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
ALCOHOLIC FERMENTATION
(Saccharomyces
CO 2
Sugars
COOH
H
C
C
cerevisiae)
NAD H + H+ NAD +
CH2OH
O
Alcool
Pyruvate
dehydrogenase
CH3 decarboxylase CH3
Ethanal
Pyruvate
O
CH3
Ethanol
Acetobacter
Lactic bacteria
COOH
L-malate
CHOH
CH2
COOH
A cetic acid
D-lactate
CO 2
COOH
CHOH
L-lactate
CH3
MALOLACTIC FERMENTATION (Oenococcus oeni)
WIDELY USED METHOD : PAPER CHROMATOGRAPHY
Legend :
T - Tartaric acid
L - Lactic acid
M - Malic acid
Suitable for any winery
Low cost but low speed and accuracy.
ENZYMATIC RECOMMENDED METHOD
Malate dehydrogenase
L-MDH
(EC 1.1.1.37)
L-malate
+
NAD+
Spectrophotometric determination
at 340nm (e = 6300 M-1.cm-1)
Oxaloacetate + NADH + H+
GOT* + L-glutamate
* Glutamate-oxaloacetate
transaminase (EC2.6.1.1)
L-aspartate + 2-oxoglutarate
Costly, not adapted to small wineries
Laboratory analysis : delays between sampling and results.
Need of easy and portable analytical devices as BIOSENSORS
PRINCIPLE OF DH-BASED BIOSENSORS
SUBSTRATE
DEHYDROGENASE
(DH)
PRODUCT
+ NADH + H+
+ NAD+
Optical
detection
(=340 nm)
AMPEROMETRIC
DETECTION
Direct oxidation
High Potential
No selectivity
Bienzymatic
systems
Mediated
oxidation
(monoenzymatic
system)
THE MALATE DEHYDROGENASE-REACTION :
DIFFERENT OPTIONS FOR SENSOR DEVELOPMENT
Malate dehydrogenase L-MDH
(EC 1.1.1.37)
L-malate
+
NAD+
High concentrations of NAD+
+ appropriate mediator
MONO-ENZYMATIC SENSOR
Oxaloacetate + NADH + H+
Enzymatic consumption of NADH
(regeneration of NAD+)
BI-ENZYMATIC SENSOR
BI-ENZYMATIC SYSTEM BASED ON DIAPHORASE (EC 1.8.1.4)
NADH + H+
NAD+
Diaphorase (Clostridium kluyverii)
2 Fe(CN)63-
2 Fe(CN)64-
250 mV
vs. SCE
2 e-
Mandatory addition of ferricyanide
Interferences 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.
BI-ENZYMATIC SYSTEM BASED ON NADH OXIDASE (EC 1.6.99)
NADH + H+
NAD+
High
stability
NADH oxidase
Dissolved in
solution
(Thermus thermophilus)
H2 O2
O2
650 mV
vs. Ag/AgCl
2 e-
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.
BI-ENZYMATIC SYSTEM INVOLVING PRUSSIAN BLUE AS
MEDIATOR
Sensing layer
Sensing layer
4 H+ + 4K+
2NADOX
2 NADH + 2 H+
2H2O2
2NADOX
2 NADH + 2 H+
Solution
Solution
(FMN)
2 NAD+
2NADOX
+
2 NAD
(FMNH2)
(FMN)
Mediator
Mediator
4 K+
4 K+
Fe4 [Fe(CN)6]3
Fe4 [Fe(CN)
]3
Prussian6 Blue
Prussian Blue
4
4 e-
e-
-150 mV
-150
vs mV
Ag/AgCl
vs Ag/AgCl
Fe4K4[Fe(CN)6]3
Fe
K
1/2
O
4 Prussian
4[Fe(CN)White
6]3
2
2NADOX
Working
4
OH
Prussian White
+ H2O2)
(FMNH
Working
electrode
+ 4K+
electrode
NAD and FMN
Precipitated on WE
must be added in solution
surface
MONO-ENZYMATIC SYSTEM INVOLVING MELDOLA’S BLUE
AS MEDIATOR
Malic acid
Incorporated in the
electrode material
N
(CH3) 2N
O
+
Oxaloacetic acid
L-MDH
In solution
NADH
NAD+
MB+
MBH
- 150 mV
vs Ag/AgCl
H
N
(CH3) 2N
O
H+ + 2e-
MB : FAST EXCHANGE OF ELECTRONS WITH NADH
MELDOLA’S BLUE-MODIFIED ELECTRODES : EVALUATION OF
INTERFERENCES DUE TO WINE PHENOLIC COMPOUNDS
I (nA) 2500
Gallic Acid
Red w ine
2000
1500
1000
500
0
-150
-100
-50
0
50
100
150
applied potential (m V)
10% MBRS-modified SPE, pyrophosphate buffer 0.1 M, pH 9.3
Gallic acid 1 mM, 50 µL red wine (Caramany)
WORKING AT -150 MV VS Ag/AgCl ALLOWS REDUCING
INTERFERENCES
PB AND MB-BASED SENSORS : COMPARATIVE TABLE
(Batch measurements in stirred buffered solution)
SENSOR DEVICE
TWO ENZYMES
SYSTEM
MONO ENZYME
SYSTEM
Electrochemical mediator
Prussian blue
MBRS 10%
Applied potential,
vs Ag/AgC l
Enzyme s
- 100 mV
-150 mV
0.14 IU-MDH
0.02 IU NADH oxidase
PVA polym er
0.14 IU MDH
Phosphate buffer
0.1 M
8
NAD+- 2 mM / FMN - 0.2 mM
3.632
0.023-0.247
4.5
Pyrophosph ate buffer
0.1 M
9.3
NAD+ - 2 mM
0.262
0-0.190
4.7
Entrapment procedure
Electrolyte solution
pH
Cofactor
Sensitivity (mA/M)
Linear Range (mM)
Detection limit (µM)
Sol-gel
REAL SAMPLES ANALYSIS
COMPARISON WITH COMMERCIALLY AVAILABLE KITS
Real samples
RQFLEX kit
(g/L)
Red wine
White wine
0.86
2.205
BOEHRINGER kit MDH/NADH oxidase
(g/L)
-based Sensor (g/L)
0.662
2.174
0.747
2.147
MDH-based Sensor
(g/L)
0.885
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)
AN ALTERNATIVE TO THE CLASSICAL MDH :
THE MALATE:QUINONE OXIDOREDUCTASE (MQO,EC 1.1.99.16).
