Tyrosinase Biosensors for the Detection of Pesticides

1
Tyrosinase Biosensors for the Detection of
Pesticides

• By Bridgett L. Steele

2
What is a biosensor?

• A miniaturized device integrating a biological
  sensing element on intimate contact with an
  appropriate transducer for conversion of the
  recognition success to a primary signal that can
  be amplified and subsequently processed.

Figure 1. Schematic of a basic biosensor.
Marco, M.-P. Barcelo, D. Meas. Sci Technol.
1996. 7, 1547-62. Gerald, M. Chaubey, A.
Malhotra, B.D. Biosens. Bioelectron. 2002. 17,
345-59.
3
What is a biosensor?

• Types of Biological Sensing Units
• Enzymes
• Antibodies
• Nucleic Acids
• Microorganisms
• Cells

• Types of Tranducers
• Amperometric
• Potentiometric
• Optical
• Piezoelectric

4
Why are These Sensors Being Developed for
Environmental Monitoring?

• Have many advantages over traditional methods
  such as High Performance Liquid Chromatography
  (HPLC) and Gas Chromatography-Mass Spectrometry
  (GC-MS), such as
• Shorter Analysis Times
• High Selectivity
• Simple
• Provide On-Line and Continuous Monitoring
• Less Expensive

5
Brief History

• Professor Leland C. Clark Jr. is credited as the
  founder of the biosensor concept.
• In 1962, Clarke addressed the New York Academy of
  Sciences Symposium.
• Later that year, Clarke published a paper on a
  glucose sensor.
• In 1969, G.G. Guilbault and J. Montalvo developed
  the first potentiometric electrode.
• In 1975, biosensors became a commercial reality.

6
Tyrosinase Biosensors

• Tyrosinase biosensors are produced by
  immobilizing tyrosinase on the surface of an
  electrode.
• This sensor can be used for the detection of
  various pollutants, such as hydrazine, atrazine,
  chlorophenols, carbamates, and organophosphorus
  pesticides.

7
Tyrosinase

• Also called monophenol monooxygenase.
• Tyrosinase is very stable and can be used in
  aqueous and non-aqueous materials.
• It has two active sites, an aromatic site and an
  metal binding site.

8
Tyrosinase

• Tyrosinase has two activities
• It catalyzes the hydroxylation of monophenols to
  ortho diphenols, reducing molecular oxygen to
  water.

9
Tyrosinase

• It also catalyzes the oxidation of
  ortho-diphenols to ortho-quinones by the
  reduction of molecular oxygen to water.

10
Amperometric Methods

• Amperometry
• Set a constant voltage and compare changes in
  current.
• The concentration of mono and diphenols is
  directly proportional to the increase in current.
• Other contaminates are detected through
  inhibition of the enzyme, which decreases
  current.

11
Amperometry
Figure 2. Current vs. time graph for successive
additions of 2.5 x 10-5 M catechol violet (right).
McArdle, F.A. Persaud, K.C. Analyst 1993. 118,
419-23.
12
Electroenzymatic Cycle
Besombes, J.-L. Cosnier, S. Labbe, P. Reverdy,
G. Anal. Chim. Acta 1995. 311, 255-63.
13
Amperometric Methods

• Chronoamperometry
• Voltage is applied for a set amount of time.
• Study the decay of current.

14
Chronoamperometry
Figure 3. Typical current decay for a tyrosinase
biosensor in the presence of 50 mM sodium 1,2
naphthoquinone-4-sulfonate (NQS) (solid line) and
50 mM NQS and 200 mM diazinon (dotted line).
Everett, R.W. Rechnitz, G.A. Anal. Chem. 1998.
70, 807-10.
15
Immobilizing the Enzyme
16
Physical Adsorption

• A carbon paste electrode was constructed by
  mixing 3 (w/w) solution of tyrosinase with a
  carbon paste made of 60 graphite and mineral
  oil.
• Amperometric and Chronoamperometric experiments
  were performed by holding the working electrode
  at -0.1V.

Wang, J. Chen, L. Anal. Chem. 1995. 67, 3824-27.
17
Current vs. Time Response
Figure 5. Current vs. time graphs for ordinary
(b) and tyrosinase electrodes (a) for successive
increments of 2.5 x 10-4 M methylhydrazine (A),
hydrazine (B) and dimethylhydrazine (C) in the
presence of 1.5 x 10-5 M phenol in a 0.05 M
phosphate buffer.
Wang, J. Chen, L. Anal. Chem. 1995. 67, 3824-27.
18
Hydrazine Detection

• Inhibitory Effect
• Dimethylhydrazinelt hydrazinelt methylhydrazine
• Coefficient of Inhibition (I0.5)
• Hydrazine 4.5 x 10-4 M
• Methylhydrazine 5.5 x 10-4 M
• Dimethylhydrazine 6.3 x 10-4 M

Wang, J. Chen, L. Anal. Chem. 1995. 67, 3824-27.
19
Hydrazine Detection

• River and drinking water samples were spiked with
  differing levels of methylhydrazine, using
  natural pH and ionic strength.
• Sensor response was fast and sensitive.
• Blank response was very low, indicating little
  interference.

