STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS

DEVOTED TO WINEMAKING MONITORING

WHY TO DETECT MALIC ACID ?THE MALOLACTIC FERMENTATION (MLF)

MLF = secondary fermentation, occurs after alcoholic fermentation,lasts from 2 weeks to several months (if T is too low).-Transformation of malic acid (diacide) in lactic acid (monoacide)- Bacterial process (Oenococcus oeni)- Deacidification: decrease in titratable acidity and increase in pH- Wine stabilisation and flavour change

MLF is usually encouraged for all dry red wines:

[Malic acid] in musts: 1-5 g/L

[Malic acid] in red wines : 0-0.1 g/L

MLF is avoided or partially performed for white wines.

MONITORING OF MLF IS FUNDAMENTAL FOR WINE PRODUCERS.

SUBSTRATES OF INTEREST

C O 2

COOH

CHOH

CH3

L-malateL-lactate

MALOLACTIC FERMENTATION (Oenococcus oeni)

COOH

CHOH

CH2

COOH

ALCOHOLIC FERMENTATION (Saccharomyces cerevisiae)

O

COOH

C

CH3

CO 2H

C

CH3

O

NAD H + H+ NAD +

CH2OH

CH3Pyruvatedecarboxylase

Alcool dehydrogenase

Sugars

Ethanal

EthanolPyruvate

Acetobacter

Acetic acidLactic bacteria

D-lactate

Legend :

T - Tartaric acidL - Lactic acidM - Malic acid

WIDELY USED METHOD : PAPER CHROMATOGRAPHY

Suitable for any winery

Low cost but low speed and accuracy.

ENZYMATIC RECOMMENDED METHOD

L-malateOxaloacetate

+ NAD++ NADH + H+

Spectrophotometric determination at 340nm ( = 6300 M-1.cm-1)

GOT* + L-glutamate

L-aspartate + 2-oxoglutarate

* Glutamate-oxaloacetate transaminase (EC2.6.1.1)

Costly, not adapted to small wineriesLaboratory analysis : delays between sampling and results.

Malate dehydrogenase L-MDH

(EC 1.1.1.37)

Need of easy and portable analytical devices as BIOSENSORS

+ NAD+

PRINCIPLE OF DH-BASED BIOSENSORS

DEHYDROGENASE(DH)

SUBSTRATE PRODUCT

+ NADH + H+

Opticaldetection(=340 nm)

AMPEROMETRIC DETECTION

Direct oxidation

High Potential

No selectivity

Bienzymatic systems

Mediatedoxidation

(monoenzymaticsystem)

+ NAD+

THE MALATE DEHYDROGENASE-REACTION : DIFFERENT OPTIONS FOR SENSOR DEVELOPMENT

Malate dehydrogenase L-MDH(EC 1.1.1.37)

L-malateOxaloacetate + NADH + H+

High concentrations of NAD+

+ appropriate mediatorEnzymatic consumption of NADH(regeneration of NAD+)

MONO-ENZYMATIC SENSOR BI-ENZYMATIC SENSOR

Diaphorase (Clostridium kluyverii)

2 Fe(CN)63-

250 mV

vs. SCE2 e-

2 Fe(CN)64-

NADH + H+ NAD+

BI-ENZYMATIC SYSTEM BASED ON DIAPHORASE (EC 1.8.1.4)

Mandatory addition of ferricyanideInterferences with wine samples

* Related papers : 1. J.-L. Marty and T. Noguer. Analusis, 21 (1993) 6-8.2. T. Noguer and J.-L. Marty. Enzyme Microb. Technol., 17 (1995) 453-456.3. T. Noguer and J.-L. Marty. Anal. Chim. Acta, 347 (1997) 63-70.

NADH oxidase (Thermus thermophilus)

O2

650 mV vs. Ag/AgCl

H2 O2

NADH + H+ NAD+

2 e-

Dissolved in solution

Highstability

BI-ENZYMATIC SYSTEM BASED ON NADH OXIDASE (EC 1.6.99)

High overpotential for H2O2 oxidation : high interferences

* Related papers : 1. T. Noguer and J.-L. Marty. Anal. Let., 30 (1997) 1069-1080.2. T. Noguer, A. Gradinaru, A. Ciucu and J.-L. Marty. Anal. Let., 32 (9) (1999) 1723-1738.

