NAD+-DEPENDENT DEHYDROGENASE SENSORS WITH

AMPEROMETRIC DETECTION

Enzyme = dehydrogenase

Great variety of substrates potentially detectable in agri-food, medical and environmental

areas

Substrate + NAD + Product + NADH + H +

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

D-lactic acidEarly indicator of a possible dysfunction

Leuconostoc sp.

Wine industry Red wines, Champagne...

Alcoholic fermentation is followed by malo-lactic fermentation

L-Malic acid

L-Lactic acid(total acicity decreases)

Lactobacillus sp.Leuconostoc sp.

A precise and real-timemonitoring is important to stop the fermentation at appropriate time

Wine industry Red wines

“Piqûre lactique”

L-Malic acid

D-Lactic acid

Proliferation of lactic bacteriaduring malo-lactic fermentation

dramatic enhancement of wine acidity

NAD+ : expensive, soluble cofactorNecessity of NAD+ addition in the reaction medium…?

Arising problems

How can we transform the biological signal into a

measurable electrical signal...?

Exception : L-Lactate DH

L-LDH

L-Lactate Pyruvate

Electrochemical oxidation

2 Fe(CN)63- 2 Fe(CN)6

4-

One enzyme system :

easy to optimize

Acts as a cosubstrateNo NAD+ is required

PVA-SbQ tridimensional matrix = DH

= NAD+

Entrapment of native NAD+ in a polymeric network

LEAKAGE of NAD, gradual loss of response intensity

= DH

= NAD+ dextran or NAD-PEG

Entrapment of « enlarged » NAD+

NO LEAKAGE, excellent operational stability

NAD-dextran extremely expensive, NAD-PEG not commercially available

How can we transform the biological signal

into a measurable electrical signal ?

Dehydrogenase (DH)

Substrate Product

NAD+ NADH + H+

Direct oxidation ?

High potential (±1V vs SCE)Oxidation of interfering substancesLoss of selectivity

H+ + 2 e-

Signal transductionUtilisation of a bi-enzyme system

Dehydrogenase (DH)

Substrate Product

NAD+ NADH + H+

E2

Oxidised mediator Reduced mediator

Electrochemical oxidation (transduction)

Diaphorase (EC 1.8.1.4, Clostridium kluyverii)

2 Fe(CN)63-

250 mV vs. SCE

2 e-

2 Fe(CN)64-

NADH + H+ NAD+

Bi-enzyme systemClassical configuration

Low stability of diaphoraseNecessary addition of ferricyanide in the medium

Relatively low potential

Commercially availableLow cost

NADH oxidase (EC 1.6.99, Thermus thermophilus)

O2

600 mV vs. SCE

H2 O2

NADH + H+ NAD+

2 e-

Bi-enzyme system« Mediatorless » configuration

High overvoltage for H2O2 oxidationEnzyme not commercially available

No mediator addition

High stability

= DH

= NAD+ dextran or NAD-PEG

Coimmobilization of DH, NOX and enlarged NAD allows to design

« reagentless » sensors

= NADH oxidase

10 µL

55 µL+ 5 µL PVA-SbQ

DH + NADH oxidase+ NAD-dextran or NAD-PEG

Electrode (Pt)

Photopolymerization

3 h under two 15 W neon lamps at 4°C

Cellophane membrane

« O » ring

Cellophane membrane

Working electrode(Pt, 2 mm diameter)

1 cmAuxiliary electrode (Pt)

Potentiostat Recorder

Thermostated cell

Water, 30°C

PVA-SbQ matrix

Buffer*

A classical enzyme-electrode device

Ethanol Acetaldehyde D-lactate

Sensitivity (mA/M) 2 1.7 4

Linear range (µM) 0.3-100 0.5-240 40-1500

Operational stability > 80 > 80 > 500(assays)

Response time (min) < 2 < 2 3

 Performance  of « reagentless » sensors

Diaphorase

2 Fe(CN)63-

250 mV vs. SCE 2 e-

2 Fe(CN)64-

NADH + H+ NAD+

3-enzyme systems(used to shift the reaction to the products ’side)

L-MDH

L-malate Oxaloacetate

GOTGlutamate-oxaloacetatetransaminase

+ glutamate

Aspartate + -ketoglutarate

Diaphorase

2 Fe(CN)63-

250 mV vs. SCE 2 e-

2 Fe(CN)64-

NADH + H+ NAD+

3-enzyme systems(used to shift the reaction to the products ’side)

D-LDH

D-lactate Pyruvate

GPTGlutamate-pyruvatetransaminase

+ glutamate

Alanine + -ketoglutarate

Monoenzymatic systemsInvolving electronic mediators for NADH

oxidation

NADH NAD+

DH

(Analyte)Substrate Product

oxidation

Mediator (ox) Mediator (red)

Electric current

-Ideally incorporatedin the electrode material-Non toxic-Fast rate for exchanging electrons with NADH

-Low oxidation potential : no interference

NADH NAD+

MB+ MBH

H+ + 2e-

DH

Substrate Product

Meldola ’s Blue : efficient mediator but soluble, leaks from the electrode surface...

