WO2001018237A1 - Enzymes inactives utilisees comme capteurs de non consommation - Google Patents

Enzymes inactives utilisees comme capteurs de non consommation Download PDF

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WO2001018237A1
WO2001018237A1 PCT/US2000/024846 US0024846W WO0118237A1 WO 2001018237 A1 WO2001018237 A1 WO 2001018237A1 US 0024846 W US0024846 W US 0024846W WO 0118237 A1 WO0118237 A1 WO 0118237A1
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enzyme
apo
label
assay
glucose
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PCT/US2000/024846
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English (en)
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Joseph R. Lakowitz
Sabato D'auria
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University Of Maryland, Baltimore
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    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase

Definitions

  • the present invention relates to an assay for the measurement of analytes such as glucose or lactate using an inactive enzyme that binds to the analyte such as an apo- enzyme.
  • analytes such as glucose or lactate
  • an inactive enzyme that binds to the analyte
  • apo-enzymes include inactive forms of glucose oxidase, glucose dehydrogenase, lactate oxidase or lactate dehydrogenase.
  • the invention more specifically relates to the measurement of such analytes, such as by fluorescent measurement.
  • Blood glucose is a clinically important analytes for diabetic health care, because close control of blood glucose is necessary to avoid the long term health effects of diabetes. Close regulation of blood glucose is essential for diabetics to minimize the long term adverse health consequences of diabetes.
  • the Diabetes Control and Complications Trial Research Group (1997) Diabetes 4, 271- 286; The Diabetes Control and Complications Trial Research Group (1993), N Engl. J Med. 329, 977-986.
  • Measurement of blood glucose requires a finger stick, which is done by most diabetics. As a result there is a substantial worldwide effort to develop non-invasive and minimally invasive methods for frequent or continuous monitoring of blood glucose. A wide variety of methods have been tested, including optical rotation (Rabinovitch, B., et al. (1982) Diabetes Care 5(3), 254-258;
  • RET fluorescence resonance energy transfer
  • GGBP glucose-galactose binding protein
  • Glucose oxidase (EC 1.1.3.4) from Aspergillus niger (GO) is an oxidoreductase enzyme that catalyzes the conversion of ⁇ -D-glucose and oxygen to D-glucono-l,5-lactone and hydrogen peroxide, and as probes to monitor the concentrations of glucose.
  • GO Aspergillus niger
  • This GO is a flavoprotein, highly specific for ⁇ -D-glucose (Manstein, D. J., et al.
  • thermostable glucose dehydrogenase as a glucose sensor.
  • biotechnological applications of enzymes are often hampered by their low stability to heat, pH, organic solvents, and proteolysis. Sthal, S. (1993) In Thermostability of Enzymes (Gupta M. N. ed pp 45-74), Springer- Verlag, Berlin; Shoichet,
  • thermophilic enzymes are not only stable and active at high temperature, but they are often stable in the presence of organic solvents and detergents. D'Auria, et al. (1997) Biochem J 323, 833-840.
  • Glucose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum is a tetramer of about 160 kDa composed of four similar subunits of about 40 kDa each.
  • the enzyme shows a Km value of 10 mM for glucose, and it is resistant to high temperatures and organic solvents. At 55°C, full activity is retained after 9 hours, and at 75°C the half-life is approximately 3 hours. Moreover, incubation of the enzyme for up 6 hours at room temperature with 50% (v/v) methanol, acetone or ethanol without any appreciable loss of activity. Smith, L.D., et al. (1989) Biochem J 261, 973-977.
  • L-lactate is removed by gluconogenesis in the liver and to a smaller extent by oxidation in skeletal muscle and the renal cortex. Burtis, C. A., et al. (1999) Tietz Textbook of Clinical Chemistry, W.B. Saunders Co., London, pp. 1917. Normal resting concentrations of blood lactate range from 0.36 to 0.75 mM at rest with somewhat higher values of 0.36 to 1.7 mM for hospitalized patients. Id. Elevated concentrations of blood lactate are indicators of a considerable number of medical conditions.
  • serum lactate levels are predictive of survival in children after open heart surgery (Siegel, L. B., et al. (1996) Intensive Care Med. 22:1418-1423.), mortality in ventilated infants (Deshpande, S. A., et al. (1997) Disease in Childhood 76: FI 5-F20) and may be preferable to pH for evaluating fetal intraparteum asphyxia (Westgren, M., et al. British Journal of Obstetrics and Gynaecology 105:29-33). In adults elevated blood lactate can predict multiple organ failure and death in patients with septic shock (Bakker, J., et al.
  • Lactate determinations are typically performed by enzymatic oxidation to pyruvate by lactate dehydrogenase (LDH) or lactate oxidase, followed by detection of NADH or H O , respectively (Burtis, C.
  • LDH lactate dehydrogenase
  • lactate oxidase lactate oxidase
  • Lactate dehydrogenase is a tetramer with a molecular weight of 136,700 ⁇ 2,100 daltons.
  • a number of isozymes are known to occur as mixed tetramers of the muscle and heart isozymes.
  • the present invention includes a method for assaying a sample suspected of containing an analyte comprising: (a) contacting the sample with a composition comprising an inactive enzyme to form a complex; wherein the analyte is not consumed in the complex, and wherein the enzyme is coupled to the analyte during the contacting step; and
  • Another embodiment of the invention is an assay for assaying a sample suspected of containing an analyte.
