inhibition based biosensors: environmental applications

II WORKSHOP ON CHEMICAL SENSORS AND BIOSENSORS

151

INHIBITION BASED BIOSENSORS: ENVIRONMENTAL APPLICATIONS

F. Mazzei1, F. Botrè2

1Università degli Studi di Roma "La Sapienza" - Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, P.le Aldo Moro, 5 – 00185 Roma 2Università degli Studi di Roma "La Sapienza" - Dipartimento di controllo e gestione delle merci e del loro impatto sull'ambiente, P.le Aldo Moro, 5 – 00185 Roma

Abstract: Kinetic assays involving inhibition of specific enzymatic systems have been extensively applied as analytical methods for the detection of food and environmental contaminants, primarily among them pesticides, herbicides and heavy metals. A particular form of enzymatic inhibition assays is represented by enzymatic inhibition bioelectrodes, whose flexibility can ensure the analysis of huge population of samples at very reduced costs. An "ideal" inhibition biosensor should, in principle, ensure the rapid detection of all contaminants endowed with the same biological effect, without the need for an extensive sample pretreatment. In this work data are reported on the possibility to detect environmental contaminants endowed with toxic effects. A new biosensor is proposed and discussed for the direct determination of 2,4 dichloro phenoxyacetic acid (2,4 D), i.e. one of the most powerful and diffused defoliant, also endowed with estrogenic properties. Keywords: enzymatic inhibition, bioelectrode, environmental applications. INTRODUCTION

The development of analytical systems and devices for the

control of environmental contaminations is by now an inalienable

requirement for the safeguard of the ecosystem [1].

In the course of the years several analytical techniques, mainly

chromatographic-spectrometric (HPLC/MS and GC/MS) have been

developed to being employed for the determination of the

environmental pollutants [2-10]. These traditional techniques of

analysis are extremely powerful in terms of sensitivity, selectivity and

specificity, but, apart from economical consideration, they present also

some disadvantages, due to the need of qualified staff, and to the

F. MAZZEI AND F. BOTRÈ ”INHIBITION BASED BIOSENSORS: ENVIRONMENTAL APPLICATIONS."

152

necessity to carry out an extensive pretreatment processes on the

sample to be assayed.

More important, it is not possible employ this techiniques "on the

spot", for the monitoring in real time of the area under investigation.

These reasons have given new impulse towards the

development of alternative analytical devices and methods, to be

applied in the screening of various contaminants in environmental

matrices, minimizing the pretreatment of sample, reducing the cost and

time of analysis and extending the number of sampling sites and/or of

samples per site. Biosensors represent one of these alternatives, and

their application to environmental monitoring has been continuously

growing the last years [11-15].

From a general point of view, all biosensors based on the

coupling of a biological agent with a physico-chemical signal

transducer (Fig. 1).

BIOSENSOR

Data acquisition and elaboration

Sample Biocatalytic layer Physical Chemical Transducer

- Electrochemical- Optical- Acoustic

- Enzymes- Microorganisms- Antibodies- Plant and Animal Tissues

Figure 1

Among the different biosensors employed in environmental

analysis [16-24], a leading role is played by the inhibition based

biosensors [25-47]. The principle of operation of these biosensors is


153

based on the interaction that occurs between specific chemical and

biological agents (inhibitors), present in the sample, and the biocatalyst

(an enzyme, a polyenzymatic sequence and/or even a whole tissue)

immobilized on the biosensor itself. The response of the biosensor is

therefore proportional to the reduction rate of the enzymatic reaction

which takes place at the sensor's interface.

Inhibition biosensors can be used for a double aim:

a) to study the kinetic characteristics of the inhibition process. In

this way it may be possible to gain indirect information regarding

the chemical nature and the conformational characteristics of the

active site(s) of the enzyme(s) involved in the signal production

sequence. In particular situations it may also be possible to

understand, or at least to shed light on, their catalytic

mechanisms. In these studies we have to bear in mind that the

generic kinetic formalism usually applied to the study of

equilibrium reactions refer to homogeneous phases, while in the

case of inhibition biosensors the backgroung theories and

equations have to be modified to include the presence of

heterogeneous phases. It has also to be taken in due account

that most of the “biological responses” producing the biosensor

signal refer to transient and/or local equilibria, and very seldom

they can be traced to either the Henri, Michaelis Menten (rapid

equilibrium) or the Briggs-Haldane (stationary state) models;

b) to determine the concentration of the inhibitor in the assayed

sample by measuring the percent of inhibition of the biocatalyst

immobilized on the biosensor.


