A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules

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A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules. Brian Hassler, Megan Dennis, Maris Laivenieks * , Robert Y. Ofoli, J. Gregory Zeikus * , and R. Mark Worden Chemical Engineering and Material Science * Biochemistry and Molecular Biology Michigan State University - PowerPoint PPT Presentation

Transcript of A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules

  • A Bioelectronic Sensor Interface Based on Trifunctional Linking MoleculesBrian Hassler, Megan Dennis, Maris Laivenieks*, Robert Y. Ofoli, J. Gregory Zeikus*, and R. Mark WordenChemical Engineering and Material Science*Biochemistry and Molecular BiologyMichigan State UniversityEast Lansing, Michigan

    Presented at 2004 Annual AIChE ConferenceNovember 7 - 12, 2004, Austin, TX

  • Presentation OutlineBackgroundDehydrogenase enzymeBioelectronic interfaceProject goalsSite directed enzyme mutagenesisCharacterization of bioelectronic interfaceCyclic voltammetryChronoamperometryConclusions

  • BackgroundDehydrogenase enzymesCatalyze electron transfer reactionsActivity easily measured electrochemicallyBioelectronic applications Often require cofactor (e.g., NAD(P)+)Challenge: regenerating cofactor after reaction

  • Background on EnzymeModel enzymesecondary alcohol dehydrogenase (sADH)Thermoanaerobacter ethanolicus Thermal stabilityActivity range: 7C 95C

  • Background on EnzymeCofactor specificity: NADP+Amino acids affecting NADP+ affinity binding 198, 199, 200, 203, 218

  • Background on Cofactor RegenerationElectron mediator requiredShuttles electrons between electrode and cofactorPrevents cofactor degradation

    Linear structureMediator requirementsTwo unique functional groupsBind to electrodeBind to cofactorFew suitable mediators(Zayats, et al., J. Am. Chem. Soc. 2002, 124, 14724-14735)

  • Research Goals

    Enhance enzyme activity with NAD+Retain thermal stabilityGenerate a unique electron transfer scaffoldUsing a hetro-trifunctional linking moleculeSuitable for wider range of electron mediators

  • Enzyme Mutagenesis5 primerMutant primer3 primerWild type templateWild type templateendMutant 5-PCR amplification 1PCR amplification 2Complete mutant5 primer3 primer5 primer3 primerMutant primer

  • Clone adhB Gene Into pCR 2.1 VectorInsert mutant gene into lacZ gene

    Cells with plasmid will have ampicillin & kanamycin resistanceTransformed cell containing the PCR product will grow white on X-gal

  • Enzymatic Activities of Wild Type, Mutant StrainsNADP+

    NAD+

  • Cofactor Regeneration by ElectrodeLinear structureMediator requirementsTwo unique functional groupsFew suitable mediators

    Branched structureMediator requirementsSingle functional groupMany suitable mediators

  • Enzyme Interface AssemblyCysteine: branched, trifunctional linkerThiol group: self assembles on goldCarboxyl group: binds to electron mediatorAmine group: binds to phenylboronic acid

    Mediators usedToluidine Blue O (TBO)Nile Blue ANeutral Red

  • Characterization ToolsCyclic VoltammetryCalibration plotsTurnover ratioEffects of increased temperaturesChronoamperometryElectrode kinetics

  • Cyclic Voltammetry Y218F-mutant sADHCyclic voltammetry

    Substrate: Isopropanol, in phosphate buffer, pH=7.4High voltage: 400mVLow voltage: -200 mVScan rate: 100 mV/sElectrode area: 1 cm2

    Calibration plot:Slope: 1 mA/mMIsat= 42mA

    Turnover ratio:65 s-1

  • Cyclic Voltammetry Wild-Type sADHCyclic voltammetry

    Substrate: Isopropanol, in phosphate buffer, pH=7.4High voltage: 400mVLow voltage: -200 mVScan rate: 100 mV/sElectrode area: 1 cm2

    Calibration plot:Slope: 1.67 mA/mMIsat= 80mA

    Turnover ratio:450 s-1

  • ChronoamperometryProcedureStep change in potentialInitial Potential (E1): -200 mVFinal Potential (E2): 400 mVPlot current vs. time

    CharacterizationEquation Measurable variablesket= Electron transfer constantQ= Charge associated with oxidation/reduction

    I=ketQexp(-kett)+ketQexp(-kett)(Forster, R. J. Langmuir 1995, 11, 2247-2255)

  • Chronoamperometry Y218F-mutant sADH-NAD+

    Forster equation

    Best fit ket valuesket= 7.0x104 s-1ket= 5.5x103 s-1

    Surface coverage=Q/nFA

    = 9.56x10-13 mol cm-2= 7.55x10-12 mol cm-2

    I=ketQexp(-kett)+ketQexp(-kett)

  • Chronoamperometry Wild Type-sADH

    Forster equation

    Best fit ket valuesket= 7.0x104 s-1

    Surface coverage

    = 2.34x10-12 mol cm-2

    I=ketQexp(-kett)+ketQexp(-kett)I=ketQexp(-kett)

  • Determination of ThermostabilityTemperatures Measured25 C (I= 9 mA)35 C (I= 15 mA)45 C (I= 21 mA)50 C (I= 25 mA)60 C (I= 38 mA)65 C (I= 8 mA)

    Chart1

    2.1972245773

    2.7080502011

    3.0445224377

    3.2188758249

    3.6375861597

    2.0794415417

    (1/Temperature) (1/K)

    Ln Current

    Sheet1

    25298.150.003354016492.1972245773

    35308.150.0032451728152.7080502011

    45318.150.0031431715213.0445224377

    50323.150.0030945381253.2188758249

    60333.150.0030016509383.6375861597

    65338.150.002957267582.0794415417

    Sheet2

    Sheet3

  • ConclusionsMutant sADH developedIncreased activity with NAD+Novel electron transfer scaffold developedTrifunctional linking moleculeWider range of mediatorsBioelectronic interface with sADH developedElectrode kinetics measuredCalibration curves developedStable up to 60 C

  • AcknowledgementsFundingMichigan Technology Tri-Corridor Department of Education GAANN FellowshipUndergraduate students involvedJohn BaldreyTimothy Howes

  • Thank you

  • Amplification Results5-mutant3-mutantcomplete mutantPCR amplification 1PCR amplification 21 kB DNA ladder

  • Determination of Turnover RatioTRmax= Isat/(FnA)Isat= The saturated currentF= Faradays constant= Surface coverageA= Electrode Area

  • Clone adhB into pBluescript

    Add KM resistance cartridge to EcoR1 site of pBluescript

    Insert adhB mutant into vector (Apa I & Kpn I)

    transfer of e- directly to electrode required high electrical potentials that degrades cofactor. mediator allows e- transfer without degrading cofactorFIX Diagramtransfer of e- directly to electrode required high electrical potentials that degrades cofactor. mediator allows e- transfer without degrading cofactorVoltage RangeFIX Voltage RangeFix capFix Cap