  • This assay comprises the sample and an inactive enzyme forming a complex, wherein the analyte reversibly couples to the enzyme, and wherein the inactive enzyme does not consume the analyte.
  • the inactive enzyme comprises a label.
  • the label can be an intrinsic or extrinsic label, and preferably an intrinsic label. More preferably, the label is a fluorescent label, a luminescent label, an enzyme label, a radioactive label, or a chemical label, and more preferably a fluorescent label, most preferably 8-anilino-l -naphthalene sulfonic acid (ANS).
  • ANS 8-anilino-l -naphthalene sulfonic acid
  • the amount or presence of an analyte preferably is measured by emission maxima, emission intensity, spectral shift, energy transfer, anisotropy, polarization, lifetime or wavelength ratio.
  • the preferred analytes for the method or assay include sugar, preferably glucose, or lactate or a substrate for an oxidase or a dehydrogenase.
  • these analytes are found in biological samples such as whole blood, serum or plasma.
  • the inactive enzyme is a mutated enzyme, an apo-enzyme or an inhibited enzyme and is preferably an apo-enzyme.
  • the preferred inactive enzyme is an apo-enzyme such as apo- glucose oxidase, apo-glucose dehydrogenase, apo-lactate dehydrogenase or apo-lactate oxidase.
  • the enzyme is produced from a thermophilic organism.
  • the assay can be a homogenous assay or a heterogeneous assay, and preferably the assay is a homogenous assay.
  • the invention also include a kit for assaying a sample suspected of containing an analyte.
  • the kit includes a composition having an inactive enzyme wherein the analyte is not consumed upon measurement.
  • the kit can be used as an assay to measure analytes such as sugar, and more preferably glucose, or lactate.
  • the inactive enzyme is preferably an apo-enzyme in which a cofactor has been removed therefrom, such as apo-glucose oxidase, apo-glucose dehydrogenase, apo-lactate dehydrogenase or apo- lactate oxidase.
  • the enzyme is produced from a thermophilic organism.
  • the inactive enzyme comprises a label.
  • the label can be an intrinsic or extrinsic label, and preferably an intrinsic label. More preferably, the label is a fluorescent label, a luminescent label, an enzyme label, a radioactive label, or a chemical label, and more preferably a fluorescent label, most preferably 8-anilino-l- naphthalene sulfonic acid (ANS).
  • the sample is preferably a biological sample.
  • the kit also includes instructions for measuring the amount or presence of the analyte in a sample.
  • Figure 1 Absorption and emission spectra of apo-glucose oxidase. Excitation at 298 nm. The protein concentration was 0.05 mg/ml.
  • Figure 2. Emission spectra 5 ⁇ M 1,8-ANS in the presence of 3 ⁇ M apo-glucose oxidase. Excitation at 325 nm.
  • Figure 3 Glucose-dependent emission intensity of 1,8-ANS bound to apo-glucose oxidase. Excitation at 325 nm, emission at 480 nm.
  • Figure 4. Frequency-domain intensity decays of 1 ,8-ANS-apo-glucooxidase in the presence of increasing concentrations of glucose. Excitation was 335 nm (DCM dye laser), emission was observed through interference filter 535/50 nm and two Coming 3-71 cutoff filters.
  • Figure 5. Glucose-dependent lifetimes and pre-exponential factors from the lifetime- global analysis.
  • Figure 7. Polarization sensing.
  • Figure 18 Frequency-domain intensity decay of ANS-labeled LDH in the absence and presence of lactate.
  • Figure 19 Schematic of polarization sensing (top) and simulations of the expected changes in compensation angle for different values of n.
  • Figure 20 Polarization sensing of lactate bound on the emission intensity of ANS- labeled LDH.
  • the present invention provides a method for measuring the amount or presence of an analyte in a sample suspected of containing the analyte comprising: contacting the sample with a composition comprising an enzyme to form a complex; wherein the analyte is not consumed in the complex, and wherein the enzyme is coupled to the analyte during the contacting step; and measuring the amount or presence of analyte coupled to the inactive enzyme.
  • the invention includes a kit for measuring the amount or presence of an analyte in a biological sample.
  • the kit comprises a composition having an inactive enzyme wherein the analyte is not consumed upon measurement.
  • a further embodiment includes an assay for determining the amount of an analyte in a sample suspected of containing an analyte comprising a sample in combination with an inactive enzyme preferably coupled with a labeling reagent, and wherein the amount or presence of an analyte is measured as a result of the analyte binding with the inactive enzyme.
  • a sample comprises any fluid containing the analyte which is often a biological sample.
  • a biological sample is used such as a sample of blood, blood serum, plasma, urine, interstitial fluid, body secretion, tears, saliva, lymphatic or other extract taken from a mammal, preferably a human, to be tested for the presence of an analyte.
  • the sample is whole blood, plasma, or serum.
  • inactive enzyme is an enzyme that retains the ability to bind to a substrate, but that does not consume the substrate, wherein the substrate is the analyte to be measured.
  • inactive enzymes of the invention include apo-enzymes.
  • inactive enzymes can include enzymes that are engineered using site-directed mutagenesis. Mutants or analogs of the gene that produces an inactive enzyme may be prepared by the deletion, insertion or substitution of one or more nucleotides of the coding sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al, supra; DNA Cloning, Vols.