- 100 mVvs SCE

Use of Meldola ’s Blue as mediator

Meldola’s blue

Reinecke’s salt

Precipitate (MBRS)

Incorporable in the electrode material(No leaching in the working medium)Increased stability of the sensor response

Use of a Meldola ’s Blue insoluble salt as mediator

+ NH4 Cr(NH3)2(SCN)4

(CH3)2N

N

O

Incorporation of MBRS in a screen-printed carbon paste electrode

Two possible stratégies : - amperometry

- chronoamperometry

6 mm

8.5mm

42 m

m

Workingelectrode

Reference/auxiliaryelectrode

s i l v e r

c o n d u c t i n g

f i l m

c a r b o n p a d i n s u l a t i n g

l a y e r

A g / A g C l

r e f e r e n c e

e l e c t r o d e

M B R S - m o d i f i e d

c a r b o n w o r k i n g

e l e c t r o d e

MB or MBRS is mixed with graphite in the SPEEnzyme and NAD+ are simplyadsorbed on the electrode

Potentiostat Recorder

Thermostated cell

Water, 25°C

Buffer +NAD+

-150 mV

-150 mV

25 μl of sample

Amperometry Chronoamperometry

MBRS is mixed with graphite in the SPEEnzyme is entrapped in PVA-SbQNAD+ is added in the cell

Reusable sensor Disposable sensor

AlDH, 28 mU entrapped inPVA-SbQ 1700 betainApplied potential, 0V, NAD 500 µM

50040030020010000

200

400

600

800

1000

1200

[Acetaldehyde] (µM)

I (nA)

y = 1,2941 + 2,7618x R^2 = 0,999

Amperometric configurationExample : detection of Acetaldehyde

Calibration plotTypical response

Sensitivity : 2.7 mA/MLinear range : 2.5 -400 µM

Time (sec)

Intensity(nA)

∆I = a.C

Substrate injection(Concentration C)

Calibration plotTypical response

Chronoamperometric configurationExample : detection of acetaldehyde

600550500450400350300250200150100500200

400

600

800

[Acetaldehyde] (µM)

I (nA)

y = 325,70 + 0,71545x R^2 = 0,998

0

200

400

600

8 0 0

1 0 0 0

Time, seconds

I ef

I (nA)

Substrate injectionPotential application

0 40

Intensity readingSensitivity : 0.7 mA/MLinear range : 10 -500 µM

Characteristics of reusable sensors

Mandatory addition of cofactor for each assay.

0.03 0.05-1 0.280 120 > 20

0.001 0.005-0.5 3.4 120 > 30Acetaldehyde

D-lactic acid

Detection limit

Linear range

SensitivityResponse time

Operational stability (assays)(mmol.L-1)

(mA.M-1)(seconds)(mmol.L-1)

Reusable sensor, calibration possible before analysing any sample.

Detection

limit

(mmol/L)

Linear

range

(mmol/L)

Sensitivity

(mA.M-1)

Time/assay

(seconds)

Coefficient

of variation

(% ; n = 30

electrodes)

0.05 0.075-1 0.589 150 7.6

Acetaldehyde 0.006 0.010-0.25 1.1 40 8.14

D-lactic acid

Characteristics of disposable sensors

Low amounts of enzymes, no immobilization

Low reproducibility between electrodesScreen-printing step is a critical issue

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)

0 -50 -100 -1500

50

100

150

200

250

300

350

400

Applied potential (mV)

I (nA)

NO INTERFERENCE

Working at -150 mV allows to avoid interferencesBest reliability of the method

Validation of the reusable sensorfor wine analysis

Good agreement between biosensor and reference methods

Biosensor Referencemethod*

4.7 0.5 4.9 0.3D-lactic acid(mmol/L) 3.6 0.4 3.9 0.3

2.17 0.2 2.25 0.1

1.69 0.1 1.67 0.1Acetaldehyde(mmol/L)

4.12 0.3 4.5 0.3

3.0 ± 0.4 3.1 ± 0.2

2.2 ± 0.1 2.3 ± 0.1

1.5 ± 0.1 1.4 ± 0.1

D-lactic acid

(mmol/L)

2.0 ± 0.2 2.1 ± 0.2

2.1 ± 0.16 2.29 ± 0.20

1.14 ± 0.06 1.03 ± 0.17

0.14 ± 0.05 0.20 ± 0.05Acetaldehyde

(mmol/L)

0.91 ± 0.09 1.03 ± 0.14

Biosensor Reference method

Validation of the disposable sensorfor wine analysis

Good agreement between biosensor and reference methods