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INHIBITION BIOELECTRODES FOR ENVIRONMENTAL ANALYSIS

In the last 20 years many works described the realization of

inhibition biosensors for the environmental analysis [25-47]. Table I

summarizes some examples of inhibition biosensors developed for the

analysis of environmental pollutants.

Table I:

Inhibitors Enzymes

Fluoride [25]; Mercury (II) [26] Urease

Fluoride [27] Acetylcholinesterase

Organophosphate and

carbamate pesticides [28-34]

Acetylcholinesterase and

Cholinesterase

Dithiocarbammates [35] Aldehyde dehydrogenase

Atrazine [36] Tyrosinase

H2S [37], HCN [37] Cytochrome oxidase

HCN [38] Peroxidase

Herbicides [39-47] Phothosystem II

Our laboratory has focused its research interests on the

development of inhibition based electrochemical biosensors for the

determination of environmental pollutants. We have indeed designed,

realized and applied inhibition based bioelectrodes, using as

biocatalysts either purified enzymes or plant tissue slices.

The use of plant tissues instead of purified enzymes for the

realization of biocatalyst-based biosensor allows a sensible reduction

the overall costs of operation, since the substitution of the catalytic

component of the biosensor is markedly less expensive than it is in the

case of the enzymatic biosensor.

Some representative amperometric, inhibition biosensors are the

following:


155

a) hybrid, bienzymatic biosensors (purified choline oxidase and plant-

tissue cholinesterase) for the determination of carbamate and

organophosphorous pesticides: inhibition of the enzyme

cholinesterase present in the inner part of the grapefruit shell

(albedum pomi citreum) [48];

b) plant tissue biosensors for the determination of atrazine: inhibition

of tyrosinase present in potato (Solanum tuberosum) tissue [49];

c) hybrid bienzymatic biosensors (purified glucose oxidase and plant

tissue acid phosphatase) for the determination of

organophosphorous pesticides: inhibition of acid phosphatase

present in potato (Solanum tuberosum) tissue [50].

Fig. 2 shows the inhibition curves obtained with the bienzymatic

acid phosphatase/glucose oxidase biosensor in the presence of known

concentration of some organophosphorous pesticides.

0 5 10 15 20 25 30 350

15

30

45

60MalathionMethyl ParathionParaoxon

[Pesticide] (ppb)

?I (pA )

Figure 2

The advantage of this type of biosensor with respect to

cholinesterase based biosensors is that the inhibition of acid

phosphatase by organophosphorous pesticides is almost completely


156

reversible, so that no reactivating treatment between the assays is

required. The reversibility of the enzymatic inhibition is also

responsible for the relatively long lifetime of operation of the sensors,

leading to a drastic reduction of the overall costs of operation.

The acid phosphatase/glucose oxidase biosensor has now been

patented by a private Italian research center for food analysis

(Co.Ri.Al.) and it is presently being evaluated for the analysis of

pesticidue residues in wheat and derived products.

2,4 Dichlorophenoxy Acid determination

2,4 Dichlorophenoxy Acid (2,4 D), is an auxinic herbicide

with growth regulator activity, that has mainly used for the selective

control of broadleaf weeds in cereal grain crops [51-53].

Although toxicity tests are still in progress, 2,4 D is

suspected to promote some tumoral pathologies, like for instance the

Hodgkin and non-Hodgkin lymphoma, prostate and pulmonary

cancer; furthermore, 2,4 D and its analogues are classified as

environmental oestrogens” (or “hormone disruptors”), for their effects

on the endocrine system.

The wide use of 2,4 D in agriculture can result in

contamination problems not only at the level of ground and surface

waters, but also of drinking water. For these reasons a reliable and

rapid technique for the the determination of 2,4 D in aqueous

samples is increasingly necessary [54-59].