  • inactive enzymes can include enzymes that are linked to an uncompetitive inhibitor, which do not prevent analyte binding, but render the enzyme inactive.
  • uncompetitive inhibition results from a covalent reaction with inhibitors.
  • the enzymes such as dehydrogenases and oxidases are produced from thermophilic organisms.
  • lactate dehydrogenase and lactate oxidase can be produced from thermophilic organisms and analyte binding proteins such as lactate binding proteins can also be produced from normal thermophilic organisms which are stabilized by mutating their amino acid sequence or by immobilization.
  • An apo-enzyme is an enzyme that does not have a cofactor attached thereto.
  • Apo- enzymes included within the scope of the invention include those enzymes normally attached to cofactors.
  • cofactors include, but are not limited to 6-hydroxyDOPA, ammonia, ascorbate, ATP, bile salts, biotin, cadmium, calcium, cobalamin, cobamide coenzymes, cobalt, coenzyme-A, copper, dipyrromethane, dithiothreitol, divalent cation,
  • F420 flavin adenine dinucleotide (FAD), flavin, flavoprotein, flavin mononucleotide (FMN), glutathione, heme, heme-thiolate, iron, iron-molybdenum, iron-sulfur, lipoyl group, magnesium, manganese, molybdenum, monovalent cation, nicotinamide adenine dinucleotide (NAD), NAD(P)H, nickel, potassium, PQQ, protoheme IX, pterin, pyridoxal phosphate, pyruvate, reduced flavin, selenium, siroheme, sodium, tetrahydrofolate, tetrahydropteridine, thiamine pyrophosphate, tryptophan tryptophylquinone (TTQ), tungsten, vanadium, or zinc.
  • F420 flavin adenine dinucleotide
  • flavin, flavoprotein flavin mononu
  • apo-enzymes within the scope of the invention include but are not limited to those disclosed in Swiss Institute of Bioinformatics, ExPASy Molecular Biology Server, http://www.espasy.ch cgi-bin/enzyme-search-cf, which is in incorporated herein by reference.
  • the apo-enzymes of the invention are inactive and therefore they are coupled to their respective substrates without consuming the substrates and have a spectral change with respect to a substrate-bound apo-enzyme.
  • the apo-enzymes include: oxidases including amine oxidase (copper-containing), malate oxidase, glucose oxidase, cholesterol oxidase, L-gluconolactone oxidase, pyridoxine 4-oxidase, alcohol oxidase, choline oxidase, 4-hydroxymandelate oxidase, glycerol-3 -phosphate oxidase, xanthine oxidase, thiamine oxidase, L-galactonolactone oxidase, methanol oxidase, D- arabinono-l,4-lactone oxidase, vanillyl-alcohol oxidase, pyruvate oxidase, pyruvate oxidase (CoA-acetylating), dihydroorotate oxidase, protoporphyrinogen oxidase, acyl-
  • Thyroid-hormone aminotransferase Tryptophan aminotransferase, Pyridoxamine—pyruvate aminotransferase, dTDP-4-amino-4,6-dideoxy-D-glucose aminotransferase, UDP-4-amino- 2-acetamido-2,4,6-trideoxyglucose aminotransferase, Glycine—oxaloacetate aminotransferase, L-lysine aminotransferase, (2-aminoethyl)phosphonate ⁇ pyruvate aminotransferase, 2-aminoadipate aminotransferase, Branched-chain amino acid aminotransferase, Aminolevulinate aminotransferase, Alanine—glyoxylate aminotransferase, Serine—glyoxylate aminotransferase, Diaminobutyrate—pyruvate aminotransferase, Alanine—oxomalonate aminotrans
  • Taurine aminotransferase Aromatic amino acid transferase, dTDP-4-amino-4,6- dideoxygalactose aminotransferase, Adenosylmethionine ⁇ 8-amino-7-oxononanoate aminotransferase, Glutamine— phenylpyruvate aminotransferase, N6-acetyl-beta-lysine aminotransferase, Valine—pyruvate aminotransferase, 2-aminohexanoate aminotransferase, D-4-hydroxyphenylglycine aminotransferase, L-seryl-tRNA(Sec) selenium transferase; dehydratases including propanediol dehydratase, glycerol dehydratase, hydroperoxide dehydratase, myo-inosose-2 dehydratase, L-serine dehydratase, D-ser
  • Diaminopropionate ammonia-lyase Cystathionine gamma-lyase, Alliin lyase, S- alkylcysteine lyase, Cystathionine beta-lyase, Cysteine lyase, Methionine gamma-lyase, Cysteine-S-conjugate beta-lyase, Selenocysteine lyase; synthases including hydroxymethylbilane synthase, glutamate synthase (NADPH), glutamate synthase (ferredoxin)), riboflavin synthase, prostaglandin-D synthase, prostaglandin-E synthase, (S)-stylopine synthase, (S)-cheilanthifoline synthase, berbamunine synthase, salutaridine synthase, (S)-canadine synthase, prostaglan
  • Diaminopimelate decarboxylase Histidine decarboxylase, Aminobenzoate decarboxylase,
  • Tyrosine decarboxylase Aromatic-L-amino-acid decarboxylase, Sulfinoalanine decarboxylase, Phenylalanine decarboxylase, 2,2-dialkylglycine decarboxylase (pyruvate),
  • Phosphatidylserine decarboxylase Phosphatidylserine decarboxylase ; dehydrogenases including UDP-N-acetylmuramate dehydrogenase, D-lactate dehydrogenase (cytochrome), cellobiose dehydrogenase (quinone), gluconate 2- dehydrogenase, D-2-hydroxy-acid dehydrogenase, pyridoxine 5-dehydrogenase, glucose dehydrogenase (acceptor), glucoside 3 -dehydrogenase, malate dehydrogenase (acceptor),