MQO from Corynebacterium glutamicum is a FAD-dependent peripheral
membrane enzyme (FAD tightly bound).
Involved in citric acid cycle.
- Natural aceptor : ubiquinone
(ménaquinone)
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
THE PRINCIPLE OF MQO-BASED BIOSENSOR
MQO used in this work in a recombinant enzyme (E. coli)
produced by GTP Technology, Labège (France).
L-Malic acid
Oxaloacetic acid
MQO-FAD
MQO-FADH2
Medox
Medred
eNO COENZYME NEEDED, MONOENZYMATIC SYSTEM
REACTION ESSENTIALLY IRREVERSIBLE
BUT : APPROPRIATE MEDIATORS MUST BE FOUND
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)
Mediator
Form used
Working
potential
Analytical
signal
vs Ag/AgCl
DPIP
PMS
BQ
BQ
TCNQ
Co (II)
phtalocyanine
Co (II)
phtalocyanine
Potassium
hexacyanoferrate
MB
MB-RS
PB
Nile Blue
Free in solution
(0.2 mM)
Free in solution
(0.3 mM)
Free in solution
(40 M)
Free in solution
(40 M)
Incorporated in WE
+50 mV
350 nA
-50 mV
300 nA
+50 mV
No signal
High mV
Interferences
+400
320 nA
+100 mV
Negligible
Incorporated in WE
+100 mV
No signal
Incorporated in WE
+400 mV
No signal
Free in solution
(0.1 mM)
Free in solution
(0.1 mM)
Incorporated in WE
Precipitated on WE
Free in solution
(0.1 mM)
High mV
Interferences
+350
140 nA
+10 mV
+10 mV
Small
signal
No signal
No signal
-150 mV
No signal
+10 mV
MQO-BASED SENSORS PERFORMANCES
TYPE OF BIOSENSOR
DPIP-based
PMS-based
electrodes
electrodes
Immobilization method
PVA-SbQ
PVA-SbQ
Potential vs Ag/AgCl
+ 50 mV
-10 mV
Linear range
5-250 µM
5-150 µM
0.7-33.5 mg/L
0.7-20.1 mg/L
0.85 mA/M
1.7 mA/M
5 µM
5 µM
(0.7 mg/L)
(0.7 mg/L)
Sensitivity
(electrode area 18 mm2)
Limit of detection
Evaluation of interferences
AS : Analytical signal (to 1mM malate),
IS = Interference signal (to 100-fold diluted red wine or 0.05 mM gallic acid)
DPIP
Potential
malic acid
(mVvs.
1mM
PMS
gall ic acid
malic acid
0.05 mM
1 mM
gall ic acid
red wine
Ag/AgCl
red wine
Rela tive
AS (nA)
0.05 mM
Rela tive
IS (nA)
IS /AS
IS (nA)
IS /AS
AS (nA)
AS
IS (nA)
IS /AS
IS (nA)
IS /AS
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
HIGH INTERFERENCES USING DPIP AS MEDIATOR
REAL SAMPLES ANALYSIS
Wine analysis using MQO biosensors using DPIP or PMS as mediators.
Average of triplicate measurements, spiked wine samples.
Sample / sensor
type
DPIP-sensor Recovery PMS-sensor
(mM)
(mM)
White wine
9.0±2.7
9.6±3.1
White wine +10 mM
22.2±6.8
132%
18.5±5.5
Recovery
89%
malic acid
Red wine
4.1±1.1
Red wine +10 mM
15.6±4.9
malic acid
3.5±0.9
115%
11.5±3.3
80%
Advantages & Drawbacks of MQO-sensor
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