Apart form “classic” HPLC and GC/MS techniques, several

immunosensors for the determination of 2,4 D have been recently

developed [54-59].

The aim of this work is to demonstrate the possible use of enzyme


157

enzymatic inhibition electrochemical biosensor also for the

determination of 2,4 D.

The biosensor here proposed is based on the combination of two

enzymes, alkaline phosphatase (ALP) and glucose oxidase (GOD),

catalyzing the following reactions respectively:

Glucose 6 Phosphate + H2O ?? ?? ALP Glucose + H2PO4-

Glucose + O2 GOD? ?? ? Gluconolactone + H2O2

The enzymes are coupled to an amperometric hydrogen

peroxide electrode, so that the current intensity measured in the

detection of H2O2 is proportional to the concentration of G6P in the

sample.

The determination of 2,4 D has been carried out by monitoring

their inhibition of the catalytic activity of ALP by the biosensor.

EXPERIMENTAL

Materials and Reagents

The enzymes acid phosphatase (E.C 3.1.3.2 from Potato),

alkaline phosphatase (E.C 3.1.3.1 from Bovine Liver), glucose oxidase

(E.C 1.1.3.4 from Aspergillus niger) and the substrate glucose-6-

phosphate (G-6-P), were purchased by Sigma Chemical Co. (St. Louis,

MO, USA); 2,4 dichlorophenoxy acid was kindly supplied from the

Società Italiana Chimici.

Biodyne Transfer membranes, (nylon 6.6 membrane, pore size

0.45 µm) with carboxylic groups on the surface, were supplied by Pall


158

Italia s.r.l. (Milano, Italy). Cellulose acetate H2O2-selective membranes

(MW cutoff? 100) were prepared according to a recently described

procedure [60]. Cellulose acetate dialysis membrane (0.0254 mm thick;

MW cutoff = 12000 D), were supplied by Sigma Chemical Co. (St.

Louis, MO, USA). The polyazetidine prepolymer (PAP) solution

(Hercules Polycup 172, 12% solids in water), used for the physico-

chemical immobilization of the enzymes, was obtained from Hercules

Inc. (Wilmington, Del. USA).

All other chemicals used were analytical grade. Twice distilled

water was used for the preparation of all solutions.

Preparation of the bienzymatic sensor

The biosensor was prepared by immobilizing an adequate amount

of ALP on a dialysis membrane using the polyazetidine prepolymer as

immobilizing agent. ALP and PAP (1 mg ALP/10 µl PAP) were spread

uniformly on the membrane. Glucose oxidase was immobilized on the

nylon 6.6 membrane with carboxyl groups on the surface, by means of

polyazetidine. GOD and PAP (1 mg GOD/10 µl PAP) were spread

uniformly on a disk of the membrane (0.8 cm diameter; density of

enzyme ? 0.135 mg/cm2). Both enzymatic membranes were left for 24

hours at room temperature, washed out with glycine buffer 0.1 M, pH

8.0 and then stored in a dry state at 0 °C.

Assembly of the sensor.

The sensor was assembled by placing on the platinum surface of

a H2O2 electrode (Universal Sensors Inc., New Orleans, LA, USA) a

sequence of three different membranes, in the following order:


159

1) a cellulose acetate membrane (to eliminate possible interferences

with other electroactive chemical species);

2) the GOD-nylon membrane;

3) the ALP-dialysis membrane (that have also the aim to prevent

microbial attack of the enzymes and the leaking of the enzymes

themselves out of the membrane).

A rubber O-ring was used to fix the above mentioned three

successive layers on the tip of the H2O2 electrode.

Storage of the sensor.

For short-term storage (up to 8 hours), the biosensor was kept

into a glycine buffer solution 0.1 M at pH=8.0. For longer term storage

the enzymatic membrane(s) were kept dried at -15 °C.

Amperometric measurements

Amperometric measurements were carried out by connecting the

biosensor to an amperometric detector (ABD, Universal Sensors Inc.,

New Orleans, LA, USA). A constant potential of +650 mV was applied

between the platinum anode and the Ag/AgCl cathode of the hydrogen

peroxide electrode. The electrode jacket was filled with an internal

filling solution of phosphate buffer and KCl both 0.01 M, pH=6.6.