  • dehydrogenases including UDP-N-acetylmuramate dehydrogenase, D-lactate dehydrogenase (cytochrome), cellobiose dehydrogenase (quinone), gluconate 2- dehydrogenase, D-2-hydroxy-acid dehydrogenase, pyridox
  • D-sorbitol dehydrogenase D-sorbitol dehydrogenase, retinal dehydrogenase, phenylglyoxylate dehydrogenase (acylating), Succinate dehydrogenase (ubiquinone), succinate dehydrogenase, butyryl-CoA dehydrogenase, acyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, beta- cyclopiazonate dehydrogenase, isovaleryl-CoA dehydrogenase, 2-methylacyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, D-amino-acid dehydrogenase, Electron-transferring-flavoprotein dehydrogenase, dimethylglycine dehydrogenase, spermidine dehydrogenase, proline dehydrogenase, NADH dehydrogenase (ubiquinone), NADPH
  • Preferred apo-enzymes include apo-oxidases and apo-dehydrogenases, such as apo- glucose oxidase, apo-glucose dehydrogenase, apo-lactate oxidase, and apo-lactate dehydrogenase.
  • An apo-enzyme for sugar is also preferred.
  • the apo-enzyme is inactive and thus does not affect the substrate to which is binds, and thus is a reversible, non- consuming sensor for the substrate.
  • An anaylte includes any molecule or molecules that can be coupled to an inactive enzyme to form a complex according to the invention, such as substrates for the apo- enzymes listed or incorporated by reference above.
  • the analyte is a substrate for an oxidase or a dehydrogenase, such as sugars or esters, preferably glucose or lactate.
  • a complex is an enzyme that is reversibly coupled to an analyte.
  • Coupled refers to a covalent or non-covalent or both a covalent and non-covalent combination, such as the coupling of an enzyme to a substrate.
  • the inactive enzyme can be labeled using a detectable label, such as a chemical label, e.g., streptavidin and biotin, an enzymatic label, e.g., LacZ and alkaline phosphatase, a radioactive label, e.g., 3 H, 14 C, 335 S, 32 P and 125 I, a fluorescent label, e.g., GFP, BFP and RFP, or a luminescent label, e.g., luciferase.
  • a detectable label such as a chemical label, e.g., streptavidin and biotin, an enzymatic label, e.g., LacZ and alkaline phosphatase, a radioactive label, e.g., 3 H, 14 C, 335 S, 32 P and 125 I, a fluorescent label, e.g., GFP, BFP and RFP, or a luminescent label, e.g., lucifera
  • an inactive enzyme preferably an apo-enzyme
  • an apparatus can be used to detect the labeled inactive enzyme and the substrate- bound labeled inactive enzyme.
  • Labels included within the scope of the invention include intrinsic and extrinsic labels.
  • an intrinsic label is a label in which a property of the sample such as mass or charge can affect a second label, e.g., the fluorescence polarization of a fluorescent label. See Huchzermeier, U.S. Pat. No. 4,476,228.
  • an inactive enzyme changes its intrinsic fluorescence in response to the analyte binding to the enzyme.
  • the inactive enzyme displays a change in a non-covalently extrinsic fluorophore upon binding with the analyte.
  • a covalently bound extrinsic probe displays a spectral change.
  • the emission, anisotropy, or polarization is dependent on the analyte binding such as lactate binding.
  • the extent of resonance energy transfer between attached donors and acceptors changes upon analyte binding.
  • a preferred method comprises shining light on a labeled coupled apo-enzyme; determining the intensity of fluorescence; mixing a sample with the labeled coupled apo-enzyme; shining light on the sample and labeled coupled apo- enzyme; and determining the change in intensity of signal, preferably fluorescence.
  • the labeled apo-enzyme is 8-anilino-l -naphthalene sulfonic acid (ANS) coupled apo-lactate dehydrogenase, ANS coupled apo-lactate oxidase, ANS coupled apo-glucose oxidase, or ANS coupled apo-glucose oxidase.
  • ANS 8-anilino-l -naphthalene sulfonic acid
  • An assay of the invention can include a heterogeneous or homogenous assay and includes any suitable methodology used for antibodies.
  • "Homogeneous assay” refers to an assay in which the presence and/or concentration of an analyte is determined without requiring the separation of sample fluid from the reaction components.
  • a homogenous assay includes formats in which a detectable signal is only generated upon specific binding of a labeled inactive enzyme to an analyte. As such, homogenous assay formats, the detection occurs without a non-bound labeled enzyme removal step. This broad classification includes many formats known to those skilled in the art.
  • Heterogeneous assay refers to an assay in which a complex is formed, which is removed from the reaction medium before measuring.
  • a kit contains instructions for performing the assay, which instructions may be printed on a package insert, packaging or label included in the kit.
