Optimization of biosensor based on interdigitated conductimetric

electrodes for the determination of polluting flux in hyporheic zones

Ph. Namour1, M. Marrakchi2, S. Dzyadevych2, F. Ruysschaert1, C. Martelet2, N. Jaffrezic-Renault2

1 Cemagref, 3 bis quai Chauveau, 69336 Lyon cedex 09, France2 CEGELY, UMR-CNRS 5005, ECL-Lyon, 69134 Ecully cedex, France

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Presentation outline• Problematic

– Objectives – Measure in hyporheic zone– A definition of organic matter

• Biosensor– Principle & structure– Optimization – First assessments

• Conclusion• Perspectives

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Objectifs

To measure OM assimilation in hyporheic zones

Organic matter & hyporheic flows

Design & optimization of a biosensor to measure protein in porous media

To quantify OM fluxes=

= to quantify

Problematic

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downwelling

upwelling

inwelling

outwelling

Harvey & Wagner 2000

Flows in the hyporheon:

influence of geomorphological units

Problematic

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surf

ace

hyp

orh

eon

high [O2]

very low or no [O2]

Aerobiosis

Anaerobiosis

NO3-

NH4+

to upstream

dm to m

m to hm

Commonly low in O2 depending on

geology, land use and organic

matter

gro

un

d-w

ate

rs

Fe2+

Fe3+

Hyporheon

Direction of ground-water flows from Winter et al. 1998

Problematic

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A definition of OM ?

No chemical definition !

Need to use a proxyProteins (30% of total COD)

Proteins as a proxy of OM

Problematic

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AmplifierElectronics

instrumentation

Biosensor

BioreceptorSample Transducer Signal Information

Enzyme

Proteinase K

Platinum interdigitat

ed thin films

electrodes

Principle

Principle of biosensorElectronic board

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Conductometric Conductometric measurementsmeasurements

( Platinum electrode )

ProteinsProteinase K

Amino-acids

Charge variation

Principle

Mechanism of bioreceptor

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Co-reticulation of the enzyme with a bi-functional agent:the glutaraldehyde.

Insoluble biopolymer

E N CH CH2 CH N P3

E N C

H

O

CH2 CO

H3 N P

albumineenzyme

H2H2

Membrane preparation

Principle

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Platinum

Glass

Protection Resin

Platinum connections

10 µm

10 µm

GlassPt electrode (Height 0.2 µm)

Preparation of biosensor

a micro-drop of mixture

Enzyme 2%

Bovine Serum Albumine 3%

Glycerol 5%

20 mM PO4 buffer pH 7.5

Structure

Micro-sensor design

20 Microelectrode insert( 8)

60 Electronic board ( 10)

10 Sensing area

Designed by PIMMA

Input signal

Converted in alternating current

Frequency: 100 kHz

Amplitude: 10 mV

Output signal

Differential measurement

Digitized by the board

Structure

Optimisation of the procedure for elaboration of the enzymatic membrane

Effect of the contact time with glutaraldehyde vapour

Twenty minutes is the optimal contact time.Optimization

Calibration curve for detection of BSA

The range of sensitivity obtained (0.2 to 8 mg/L) is in accordance with the values of the actual concentrations of proteins in hyporheic

water.Optimization

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two zones:

zone “a”: response decreasing: leaching of certain molecules of enzymes not well adhering on the surface of the membrane;

zone “b”: the response is quite stable for more than one month.

Stability

Optimization

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Sensor response versus microBCA method (river & sewer waters)

First assessments

6.6 Nmg/L

0.3 Nmg/L 0.2 Nmg/L

We have shown the feasibility of a microsensor based on interdigitated microelectrodes in the case of measurements of Organic Matter content (proteins). Concentration range for BSA between 0.2 to 8 mg/L, in accordance with the real concentrations of proteins in hyporheic water.

Conclusion

Conclusion

Repeatability = 3.3% & sensibility = 0.88 µS/mg

Stability = more than one month (in laboratory)

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ISO 5725River water & wastewaters

Standard additionsAccuracy (trueness, precision)

StabilityReproducibility

Validation in laboratory

Perspectives

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Continuous monitoring along a riffle, downstream a sewer overflow

hyporheon

micro-sensor “piezometer”perforated measurement room with micro-electrodes

riffle

Perspectives

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This work was financially supported in the framework of NMAC

concerted action of French ministry

research, priority thematic action of

Rhone-Alpes region and NATO LST.CLG.980843

Acknowledgements

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Thank you for your attention

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Field metrology

Measure in situ

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Which instrumentation ?

Spatial scale :

Constraint: To respect metric of studied phenomena

mm3-cm3

microorganisms, invertebrates

km2

catchments

transient phenomenone.g. : overflow

Long term trends

e.g. : global change

Time scale :

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Instrumented site

Sewer overflow

Measurement unit

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Problem

Lag time

the first polluting peak is missing

Downstream

Sewer Overflow

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Need for remove this lag time

But what is an “in situ measurement” ?

by an in situ measurement

The Protein Biosensor

Microconductimetric structures already used as the transducer for enzymatic microsensors

Detection of pesticides (Dzyadevych et al, 2002)

Enzyme ProteaseCatalyses hydrolysis reaction of proteins:

peptidic links are broken, amino acids are released

increasing of the ionized species in the membrane

increasing of the conductivity of the membrane

Choice of the proteaseProteinase K is chosen

Protease with a serine group

Hydrolyses the proteins of all origins, in few hours

Preferentially peptidic bonding located after hydrophobic amino acids (leucine, for instance)

Immobilisation of the enzymeon the micro-conductimetric

structureA mixture of enzyme & BSA was co-reticulated

with glutaraldehyde

Procedure of immobilisation:1-Preparation of the solution 1: phosphate buffer 1mM, pH 7.5

with 5% of glycerol

2-Enzymatic membrane: 4mg of protease mixed with 6mg of BSA in 100 L of solution 1.

3-Deposition on the surface of the micro-conductimetric structure

4-The structure is put in a saturated atmosphere of glutaraldehyde during an optimized time.

5-The sensor is then dried in air during at least 30 minutes.

Interdigited electrode

digit width 10 µm

Platinum deposit on glass substrate

interdigitated distance 10 µm

digit length 90

µm

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In situ measurement

micro-electrodes

To privilege a measurement :

local; nondisturbing;

continue;

cheap;

autonomous; reliable.