Experiments with ALP free in solution.

To quantify the inhibition degree of 2,4 D towards ALP we have

realized a set of experiments with the enzyme free in solution.

Experiments were carried out in a glass cell, thermostated at 37 °C by

forced water circulation, ensuring a uniform magnetic stirring, at a

constant rate, throughout the assay, in 2,5 mL of glycine buffer solution


160

0.1 M at pH=8.0, containing ALP (final concentrations: 1.0 U/mL) and

MgCl2 0.2 mM as cofactor of ALP. This pH value was found to be the

optimum balance between the pH values with respect to the maximum

catalytic activities of GOD and ALP. The determination of 2,4 D was

performed by dipping the GOD sensor in glycine buffer 0.1 M, pH=8.0,

at 37 °C, and adding G-6-P at a final concentration of 0.5 mM: this

value has been chosen since it represents the best compromise

between the optimal bioelectrodes responses (it lies in the middle of

the linearity range, toward G-6-P biosensor) and the optimal

concentration ratio G-6-P/inhibitor to quantitatively highlight the

inhibition of the catalytic activity of ALP. After stabilization of the

current, increasing quantities of the solution containing 2,4 D was

added under constant magnetic stirring, and the decrease of the

current, proportional to the decrease of the hydrogen peroxide

production following ALP inhibition, was followed for 30 minutes.

In this way a calibration curve reporting the decrease of current as

a function of the concentration of added inhibitor can easily be derived.

Experiments with the bienzymatic biosensor.

The measurements with the bienzymatic sensor were performed

in the same conditions described above, with the difference that no

ALP was added to the measuring chamber.

RESULTS

Fig. 3 shows the amperometric signal recorded during a typical

inhibition experiment, followed by the biosensor. The first arrow refers

to the addition of G6P, to a final concentration 0.5 mM, the following

arrows to the addition of 2,4 D to a final concentration of 3.5 ppm.


161

0 20 40 60 80 100 120Time (min)

0

2

4

6

8

10I (nA)

G-6-P

2,4 D

Figure 3

As it can be seen, the value of the current intensity increases

after the addition of G6P and decreases after the addition of the 2,4 D,

thus confirming the inhibition power of the herbicide towards the

catalytic activity of ALP.

Fig. 4 shows the calibration plot obtained with the GOD

biosensor with ALP free in solution, in presence of known

concentrations of 2,4 D.

0 1 2 3 4 50

1

2

3

4

5

6Incubation time: 60'Incubation time: 30'

[2,4 D] (ppm)

?I (nA )

Figure 4


162

Figures 5 shows a comparison between the inhibition of ALP

and cid phosphatase (AP) by 2,4 D: all experiments are carried out in

the presence of the same activity of ALP and AP in solution, and of

different concentration of 2,4 D. As we can see the inhibition degree

towards AP is negligible respect the data obtained with ALP.

0 1 2 3 4 50

1

2

3Acid PhosphataseAlkaline Phosphatase

[2,4 D] (ppm)

?I (nA )

Figure 5

Fig. 6 shows the calibration plot obtained with the bienzymatic

sensor in presence of known concentration of 2,4 D.


163

0 1 2 3 4 50

1

2

3

4Incubation time: 60'Incubation time: 30'

[2,4 D] (ppm)

?I (nA )

Figure 6

Tables I and II summarize the main electroanalytical features of

the proposed method (ALP free in solution and bienzymatic biosensor

respectively), as evaluated on standard solutions of 2,4 D.