  • the printed matter can also be included on receptacles included in the kit, and indicia of sample and reagent volumes can be indicated in the test receptacle.
  • instructions for dilution of the kit components and/or the sample if necessary directions for volume or concentration of labeled apo-enzyme used for each assay, volume of sample to add to the labeled inactive enzyme assay, directions for labeling an inactive enzyme, directions for taking measurement of labeled components, preferred temperature conditions, and timing of component addition and mixing, and use of a standard to calibrate test results.
  • the invention includes a method of glucose sensing using an inactive form of glucose oxidase from Aspergillus niger.
  • Glucose oxidase was rendered inactive by removal of the FAD cofactor.
  • the resulting apo-glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence.
  • 8- Anilino-1-naphthalene sulfonic acid (ANS) was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence.
  • the steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%.
  • apo-glucose oxidase noncovalently bound 8- anilino-1-naphthalene sulfonic acid ANS
  • the ANS bound to glucose oxidase displayed decreases in both intensity and lifetime upon addition of glucose.
  • Preferred embodiments of the invention include glucose or other sugar converting enzymes rendered inactive by removal of necessary cofactors.
  • the invention includes other substrate- converting enzymes rendered inactive by removal of necessary cofactors.
  • the present invention includes a protein biosensor for D-glucose based on a thermostable glucose dehydrogenase.
  • the glucose dehydrogenase was non-covalently labeled with 8-anilino-l-naphthalene sulfonic acid (ANS).
  • ANS 8-anilino-l-naphthalene sulfonic acid
  • the ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be used to measure the glucose concentration.
  • Our results suggest that enzymes which use glucose as their substrate can be used as reversible and non-consuming glucose sensors in the absence of required cofactors.
  • using apo-enzymes for a reversible and non-consuming sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes, which are also included in the invention.
  • an enzyme uses glucose as the substrate, but under conditions where no reaction occurs.
  • a thermophilic and thermostable GD is used that binds glucose, and catalyzes the following reaction:
  • one embodiment of the invention is a protein biosensor for L-lactate using lactate dehydrogenase non-covalently labeled with 8-anilino-l-naphthalene sulfonic acid (ANS).
  • the ANS-labeled lactate dehydrogenase displays an approximate 40% decrease in emission intensity upon binding lactate. This decrease is used to measure the lactate concentration.
  • the ANS-labeled lactate dehydrogenase is used in a new easy-to-use apparatus for lactate monitoring which can be used a variety of formats.
  • this invention includes a method for measuring lactate using lactate dehydrogenase.
  • Lactate dehydrogenase is a tetramer with a molecular weight of 136.700 ⁇ 2,100 daltons.
  • a number of isozymes are known to occur as mixed tetramers of the muscle and heart isozymes. Any of the various isozymes can be used as the sensor.
  • beef heart lactate dehydrogenase is non-covalently labeled with 8-anilino-l-naphthalene sulfonic acid (ANS).
  • ANS labeled lactate dehydrogenase displays a decrease in the ANS emission intensity upon binding lactate. This decrease in the ANS fluorescence occurs without consumption of lactate.
  • a change of emission intensity is also expected for other labeled apo-enzymes upon binding to a substrate.
  • a simple, easy-to-use apparatus can contain the ANS labeled lactate dehydrogenase and can measure the changes in intensity. Thus, this apparatus can be used to quantify the amount of lactate in a sample of blood, serum, or other bodily fluid.
  • This apparatus can be used at a patients' bedside, doctor's office, at gymnasium, at a sporting event, or other forum.
  • Inactive enzyme sensors preferably apo- enzyme sensors may be used in a variety of formats including a central clinical lab, a doctor's office testing, home healthcare or a portable device which is worn by a patient.
  • Glucose and all other chemicals were of reagent grade and purchased from Sigma. 8-anilino-l -naphthalenesulfonic acid (ANS) and glucose oxidase from Aspergillus niger were also obtained from Sigma. Steady state fluorescence measurements were carried out on a SLM AMINCO spectrofluorometer at room temperature, by using quartz cuvettes.
  • Frequency-domain intensity decay measurements were performed using instrumentation described previously. Lakowicz, J. R., et al. (1991) Frequency-domain fluorescence spectroscopy in Topics in Fluorescence Spectroscopy, Vol. 1 : Techniques (J. R. Lakowicz, Ed.), Plenum Publishing, New York, pp. 293-355. The intensity decay data were analyzed in terms of the multiexponential model.
  • ⁇ values are the initial amplitudes of the component with a decay times ( ⁇ i).
  • the subscript j refers to different concentrations of glucose.
  • TJ decay times
  • a saturated ammonium sulfate solution at 25% was acidified to pH 1.4 with H 2 SO 4 .
  • 5 mg of glucose oxidase in 0.5 ml solution was added drop-wise with stirring to 5.0 ml of the acidified salt solution at 4°C.
  • the FAD was split off from the enzyme, and the yellow supernatant was removed after centrifugation at 13,000 rpm for 15 min. Swoboda, B. E. P. (1969) Biochim. Biophys. Acta. 175, 364-379.
  • the precipitate was re-dissolved and neutralized by adding of sodium acetate.
  • the neutralized solution was subjected two more cycles of acidified salt treatment, centrifugation and neutralization. Finally, the protein was washed three times on Centricon tube (Amicon) in 10 mM sodium phosphate, pH 6.8.
