STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS DEVOTED TO WINEMAKING MONITORING
description
Transcript of STRATEGIES FOR THE DEVELOPMENT OF MALATE SENSORS DEVOTED TO WINEMAKING MONITORING
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