Table I

Incubation time: 60’ Incubation time: 30’ Temperature of analysis: 37 °C 37 °C pH: 8.0 8.0 Buffer: Glycine 0.1 mol/L Glycine 0.1 mol/L Equation of the calibration graph: Y = ? i (nA); X = [2,4 D concentration] (ppm)

Y = 0.5 +1. 36 X

Y = 0.22 + 0.82 X

Linearity range (ppm): 0.015 – 4.2 0.035 – 4.2

Correlation coefficient: 0.9971 0.9880 Lower detection limit (ppm): 0.01 0.05 Repeatibility of the measurements (as pooled standard deviation in the linearity range):

2.3

3.2

Table II


164

Incubation time: 60’ Incubation time: 30’ Temperature of analysis: 37 °C 37 °C pH: 8.0 8.0 Buffer: Glycine 0.1 mol/L Glicina 0.1 mol/L Equation of the calibration graph: Y = ? i (nA); X = [2,4 D concentration] (ppm)

Y = 0.18 + 0.81 X

Y = 0.09 + 0.51 X

Linearity range (ppm): 0.015 – 4.2 0.035 – 4.2 Correlation coefficient: 0.9980 0.9984 Lower detection limit (ppm): 0.01 0.05 Repeatibility of the measurements (as pooled standard deviation in the linearity range):

1.5

2.0

Fig. 7 reports the variation of the biosensor performance with

time, expressed as the percentage of the biosensor original response,

and evaluated on a 0.5 mM solution of G6P. The biosensor was used

for 4-5 determinations per day. The decrease of biosensor activity

recorded after the first few assays is due to an excess of enzyme,

unspecifically adsorbed on the catalytic membrane at the end of the

physico-chemical immobilization procedure: usually this excess is

completely washed out when the percent of initial activity reaches 70-

75%. The decrease of biosensor response is then more regular,

retaining about 50% of initial activity after 2-3 weeks.


165

0 2 4 6 8 10 12 14 16 18 20 22 240

25

50

75

100

Time (days)

% of activit y

Figure 7

DISCUSSION AND CONCLUSIONS

According to the obtained results, the biosensor described in

this work shows interesting electroanalytical properties, presenting the

same advantages of analogous analyitical devices reported in literature

(basically its simplicity of preparation and use and the reduced time

and costs of operation). Our research is presently directed towards the

improvement of the biosensor performance: evaluation of different

sample preconcentration techniques are in progress to reduce the

sensitivity gap between the detection limit of our method (15 ppb for 60'

of incubation time using the bienzymatic sensor) and the maximum

concentration for pesticides allowed in drinking water by European

Community (0.1 ppb) [61].

Should this problem be solved, the biosensor could be used for

the immediate and continuous detection of 2,4 D, while other and more

sophisticated techniques, like HPLC, GC-MS and/or LC-MS-MS and

GC-MS-MS should be used as confirmation techniques whenever a


166

“positive” sample is detected.

The use of this and analogous inhibition biosensors in

environmental field can represent an important tool for the

environmental monitoring activity. In particular the inhibition based

bioelectrodes, for their simplicity of realization and use are to take in

serious consideration. In recent years the research in the field of the

biosensors was direct to the development of highly selective and

sensitive devices, but it is our personal opinion that in the

environmental analysis, as well as in food analysis, the use of

biosensor is not going to be “exclusive” (like it may be for instance in

the case of biocompatible implantable devices to be used in clinical

chemistry).

The role of biosensors in the determination of environmental

pollutants is in our opinion not to completely replace the traditional,

more sophisticated instrumental techniques, but to represent a valid

complement to them, especially in all those situations where it is

necessary to carry out measures “on the spot”, reducing the overall

times of analysis and minimizing the sample pretreatment process. In

such situations a biosensor (or better an array of biosensors) would be

the analytical tool supplying all the necessary information to monitor, in

real time, the state of pollution of the matrix under investigation. In the

case of a positive response of one or more biosensors a “traditional”

sampling procedure will be activated to carry out more specific assays

aimed to confirm and quantify more precisely the extent of each case of

environmental contamination.

In this field the development of inhibition based biosensors

would be a very powerful tool for the screening of huge populations of

samples for different classes of pollutants, thus detecting any


167

compound belonging to the same class on the basis of the common

biological effects, and representing an effective and powerful aid for the

early detection of environmental contamination.