  • the apo-glucose oxidase (GO) retained its capacity for reactivation by FAD for several weeks when stored at O°C, and pH 6.8.
  • the absorbance spectrum shows the characteristic shape of the coenzyme-free proteins, with a maximum of absorbance at 278 nm due to the aromatic amino acid residues.
  • the absence of absorption at wavelengths above 300 nm indicates the FAD has been completely removed.
  • the fluorescence emission spectrum of apo-GO at room temperature upon excitation at 298 nm run displays an emission maximum at 340 nm, which is characteristic of partially shielded tryptophan residues.
  • the addition of 20 mM glucose to the enzyme solution resulted in a quenching of the tryptophanyl fluorescence emission about 18%. This result indicates that the apo-GO is still able to bind glucose.
  • the observed fluorescence quenching may be mainly ascribed to the tryptophanyl residue 426.
  • the glucose-binding site of GO is formed by Asp 584, Tyr 515, His 559 and His 516.
  • Phe 414, Tip 426 and Asn 514 are in locations where they might form additional contacts to the glucose. Hecht, H. J., et al. (1993) J Mol Biol
  • Glucose dehydrogenase (GD), ANS and D-glucose were obtained from Sigma. GD was placed in 10 mM sodium phosphate buffer, pH 6.0. This enzyme solution represents the starting material for the fluorescence measurements. For all fluorescence measurements the final concentrations of ANS and GD were 4 ⁇ M and 3 ⁇ M, respectively. Steady state fluorescence measurements were performed in quartz cuvettes in an ISS spectrofluorometer using magic angle polarizer conditions. Frequency-domain (FD) measurements were performed using instrumentation described previously. Lakowicz, J. R., et al. (1991) In Topics In Fluorescence Spectroscopy, Volume T.
  • the light source was a frequency doubled pyridine 2 dye laser and the emission observed through a 465 nm interference filter.
  • the FD measurements were also performed using magic angle polarizer conditions.
  • the FD intensity decay data were analyzed by non-linear least squares in terms of the multi- exponential model, also described above.
  • the intensity-weighted lifetime is given by
  • Polarization sensing provides a method by which a change in intensity is observed as a change in polarization. This polarization is proportional to relative intensities of the sample and the reference. Reference displays a constant intensity and the sample intensity depends on the glucose concentration.
  • the sample (S) and reference (R) sides of the sensor are illuminated with a UV hand lamp.
  • the observed polarization P is given by
  • the initial polarization of the sample will be P 0 .
  • Equation (9) describes the dependence of observed changes in polarization ( ⁇ P) on the values of n and k. It is interesting to consider values of k needed to obtain the maximum change of ⁇ P for different values of n.
  • ANS is known to be a polarity-sensitive fluorophore which displays an increased quantum yield in low polarity environments. Weber, G. (1951). Biochem J. 51 :155-167; Slavik, J. (1982). Biochim. Biophys. Acta 694:1-25. Additionally, ANS frequently binds to proteins with an increase in intensity. We examined the effects of GD on the emission intensity of ANS. A moderate enhancement was found but the ANS intensity remained low compared to other ANS-protein complexes. Also, addition of glucose to this GD-ANS complex did not change upon addition of glucose.
  • thermophilic protein is a thermophilic protein and can be expected to be rigid under mesophilic conditions.
  • thermophilic proteins often display increased activity at higher temperatures or the presence of non-polar solvents (D'Auria, S., et al. (1999) Biophys. Chem 81, 23-31; D'Auria, S., et al. (1999) J. Biochem. 126, 545-552) which are conditions expected to increase the protein dynamics.
  • Addition of acetone to the solution containing ANS and GD resulted in a dramatic increase in the ANS intensity, ( Figure 8), as well as in a blue-shift of the emission maximum.
  • Addition of similar amounts of acetone to ANS in the absence of the protein produced modest fluorescence increase.
  • the increase in the ANS intensity reflects a change in the local protein environment which is due to acetone.
  • Figure 12 shows the emission polarized spectra of ANS-GD at various concentrations of glucose. The polarization decreases at higher glucose concentrations because the emission from this solution is observed through the horizontal polarizer.
  • acetone may be eliminated by selecting proteins which are less thermophilic.
  • the proteins can be engineered for covalent labeling by insertion of cysteine residues at appropriate locations in the sequence.
  • the glucose induced spectral changes may be larger with other polarity sensitive probes or by the use of RET between two fluorophores on the protein.
  • apo-enzymes are found to be a valuable source of protein sensors.
  • beef heart lactate dehydrogenase, ANS and L-lactate were obtained from Sigma. Lactate dehydrogenase was extensively dialyzed against mM sodium phosphate buffer, pH 6.0 at 4°C. After dialysis the enzyme solution was centrifuged at 12,000 rpm for 30 min at 4°C and the supernatant was recovered. The supernatant was filtered by utilizing an inorganic membrane filter, Anotop 10 (Whatman). The obtained enzyme solution represents the starting material for the fluorescence measurements. For all fluorescence measurements the final concentrations of ANS and lactate dehydrogenase were 4 ⁇ M and 3 ⁇ M, respectively.