Fe4 [Fe(CN)6]3 Prussian Blue

Fe4K4[Fe(CN)6

]3Prussian White

2NADOX (FMNH2)

2 NADH + 2 H+

2 NAD+

Mediator

Working electrode

Sensing layer

4 e-

Solution

-150 mV vs Ag/AgCl

2NADOX (FMN)

4 K+4 H+ + 4K+

BI-ENZYMATIC SYSTEM INVOLVING PRUSSIAN BLUE AS MEDIATOR

1/2 O2

+ H2O

Fe4 [Fe(CN)6]3 Prussian Blue

Fe4K4[Fe(CN)6

]3Prussian White

2NADOX (FMNH2)

2 NADH + 2 H+

2 NAD+

2H2O2

4 OH-

+ 4K+

Mediator

4 e-

Solution

2NADOX (FMN)

4 K+

Working electrode

Sensing layer

-150 mV vs Ag/AgCl

Precipitated on WE

surface

NAD and FMN

must be added in solution

NADH NAD+

MB+ MBH

H+ + 2e-

Malic acid Oxaloacetic acid

- 150 mVvs Ag/AgCl

O+

N

(CH3)2N O

HN

(CH3)2N

L-MDH

MONO-ENZYMATIC SYSTEM INVOLVING MELDOLA’S BLUE AS MEDIATOR

In solutionIncorporated in the

electrode material

MB : FAST EXCHANGE OF ELECTRONS WITH NADH

10% MBRS-modified SPE, pyrophosphate buffer 0.1 M, pH 9.3Gallic acid 1 mM, 50 µL red wine (Caramany)

WORKING AT -150 MV VS Ag/AgCl ALLOWS REDUCING INTERFERENCES

MELDOLA’S BLUE-MODIFIED ELECTRODES : EVALUATION OF INTERFERENCES DUE TO WINE PHENOLIC COMPOUNDS

(Batch measurements in stirred buffered solution)

PB AND MB-BASED SENSORS : COMPARATIVE TABLE

SENSOR DEVICE TWO ENZYMES

SYSTEM

MONO ENZYME

SYSTEM

Electrochemical mediator Prussian blue MBRS 10%

Applied potential,

vs Ag/AgCl

- 100 mV -150 mV

Enzymes 0.14 IU-MDH

0.02 IU NADH oxidase

0.14 IU MDH

Entrapment procedure PVA polymer Sol-gel

Electrolyte solution Phosphate buffer

0.1 M

Pyrophosphate buffer

0.1 M

pH 8 9.3

Cofactor NAD+- 2 mM / FMN - 0.2 mM NAD+ - 2 mM

Sensitivity (mA/M) 3.632 0.262

Linear Range (mM) 0.023-0.247 0-0.190

Detection limit (µM) 4.5 4.7

REAL SAMPLES ANALYSIS

COMPARISON WITH COMMERCIALLY AVAILABLE KITS

Real samples RQFLEX kit

(g/L)

BOEHRINGER kit

(g/L)

MDH/NADH oxidase

-based Sensor (g/L)

MDH-based Sensor

(g/L)

Red wine 0.86 0.662 0.747 0.885

White wine 2.205 2.174 2.147 1.805

GOOD CORRELATIONS BUT NAD (and FMN) MUST BE ADDED IN REACTIONAL MEDIUM

RESEARCHS FOCUS ON OBTENTION OF A FAD-BOUND NADH OXIDASE (GTP Technology, Labège, France)

MQO from Corynebacterium glutamicum is a FAD-dependent peripheral membrane enzyme (FAD tightly bound).Involved in citric acid cycle. - Natural aceptor : ubiquinone (ménaquinone)

AN ALTERNATIVE TO THE CLASSICAL MDH : THE MALATE:QUINONE OXIDOREDUCTASE (MQO,EC 1.1.99.16).

Alternative metabolic pathway (PEP shunt) for the conversion of malate to oxaloacetate in E. coli. Van der Rest et al., J Bacteriol. 182(24) (2000) 6892-6899

MQO-FAD MQO-FADH2

Medox Medred

e-

L-Malic acid Oxaloacetic acid

MQO used in this work in a recombinant enzyme (E. coli) produced by GTP Technology, Labège (France).