REFERENCES

1. T.R. Roberts, in Chemistry, Agriculture and the Environment, (M.L.

Richardson Ed.), The Royal Society of Chemistry, Cambridge

(1991) 249

2. J. Sherma, Anal. Chem., 61 (1989) 153R

3. D. Barcelò in Environmental Analysis: Techniques, Applications and

Quality Assurance (D. Barcelò Ed.), Elsevier Science Publishers,

Amsterdam, Vol. 13 (1993) 149



6. S. Butz and H.J. Stan, Anal. Chem., 67 (1995) 620

7. Analysis of Pesticides Residues, (H.A. Moye Ed.), Wiley, New York

(1981)

8. DFG Manual of Pesticide Residue Analysis, (H.P. Thier, H. Zeumer

Eds.), VCH, Veinheim, Germany (1987)

9. D. Volmer, K. Levsen, G. Wunsch, J. Chromatogr., 647 (1993) 235

10. D. Barcelo, G. Durand, Anal. Chem., 62 (1990) 1696

11. Biosensors,(C. Tran Minh Ed.) Chapman & Hall, London (1993)

12. Biosensors Fundamentals and Applications (A.P.F. Turner, I.

Karube and G.S. Wilson Eds.), Oxford University Press, Oxford

(1987)

13. Biosensors: An introduction, (B. Eggins Ed.), Wiley & Teubner

Publishers (1996)

14. Biosensors: A Practical Approach, (A.E.G. Cass Ed.) Oxford


168

University Press, Oxford (1990)

15. Biosensors, (E.A.H. Hall Ed.) Open University Press, Milton Keynes

(1990)