  • the FD measurements were a performed using magic angle polarizer conditions. As discussed above, the FD intensity decay data were analyzed by non-linear least squares in terms of multi-exponential model
  • the intensity-weighted lifetime is given by
  • Figure 14 shows the intrinsic tryptophan emission of ANS-coupled lactate dehydrogenase. Addition of micromolar concentrations of lactate resulted in an approximate 30% decrease in the tryptophan intensity, consistent with an earlier report on the C isozyme of lactate dehydrogenase. Gupta, G. S., et al. (1997) Indian J. of Biochemistry & Biophysics 34:307-312. An increase of lactate concentration over 200 ⁇ M does not introduce further changes in fluorescence intensity. Thus, the enzyme is mostly saturated by 200 ⁇ M lactate. As shown in Figure 15 and Table 2, lactate binding did not significantly change nor affect the mean lifetime, intensity-weighted lifetime or intensity decay of lactate dehydrogenase.
  • UV output near 370 nm has become available from light emitting diodes (LED) (Sipior, J., et al. (1995) Anal. Biochem.
  • lactate dehydrogenase is labeled with the fluorophore, 8-anilino-l-naphthalene sulfonic acid, which is suitable for LED excitation.
  • the emission intensity of ANS solution with lactate dehydrogenase was about 30-fold higher and displayed a blue shift from 525 to 465 nm.
  • ANS concentrations significantly higher than the lactate dehydrogenase concentration did not appear to bind to lactate dehydrogenase.
  • Others have reported that tetrameric lactate dehydrogenase binds 4 to 6 ANS molecules, and further addition of ANS does not result in further ANS binding. Kube, D. et al.
  • Polarization sensing is accomplished by constructing a sensor such that a stable intensity reference is observed through one polarizer and the sample is observed through a second orthogonal polarizer.
  • a stable intensity reference is observed through one polarizer and the sample is observed through a second orthogonal polarizer.
  • the reference is a ANS-lactate dehydrogenase solution in the absence of lactate, which can be expected to display similar temperature, time or illumination-dependent changes as the sample.
  • This reference is observed through a vertically oriented polarizer.
  • the sample contains ANS-lactate dehydrogenase and various concentrations of lactate, and is observed through a horizontally oriented polarizer.
  • the emission from both sides of the sensor is then observed through an analyzer polarizer.
  • the analyzer polarizer is rotated until the emission from both sides is equalized, which can be measured visually (Gryczynski, I., et al. (1999) Anal. Chemistry 71 : 1241 -1251) or with a simple photocell or photodiode circuit shown in Figure 19. Gryczynski, Z., et al. (2000) Optical Engineering in press.
  • the analyzer polarizer is rotated until the voltage across the differential electronics (Watson Bridge) is zero.
  • the angle of polarizer rotation can then be used to determine the lactate concentration. This angle is called the "compensation angle.”
  • the intensity change induced by analyte results in a change of the compensation angle, ⁇ , which is related to the concentration of analyte.
  • ⁇ the compensation angle
  • the total intensity change -40% (n-1.7) the change in compensation angle was about 6° for the entire range of lactate concentrations. While the range seems small, the compensation angles are readily measured to about 0.1 degrees, so that a 6° change corresponds to an accuracy of 2% in the lactate concentration.
  • the device shown in Figure 19 was battery powered and could be easily designed as a portable instrument for bedside use.
  • the simulations in Figure 19 are for intensity changes comparably to those observed for ANS-lactate dehydrogenase.
  • the beef heart lactate dehydrogenase was only moderately stable at room temperature and had to be used within several days following removal from the ammonium sulfate solution.
  • One manner is by immobilization of the lactate dehydrogenase into a matrix which often stabilizes proteins.
  • An alternative embodiment may involve using lactate dehydrogenase from thermophilic organisms or mesophilic organisms.
  • Another alternative embodiment may include modifying lactate dehydrogenase to obtain a larger spectral change, preferably with a change in lifetime or a useful spectral shift.
  • Site directed mutagenesis of lactate dehydrogenase could result in lactate dehydrogenase with more stability and larger and more useful spectra changes.
  • An apparatus which uses the ANS coupled lactate dehydrogenase to measure lactate in blood, serum, or other bodily fluids is possible. This apparatus is easy-to-use and provides quick results.
  • This apparatus uses, in its preferred embodiment, polarization sensing to quantify the amount of lactate present in the sample.
  • Other types of sensors can be used which can use various fluorescence measurements including emission intensity, emission maxima, spectral shift, wavelength-ratio, energy transfer and lifetime.
  • Light sources can include laser diodes, LEDs, and similar devices.
  • the sample (S) and reference (R) sides of the sensor are illuminated with a UV hand lamp.
  • the emission from the reference passes through a vertically oriented polarizer, and the emission from the sample passes through a horizontally oriented polarizer.
  • the emission from S and R is observed through an analyzer polarizer (AP) using a dual photocell.
  • AP analyzer polarizer
  • I H I ⁇ s sin 2 ⁇ (11) where ⁇ is the angular displacement of the analyzer polarizer from the vertical position.
  • a change in the sample intensity induced by the analyte results in changes of the analyzer polarizer angle needed to equalize the intensities. This difference is shown as the compensation angle, ⁇ .
  • ⁇ ⁇ 0 - ⁇ (14)
  • ⁇ 0 refers to the rotation angle of analyzer polarizer (AP) needed to equalize the intensities in the absence of analyte and ⁇ is the angle of AP needed to equalize the reference and sample intensities in the presence of given analyte concentration.
  • the ANS-coupled lactate dehydrogenase does not consume nor alter the lactate in the sample. While this aspect of not changing the lactate is preferable, it is possible that the fluorescent molecule coupled enzyme alters or changes the lactate in the sample.
  • Figure 19 shows simulations of the expected changes in compensation angle ⁇ as a function of initial ratio of the reference to sample fluorescence, k, for different values of n.
  • lactate oxidase coupled to a fluorescent molecule instead of lactate dehydrogenase.
  • the lactate oxidase or the lactate dehydrogenase can be derived from thermophilic or mesophilic organisms.
  • one can immobilize the enzyme to increase its ability to withstand temperatures higher than 37 degrees Celsius.

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Abstract

Selon cette invention, des enzymes inactives telles que des apo-enzymes sont utilisées comme capteurs de non consommation qui se fixent à des analytes sans les détruire. Ces capteurs peuvent être utilisés dans des procédés, des dosages ou des kits pour mesurer la concentration des analytes. Des formes inactives de diverses enzymes telles que glucose oxydase, glucose déshydrogénase ou lactate déshydrogénase qui se fixent au glucose ou au lactate peuvent être utilisées comme biocapteurs de glucose ou lactate.
PCT/US2000/024846 1999-09-10 2000-09-11 Enzymes inactives utilisees comme capteurs de non consommation WO2001018237A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
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US6681127B2 (en) 1999-08-26 2004-01-20 Novartis Ag Ocular analyte sensor
US7041063B2 (en) 1996-09-04 2006-05-09 Marcio Marc Abreu Noninvasive measurement of chemical substances
EP1856138A1 (fr) * 2005-03-04 2007-11-21 Carnegie Institution Of Washington Capteurs stables dans l environnement et leurs procédés d utilisation
US8173863B2 (en) 2005-10-14 2012-05-08 Carnegie Institution Of Washington Sucrose biosensors and methods of using the same
US8530633B2 (en) 2004-10-14 2013-09-10 Carnegie Institution Of Washington Development of sensitive FRET sensors and methods of using the same
US8846365B2 (en) 2005-10-14 2014-09-30 Carnegie Institution Of Washington Nucleic acids encoding phosphate fluorescent indicators and methods of using the same
WO2019045331A1 (fr) * 2017-08-31 2019-03-07 단국대학교 산학협력단 Nanosonde hybride à base de silsesquioxane oligomère polyédrique, et capteur comprenant celle-ci
KR20190024686A (ko) * 2017-08-31 2019-03-08 단국대학교 산학협력단 다면체 올리고머 실세스퀴옥산을 기반의 하이브리드 나노 프로브 및 이를 포함하는 센서
KR20200069010A (ko) * 2018-12-06 2020-06-16 단국대학교 산학협력단 Poss-마이크로 니들 및 이를 포함하는 마이크로 니들 패치

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7041063B2 (en) 1996-09-04 2006-05-09 Marcio Marc Abreu Noninvasive measurement of chemical substances
US6850786B2 (en) 1999-08-26 2005-02-01 Novartis Ag Ocular analyte sensor
US6681127B2 (en) 1999-08-26 2004-01-20 Novartis Ag Ocular analyte sensor
US7403805B2 (en) 2001-02-23 2008-07-22 Marcio Marc Abreu Apparatus and method for noninvasive measurement of analytes from the conjunctiva using mid-infrared radiation
US8530633B2 (en) 2004-10-14 2013-09-10 Carnegie Institution Of Washington Development of sensitive FRET sensors and methods of using the same
EP1856138A1 (fr) * 2005-03-04 2007-11-21 Carnegie Institution Of Washington Capteurs stables dans l environnement et leurs procédés d utilisation
EP1856138A4 (fr) * 2005-03-04 2008-12-10 Carnegie Inst Of Washington Capteurs stables dans l environnement et leurs procédés d utilisation
US8357505B2 (en) 2005-03-04 2013-01-22 Carnegie Institution Of Washington Environmentally stable sensors and methods of using the same
US8173863B2 (en) 2005-10-14 2012-05-08 Carnegie Institution Of Washington Sucrose biosensors and methods of using the same
US8846365B2 (en) 2005-10-14 2014-09-30 Carnegie Institution Of Washington Nucleic acids encoding phosphate fluorescent indicators and methods of using the same
WO2019045331A1 (fr) * 2017-08-31 2019-03-07 단국대학교 산학협력단 Nanosonde hybride à base de silsesquioxane oligomère polyédrique, et capteur comprenant celle-ci
KR20190024686A (ko) * 2017-08-31 2019-03-08 단국대학교 산학협력단 다면체 올리고머 실세스퀴옥산을 기반의 하이브리드 나노 프로브 및 이를 포함하는 센서
KR102098551B1 (ko) * 2017-08-31 2020-04-08 단국대학교 산학협력단 다면체 올리고머 실세스퀴옥산을 기반의 하이브리드 나노 프로브 및 이를 포함하는 센서
KR20200069010A (ko) * 2018-12-06 2020-06-16 단국대학교 산학협력단 Poss-마이크로 니들 및 이를 포함하는 마이크로 니들 패치
KR102198384B1 (ko) 2018-12-06 2021-01-05 단국대학교 산학협력단 Poss-마이크로 니들 및 이를 포함하는 마이크로 니들 패치

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