strategies for the development of malate sensors devoted to winemaking monitoring
TRANSCRIPT
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