Wang, J. Chen, L. Anal. Chem. 1995. 67, 3824-27.
20
Sensor Performance

• Reversibility
• Sensor regained original response after being
  rinsed with buffer, which is consistent with
  competitive inhibition.
• Reproducibility
• Sensors response only deviated 3.8 over 5
  trials.
• Shelf Life
• 30 days in air storage at 4oC

Wang, J. Chen, L. Anal. Chem. 1995. 67, 3824-27.
21
Entrapment

• A 30 mL mixture of 0.06 mg of tyrosinase and 18
  nmol of monomer 1 were spread on a glassy carbon
  disk and dried under vacuum.
• Electropolymerization of the monomer was carried
  out for 30 minutes at 0.75 V v. SCE.

Monomer 1.
Besombes, J.-L. Cosnier, S. Labbe, P. Reverdy,
G. Anal. Chim. Acta 1995. 311, 255-63.
22
Entrapment

• Tyrosinase has an isoelectric point of 4.7, and
  at the experimental pH of 6.5 carries a negative
  charge.
• Therefore in aqueous solutions it has
  electrostatic interactions with the quaternary
  amine.

Monomer 1.
Besombes, J.-L. Cosnier, S. Labbe, P. Reverdy,
G. Anal. Chim. Acta 1995. 311, 255-63.
23
Detection of Pollutants
Chloroaniline
Atrazine
Chloroisopropylphenylcarbamate
3,4 Dichlorophenol
24
Detection of Pesticides
Besombes, J.-L. Cosnier, S. Labbe, P. Reverdy,
G. Anal. Chim. Acta 1995. 311, 255-63.
25
Sensor Performance

• Over 200 determinations could be performed with
  one sensor.
• Shelf Life
• When stored at 4oC, sensor retained 85 of its
  activity after seven days and 20 of its activity
  after 54 days.
• Reproducibility
• Maximum deviation of 23 for ten sensors.

Besombes, J.-L. Cosnier, S. Labbe, P. Reverdy,
G. Anal. Chim. Acta 1995. 311, 255-63.
26
Cross-Linking

• A 20 ml solution of 20 mg/mL of tyrosinase was
  applied to the surface of a pre-activated glassy
  carbon electrode and dried under vacuum.
• A 10 mL solution of 1 glutaraldehyde was added
  to the surface, reacted for 30 min and dried
  under vacuum.

Everett, R.W. Rechnitz, G.A. Anal. Chem. 1998.
70, 807-10.
27
Method

• A 10 s reductive pulse of -150 mV was applied to
  the electrode, in order to determine the rate of
  current decay.
• Experiments were run in 10 mL of phosphate buffer
  containing 50 mL of sodium 1,2
  napthoquinone-4-sulfonate (NQS).

Everett, R.W. Rechnitz, G.A. Anal. Chem. 1998.
70, 807-10.
28
Detection of Diazinon

• Figure 4. From Top to Bottom 0, 50, 100, 200,
  300, 500, 1500 mM of diazonin in 0.05 M phospahte
  buffer containing 50 mL of NQS.

Everett, R.W. Rechnitz, G.A. Anal. Chem. 1998.
70, 807-10.
29
Detection of Diazinon and Dichlorvos

• Dichlorvos
• Detection Limit of 75 nM
• I0.5 of 50 mM
• Diazinon
• Detection Limit of 5 uM
• I0.5 of 1000 mM

Everett, R.W. Rechnitz, G.A. Anal. Chem. 1998.
70, 807-10.
30
Ceramic Chip Biosensor

• The anode was polymerized in a solution of 0.1 M
  pyrrole and 0.1 M tetraethylammonium
  p-toluenesulfonate at 2.0 V for 2 min.
• Two applications of 10 g/L tyrosinase and 20 mL
  of glutaraldehyde were added to the surface and
  dried.

McArdle, F.A. Persaud, K.C. Analyst 1993. 118,
419-23.
31
Ceramic Chip Biosensor

• A detection level of 1.5 x 10-6 M of atrazine was
  achieved.
• The enzyme electrode was insensitive to
  monophenols, due to covalent cross-linking, but
  retained its sensitivity to o-diphenols and
  triphenols.

McArdle, F.A. Persaud, K.C. Analyst 1993. 118,
419-23.
32
Conclusion

• Biosensors have many advantages over traditional
  methods of detection for pesticides.
• Use of these sensors are limited, due to
• Availability
• Stability
• Inability to Sense Reaction Products

33
References

• Marco, M.-P. Barcelo, D. Meas. Sci Technol.
  1996. 7, 1547-62.
• Gerald, M. Chaubey, A. Malhotra, B.D. Biosens.
  Bioelectron. 2002. 17, 345-59.
• Marty, J.L Garcia, D. Rouillon, R. Trends Anal.
  Chem. 1995. 14, 329-333.
• Besombes, J.-L. Cosnier, S. Labbe, P. Reverdy,
  G. Anal. Chim. Acta 1995. 311, 255-63.
• Turner, A.P.F. