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NAD + -DEPENDENT DEHYDROGENASE SENSORS WITH AMPEROMETRIC DETECTION

NAD + -DEPENDENT DEHYDROGENASE SENSORS WITH AMPEROMETRIC DETECTION. +. +. Substrate +. NAD. Product +. NADH. + H. Enzyme = dehydrogenase. >250 NAD-dependent dehydrogenases. Great variety of substrates potentially detectable in agri-food, medical and environmental areas.

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NAD + -DEPENDENT DEHYDROGENASE SENSORS WITH AMPEROMETRIC DETECTION

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  1. NAD+-DEPENDENT DEHYDROGENASE SENSORS WITH AMPEROMETRIC DETECTION

  2. + + Substrate + NAD Product + NADH + H Enzyme = dehydrogenase >250 NAD-dependent dehydrogenases Great variety of substrates potentially detectable in agri-food, medical and environmental areas

  3. Sugar Industry (sugar beet, sugar cane) Lactic bacteria (Leuconostoc mesenteroides and L. dextranicum) produce polysaccharidic gums which perturbs the process (obstruction of pipes…) Sugar fermentation Leuconostoc sp. D-lactic acid Early indicator of a possible dysfunction

  4. Wine industry Red wines, Champagne... Alcoholic fermentation is followed by malo-lactic fermentation L-Malic acid A precise and real-time monitoring is important to stop the fermentation at appropriate time Lactobacillus sp. Leuconostoc sp. L-Lactic acid (total acicity decreases)

  5. Wine industry Red wines “Piqûre lactique” L-Malic acid dramatic enhancement of wine acidity Proliferation of lactic bacteria during malo-lactic fermentation D-Lactic acid

  6. Arising problems NAD+ : expensive, soluble cofactor Necessity of NAD+ addition in the reaction medium…? How can we transform the biological signal into a measurable electrical signal...?

  7. Exception : L-Lactate DH L-Lactate Pyruvate Acts as a cosubstrate No NAD+ is required One enzyme system : easy to optimize L-LDH 2 Fe(CN)63- 2 Fe(CN)64- Electrochemical oxidation

  8. = DH = NAD+ Entrapment of native NAD+ in a polymeric network PVA-SbQ tridimensional matrix LEAKAGE of NAD, gradual loss of response intensity

  9. = DH = NAD+ dextran or NAD-PEG Entrapment of « enlarged » NAD+ NO LEAKAGE, excellent operational stability NAD-dextran extremely expensive, NAD-PEG not commercially available

  10. How can we transform the biological signal into a measurable electrical signal ? Substrate Product Dehydrogenase (DH) NAD+ NADH + H+ H+ + 2 e- Direct oxidation ? High potential (±1V vs SCE) Oxidation of interfering substances Loss of selectivity

  11. Signal transduction Utilisation of a bi-enzyme system Substrate Product Dehydrogenase (DH) NAD+ NADH + H+ E2 Oxidised mediator Reduced mediator Electrochemical oxidation (transduction)

  12. NADH + H+ NAD+ Bi-enzyme system Classical configuration Commercially available Low cost Diaphorase(EC 1.8.1.4, Clostridium kluyverii) Relatively low potential 2 Fe(CN)63- 2 Fe(CN)64- 250 mV vs. SCE 2 e- Low stability of diaphorase Necessary addition of ferricyanide in the medium

  13. NADH + H+ NAD+ High stability Bi-enzyme system « Mediatorless » configuration No mediator addition NADH oxidase(EC 1.6.99, Thermus thermophilus) H2 O2 O2 600 mV vs. SCE 2 e- High overvoltage for H2O2 oxidation Enzyme not commercially available

  14. = NAD+ dextran or NAD-PEG Coimmobilization of DH, NOX and enlarged NAD allows to design « reagentless » sensors = DH = NADH oxidase

  15. 55 µL + 5 µL PVA-SbQ 10 µL DH + NADH oxidase + NAD-dextran or NAD-PEG « O » ring Cellophane membrane Photopolymerization 3 h under two 15 W neon lamps at 4°C Electrode (Pt)

  16. A classical enzyme-electrode device

  17.  Performance  of  « reagentless » sensors EthanolAcetaldehydeD-lactate Sensitivity (mA/M) 21.74 Linear range (µM) 0.3-100 0.5-24040-1500 Operational stability > 80> 80> 500 (assays) Response time (min) < 2< 23

  18. NADH + H+ NAD+ 3-enzyme systems (used to shift the reaction to the products ’side) L-malate Oxaloacetate GOT Glutamate-oxaloacetate transaminase L-MDH + glutamate Aspartate + a-ketoglutarate Diaphorase 2 Fe(CN)64- 2 Fe(CN)63- 250 mV vs. SCE 2 e-

  19. NADH + H+ NAD+ 3-enzyme systems (used to shift the reaction to the products ’side) D-lactate Pyruvate GPT Glutamate-pyruvate transaminase D-LDH + glutamate Alanine + a-ketoglutarate Diaphorase 2 Fe(CN)64- 2 Fe(CN)63- 250 mV vs. SCE 2 e-

  20. (Analyte) Substrate Product DH NADH NAD+ Mediator (ox) Mediator (red) oxidation Electric current Monoenzymatic systems Involving electronic mediators for NADH oxidation -Ideally incorporated in the electrode material -Non toxic -Fast rate for exchanging electrons with NADH -Low oxidation potential : no interference

  21. Use of Meldola ’s Blue as mediator Substrate Product DH NADH NAD+ MB+ MBH H+ + 2e- - 100 mV vs SCE Meldola ’s Blue : efficient mediator but soluble, leaks from the electrode surface...

  22. Use of a Meldola ’s Blue insoluble salt as mediator Reinecke’s salt Meldola’s blue Precipitate (MBRS) Incorporable in the electrode material (No leaching in the working medium) Increased stability of the sensor response

  23. 6 mm 42 mm 8.5 mm • Working • electrode Reference/auxiliary electrode Incorporation of MBRS in a screen-printed carbon paste electrode Two possible stratégies : - amperometry - chronoamperometry

  24. Amperometry Chronoamperometry MBRS is mixed with graphite in the SPE Enzyme is entrapped in PVA-SbQ NAD+ is added in the cell MB or MBRS is mixed with graphite in the SPE Enzyme and NAD+ are simply adsorbed on the electrode Reusablesensor Disposable sensor

  25. 1200 1000 800 600 400 200 0 0 100 200 300 400 500 Amperometric configuration Example : detection of Acetaldehyde Typical response Calibration plot AlDH, 28 mU entrapped in PVA-SbQ 1700 betain Intensity I (nA) (nA) Applied potential, 0V, NAD 500 µM y = 1,2941 + 2,7618x R^2 = 0,999 ∆I = a.C Time (sec) [Acetaldehyde] (µM) Sensitivity : 2.7 mA/M Linear range : 2.5 -400 µM Substrate injection (Concentration C)

  26. 1 0 0 0 8 0 0 6 0 0 4 0 0 2 0 0 800 0 600 400 200 0 50 100 150 200 250 300 350 400 450 500 550 600 Chronoamperometric configuration Example : detection of acetaldehyde Typical response Calibration plot I (nA) I (nA) y = 325,70 + 0,71545x R^2 = 0,998 I e f 0 40 [Acetaldehyde] (µM) T i m e , s e c o n d s Intensity reading Sensitivity : 0.7 mA/M Linear range : 10 -500 µM Substrate injection Potential application

  27. Characteristics of reusable sensors Detection limit Linear range Response time Operational stability (assays) Sensitivity (mA.M-1) (mmol.L-1) (seconds) (mmol.L-1) D-lactic acid 0 . 03 0 . 05-1 0 . 280 120 > 20 Acetaldehyde 0 . 001 0 . 005-0 . 5 3 . 4 120 > 30 Reusable sensor, calibration possible before analysing any sample. Mandatory addition of cofactor for each assay.

  28. Characteristics of disposable sensors C o e ffi c i e n t De te c ti o n Li ne ar S e n s i t ivi t y Ti me / a s sa y o f v a ri a ti o n - 1 li m it r a n g e ( mA .M ) ( se c o n ds ) ( % ; n = 3 0 ( m m o l/L) ( m m o l/L) e l e ctr o d es ) 0 . 0 5 0 . 0 7 5- 1 0 . 5 8 9 1 5 0 7 . 6 Ac et a ld e h y d e 0 . 0 0 6 0 . 0 1 0- 0 . 2 5 1 . 1 4 0 8 . 1 4 Low amounts of enzymes, no immobilization Low reproducibility between electrodes Screen-printing step is a critical issue

  29. I (nA) 400 350 300 250 200 NO INTERFERENCE 150 100 50 0 Applied potential (mV) 0 -50 -100 -150 Influence of applied potential on interferences due to phenolic compounds (gallic acid, 17 mg/L*) (* injection of 50 µl gallic acid 3,5 g/L) Working at -150 mV allows to avoid interferences Best reliability of the method

  30. Re fer en c e B i o s en s o r m e t h o d * ± ± 4 . 7 0 .5 4 . 9 0 .3 D -l a c t ic a c i d ± ± ( m m o l/L) 3 . 6 0 .4 3 . 9 0 .3 ± ± 2 . 1 7 0 .2 2 . 2 5 0 .1 Ac et a l de h y d e ± ± 1 . 6 9 0 .1 1 . 6 7 0 .1 ( m m o l/L) ± ± 4 . 1 2 0 .3 4 . 5 0 .3 Validation of the reusable sensor for wine analysis Good agreement between biosensor and reference methods

  31. Biosensor Reference method ± ± 3 . 0 0 .4 3 . 1 0 .2 ± ± 2 . 2 0 .1 2 . 3 0 .1 D -l a c t ic a c i d ± ± ( m m o l/L) 1 . 5 0 .1 1 . 4 0 .1 ± ± 2 . 0 0 .2 2 . 1 0 .2 ± ± 2 . 1 0 .1 6 2 . 2 9 0 .2 0 ± ± 1 . 1 4 0 .0 6 1 . 0 3 0 .1 7 Ac et a l de h y d e ± ± 0 . 1 4 0 .0 5 0 . 2 0 0 .0 5 ( m m o l/L) ± ± 0 . 9 1 0 .0 9 1 . 0 3 0 .1 4 Validation of the disposable sensor for wine analysis Good agreement between biosensor and reference methods

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