NO COENZYME NEEDED, MONOENZYMATIC SYSTEMREACTION ESSENTIALLY IRREVERSIBLE BUT : APPROPRIATE MEDIATORS MUST BE FOUND

THE PRINCIPLE OF MQO-BASED BIOSENSOR

Mediator Form used Workingpotentialvs Ag/AgCl

Analyticalsignal

DPIPFree in solution

(0.2 mM)+50 mV 350 nA

PMSFree in solution

(0.3 mM)-50 mV 300 nA

BQFree in solution

(40 μM) +50 mV N o signal

BQFre e i n solution

(40 μM) +400 mV 320 nA

TCNQ Incorporate d i nWE +100 mV NegligibleC (o II)

phtalocyanine Incorporate d i nWE +100 mV N o signal

C (o II)phtalocyanine Incorporate d i nWE +400 mV N o signal

Potassiumhexacyanoferrate

Fre e i n solution(0.1 m )M +350 mV 140 nA

MB Fre e i n solution(0.1 m )M +10 mV Small

signalMB-RS Incorporate d i nWE +10 mV N o signalPB Precipitate d o nWE +10 mV N o signal

Nil e Blue Fre e i n solution(0.1 m )M -150 mV N o signal

SELECTION OF MEDIATOR(S) FOR MQO

Analytical responses of the sensors to 1 mM malic acid (0,134g/L)(Working potentials were selected by cyclic voltammetry)

High Interferences

High Interferences

TYPE OF BIOSENSOR DPIP-based

electrodes

PMS-based

electrodes

Immobilization method PVA-SbQ PVA-SbQ

Potential vs Ag/AgCl + 50 mV -10 mV

Linear range 5-250 µM

0.7-33.5 mg/L

5-150 µM

0.7-20.1 mg/L

Sensitivity

(electrode area 18 mm2)

0.85 mA/M 1.7 mA/M

Limit of detection 5 µM

(0.7 mg/L)

5 µM

(0.7 mg/L)

MQO-BASED SENSORS PERFORMANCES

DPIP PMS

malic acid

1mMred wine

gallic acid

0.05 mM

malic acid

1 mMred wine

gallic acid

0.05 mM

Potential

(mVvs.

Ag/AgClAS (nA)

Relative

ASIS (nA) IS/AS IS (nA) IS/AS AS (nA)

Relative

ASIS (nA) IS/AS IS (nA) IS/AS

100 417±52 1 105±15 0.25 355±48 0.85 - - - - - -

50 405±63 0.97 100±9 0.25 345±34 0.85 490±81 1 25±5 0.05 55±13 0.11

10 342±41 0.82 95±10 0.28 337±42 0.99 460±88 0.94 15±5 0.03 14±13 0.03

-10 205±26 0.49 75±9 0.37 300±40 1.46 400±72 0.82 0±3 0 7±15 0.02

-50 - - - 315±65 0.64 -10±7 -0.03 -17±12 -0.05

-100 - - - 275±57 0.56 -10±7 -0.04 3±15 0.00

Evaluation of interferences

HIGH INTERFERENCES USING DPIP AS MEDIATOR

AS : Analytical signal (to 1mM malate), IS = Interference signal (to 100-fold diluted red wine or 0.05 mM gallic acid)

REAL SAMPLES ANALYSIS

Sample / sensor

type

DPIP-sensor

(mM)

Recovery PMS-sensor

(mM)

Recovery

White wine 9.0±2.7 9.6±3.1

White wine +10 mM

malic acid

22.2±6.8 132% 18.5±5.5 89%

Red wine 4.1±1.1 3.5±0.9

Red wine +10 mM

malic acid

15.6±4.9 115% 11.5±3.3 80%

Wine analysis using MQO biosensors using DPIP or PMS as mediators. Average of triplicate measurements, spiked wine samples.

MQO : cofactorless enzyme, irreversible conversion of malate

BUT : Poor stability, supplied in (NH4)2SO4 by GTP technology, must be desalted before immobilization : loss of activity

Mediators : DPIP and PMS were used in solution, high interferences with DPIP,low stability of PMS (light sensitive).

Appropriated mediators still must be found :

* Efficient electronic transfert with FADH2,

* Low detection potential, reduced interactions with polyphenolic compounds

* Incorporable in screen-printed electrodes

Advantages & Drawbacks of MQO-sensor