16. K. R. Rogers, Biosensors Bioelectronics, 10 (1995) 533

17. M. Minunni, P. Skladal, and M. Mascini, Anal. Lett., 27 (1994) 1475

18. K.R. Rogers and L.R. Williams, Trends Anal. Chem., 7 (1995) 289

19. F. Jung, S.J. Gee, R.O. Morrison, M.H. Goodrow, A.E. Karu, A.L.

Brown, Q.X. Li, B. Hammock, Pestic. Sci., 26 (1989) 303

20. J. M. Van Emon and V. Lopez-Avila, Anal. Chem., 64 (1992) 79A

21. J.L. Marty, D. Garcia, R. Roullion, Trends Anal. Chem., 14(7)

(1995) 329

22. M.P. Marco, S. Gee, B.D. Hammock, Trends Anal. Chem., 14(7)

(1995) 341

23. P.M. Kramer, J. AOAC Int., 79(6) (1996) 1245

24. H.Y. Neujahr, Biotechnol. Ge. Eng. Rev., 1 (1985) 167

25. C. Tran Minh, J. Beaux, Anal. Chem., 51 (1979) 91

26. R.B. Shi, K. Stein, and G. Schwedt, Fresenius J. Anal. Chem., 357

(1997) 752

27. C. Tran Minh, J. Beaux, C.R. Acad. Sci. Paris, Ser. C, 288 (1979)

545

28. L. Campanella, M. Achilli, M.P. Sammartino, M. Tomassetti,

Bioelectrochem. Bioenerg., 26 (1991) 237

29. P. Sklàdal, M. Mascini, Biosens. Bioelectron., 7 (1992) 335

30. J.L. Marty, N. Mionetto, T. Noguer, F. Ortega, C. Roux, Biosens.

Bioelectron., 8 (1993) 273

31. D. Martorell, F. Cespedes, E. Martinez-Fabregas, S. Alegret, Anal.

Chim. Acta, 290 (1994) 343

32. J.L. Marty, K. Sode, I. Karube, Electroanalysis, 4 (1992) 249


169

33. P. Durand, D. Thomas, JEPTO, 5-4/5 (1984) 51

34. M. Stoytcheva, Anal. Lett., 27(15) (1994) 3065

35. J.L Marty, T. Noguer, Analusis, 21 (1993) 231

36. F.A. Mc Ardle, K.C. Persaud, Analyst, 118 (1993) 419

37. W.J. Albery, A.E.G. Cass, Z.X. Shu, Biosens. Bioelectron., 5 (1990)

367

38. V. Volotovsky, N. Kim, Biosens. Bioelectron., 13(9) (1998) 1029

39. Brewster JD, Lightfield AR and Bermel PL (1995) Storage and

immobilization of Photosystem II reaction centers used in an assay

for herbicides. Analytical Chemistry 67: 1296-1299

40 Carpentier R, Loranger C, Chartrand J, Purcell M (1991)

Photoelectrochemical cell containing chloroplast membranes as a

biosensor for phytotoxicity measurements. Analytica Chimica Acta

249(1): 55-60

41 Jockers R, Bier FF, Schmid RD (1993) Specific binding of

photosynthetic reaction centers to herbicide-modified grating

couplers. Analytica Chimica Acta 280(1): 53-59

42 Marty J-L, Garcia D, Rouillon R (1995) Biosensors: potential in

pesticide detection. Trends in Analytical Chemistry 14(7): 329-333

43 Merz D, Geyer M, Moss DA and Ache H-J (1996) Chlorophyll

fluorescence biosensor for detection of herbicides. Fresenius

Journal of Analytical Chemistry 354: 299-305

44 Pandard P, Rawson DM (1993) An amperometric algal biosensor

for herbicide detection employing a carbon cathode oxygen-

electrode. Environmental Toxicology and Water Quality 8(3): 323-

333

45 Rouillon R, Tocabens M and Marty J-L (1994) Stabilization of

chloroplasts by entrapment in polyvinylalcohol bearing


170

styrylpyridinium groups. Analytical Letters 27(12): 2239-2248

46 Rouillon R, Mestres J-J, Marty J-L (1995) Entrapment of

chloroplasts and thylakoids in polyvinylalcohol-SbQ. Optimization

of membrane preparation and storage conditions. Analytica

Chimica Acta 311: 437-442

47 Rouillon R, Sole M, Carpentier R, Marty J-L (1995) Immobilization

of thylakoids in polyvinylalcohol for the detection of herbicides.

Sensors and Actuators B 26-27: 477-479

48 C. Botrè, F. Botrè, G. Lorenti, F. Mazzei, F. Porcelli, G. Scibona

and G. Simonetti, Sensors and Actuators, B, 19 (1994) 689

49 F. Mazzei, F. Botrè, G. Lorenti, F. Porcelli, G. Scibona, G.

Simonetti and C. Botrè. Anal. Chim. Acta, , 16 (1995) 79

50 F. Mazzei, F. Botrè, C. Botrè, Anal. Chim. Acta, 336 (1996) 67

51 Biochemistry and Physiology of Herbicide Action,( C. Fedtke Ed.)

Springer-Berlag, New York (1982)

52 Physiology of Herbicide Action, (M.D. Devine, S.O. Duke and C.

Fedtke Eds.) Prentice Hall Inc., Englewood Cliffs, NJ (1993)

53 T.M. Sterling and J.C. Hall, in Herbicide Activity: Toxicology,

Biochemistry and Moleculare Biology (R. Michael Roe, J.D. Burton,

R.J. Kuhr Eds.), IOS Press, (1997) 111

54 C. G. Bauer, A. V. Erementko, E. Ehrentreich, F. F. Bier, A.

Makower, H. B. Halsall, W. R. Heineman, and F. W. Scheller, Anal.

Chem., 68 (1996) 2453

55 E. P. Medyantseva, M. G. Vertlib, M. P. Kutyreva, E. I. Khaldeeva,

G. K. Budnikov, and S. A. Eremin, Anal. Chim. Acta, 347 (1997) 71

56 T. Kalab and P.A. Skladal, Anal. Chim. Acta, 304 (1995) 361

57 B. B. Dzantiev and A.V. Zherdev, Biosensors Bioelectronics, 11

(1996) 179


171

58 P. Skladal and T. A. Kalab, Anal. Chim. Acta, 316 (1995) 73

59 K. R. Rogers, S. D. Kohl, L. A. Riddick, and T. Glass, Analyst 122

(1997) 1107-1111

60 M. Mascini, F. Mazzei, D. Moscone, G. Calabrese, M. Massi

Benedetti, Clin. Chem. 33 (1987) 591

61 C. Wittmann, B. Hock, J. Agric. Food. Chem., 39 (1991) 1194

inhibition based biosensors: environmental applications

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