phosphorylation of their corresponding substrates with a simultaneous transformation of ATP to ADP which is determined as described above. So the determination of said substrates of kinases according the present invention requires the presence of said corresponding kinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron-acceptors, other enzymes.
  • the invention is suitable also for the determination of inhibitors of kinases.
  • the method of the ADP determination is suitable also for the determination of substrates and inhibitors of the enzymes producing AMP by their catalytic action, e.g. cyclic-3', 5 '-nucleotide phosphodiesterase and acyl-CoA synthetase.
  • the determination of said substrates requires the presence of said corresponding enzyme producing AMP, myokinase, ATP, creatine kinase, creatine phosphate, creatinase, sarcosine oxidase and optionally said substances improving the measurement, such as enzyme activators, enzyme stabilisers, surfactants, electron- acceptors, other enzymes.
  • a concentration of ADP was determined by a chemiluminiscent measurement.
  • the reaction mixture (1.8 ml) contained the reagents in 0.05 M phosphate buffer (pH 7.5) as follows: 2.5 U of creatine kinase (Sigma, St. Louis, USA, Cat. No. C 3755), 0.8 U of creatinase (Sigma, St. Louis, USA, Cat. No. C 2409), 1.5 U of sarcosine oxidase (Sigma, St. Louis, USA, Cat. No. S 7897 ), 5 U of peroxidase (Sigma, St. Louis, USA, Cat. No.
  • a concentration of ADP was determined by a measurement of oxygen depletion using an electrochemical oxygen electrode.
  • the reaction mixture (2.9 ml) contained the reagents in 0.05 M phosphate buffer (pH 7.5) as follows: 4 U of creatine kinase, 1.2 U of creatinase, 2.5 U of sarcosine oxidase, 2 mM creatine phosphate, 1.5 mM magnesium chloride.
  • the reaction mixture was placed into the 3-ml amperometric cell equipped with the Clark oxygen electrode (Amel, Milan, Italy). After the addition the ADP solution (100 ⁇ ) the cell was closed and the current change was measured.
  • the calibration curve for ADP is illustrated in Fig. 2.
  • a concentration of acetate (kinase substrate) was determined by a spectrophotometric measurement.
  • the reaction mixture (1.9 ml) contained the reagents in 0.05 M phosphate buffer (pH 7.5) as follows: 2 U of acetate kinase (Sigma, St. Louis, USA, Cat. No.
  • the biosensor was immersed in 10 ml of 0.2M Tris buffer (pH 9.8) containing 5 mM ATP, 5 mM creatine phosphate, 2 mM magnesium acetate, 5 mM glycerol and a current was monitored at the constant potential of 700 mV against a reference electrode (standard calomel electrode, SCE).
  • the biosensor was calibrated by successive additions of the ADP solution.
  • the measurement of the activity of glycerokinase was performed at the same conditions.
  • the current change was monitored kinetically and activity was expressed in ⁇ of ADP forming by glycerokinase per 1 min.
  • the measured activities of glycerokinase preparations (12.3 U/mg and 475 U/ml, respectively) agreed with th declared by the supplier (11.9 U/mg and 493 U/ml, respectively).
  • a concentration of free fatty acids was determined by a fluorometric measurement.
  • the reaction mixture (0.45 ml) contained the reagents in 0.1 M Tris buffer (pH 7.8) as follows: 0.1 U of acyl-CoA-synthetase (Sigma, St. Louis, USA, Cat. No. A 3352), 1 U of myokinase (Sigma, St. Louis, USA, Cat. No. M 3003), 0.5 U of creatine kinase, 0.2 U of creatinase, 0.4 U of sarcosine oxidase, 1.5 U of peroxidase, 0.5 mM coenzyme A trilithium salt (Sigma, St.
  • a concentration of hexoses was determined by an multienzyme biosensor.
  • a palladium electrode (2 mm diameter; Amel, Milan, Italy) was cleaned and covered with an film of polyvinyalcohol.
  • a solution of enzymes was prepared by mixing hexokinase (10 mg/ml, Sigma, St. Louis, USA, Cat. No. H 6380), creatine kinase (8 mg/ml), creatinase (12 mg/ml), sarcosine oxidase (35 mg/ml ), and glutaraldehyde (0.5 mg/ml). 2 ⁇ of this solution were placed into the surface of the electrode and left to dry for 3 h at room temperature followed by 15 min at 40°C.
  • the biosensor was immersed in 10 ml of phosphate buffer (pH 7.3) containing. 10 mM ATP, 5 mM creatine phosphate, 2 mM magnesium acetate, 4 mM ferricyanide and a current was monitored at the constant potential of 300 mV against a reference electrode (SCE).
  • SCE reference electrode
  • the biosensor was calibrated by successive additions of the equimolar solution of glucose and fructose.
  • the calibration curve for hexoses is illustrated in Fig. 5.
  • a concentration of caffeine (inhibitor of AMP producing enzyme) was determined by an multienzyme biosensor.
  • the planar carbon screen printed electrode (1.6 mm diameter, prepared using carbon printing ink of Gwent Electronic Materials, Ltd., UK, Cat. No. C2000802D2) was cleaned and modified with ferrocene by adsorption.
  • a solution of enzymes with an activator was prepared by mixing cyclic-3', 5 '-nucleotide phosphodiesterase (10 mg/ml, Sigma, St. Louis, USA, Cat. No. P 0520), calmodulin (12,500 U/ml, Sigma, St. Louis, USA, Cat. No.

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Abstract

La présente invention concerne une méthode innovante de détermination de l'ADP. La méthode est basée sur la phosphorylation de l'ADP avec la créatine kinase et la créatine phosphate pour former de la créatine, qui est ensuite transformée par la créatinase en sarcosine, suivie de l'oxydation de la sarcosine par la sarcosine oxydase en présence d'un accepteur d'électrons. La méthode convient à la détermination de l'ADP en présence d'ATP, à la détermination de l'AMP après sa transformation en ADP par la myokinase, à la détermination de l'AMP cyclique après sa transformation en ADP par la cyclic-3',5'-nucléotide phosphodiestérase et la myokinase, à la détermination d'activités, de substrats et d'inhibiteurs des kinases et autres enzymes impliquées. Les enzymes utilisées dans cette invention sont stables et disponibles et peuvent être appliquées sous forme libre ou immobilisée. Le signal proportionnel à la quantité d'ADP est détecté par des méthodes électrochimiques ou optiques.
PCT/SK2010/050018 2010-10-15 2010-10-15 Méthode de détermination quantitative de l'adénosine diphosphate WO2012050536A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN107236785A (zh) * 2017-07-19 2017-10-10 王贤俊 一种肌酸激酶检测试剂盒
CN107267595A (zh) * 2017-07-19 2017-10-20 王贤俊 一种肌酸激酶同工酶检测试剂的制备方法
CN107402305A (zh) * 2017-07-19 2017-11-28 王贤俊 一种肌酸激酶同工酶的定量检测试剂盒

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US4923796A (en) 1978-08-08 1990-05-08 Boehringer Mannheim Gmbh Method for the quantitative enzymatic determination of ADP
US5916761A (en) 1995-12-27 1999-06-29 Asahi Kasei Kogyo Kabushiki Kaisha Method for assaying vital sample
US5891659A (en) 1996-03-04 1999-04-06 Kikkoman Corporation Bioluminescent adenosine phosphate ester assay and reagent
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107236785A (zh) * 2017-07-19 2017-10-10 王贤俊 一种肌酸激酶检测试剂盒
CN107267595A (zh) * 2017-07-19 2017-10-20 王贤俊 一种肌酸激酶同工酶检测试剂的制备方法
CN107402305A (zh) * 2017-07-19 2017-11-28 王贤俊 一种肌酸激酶同工酶的定量检测试剂盒

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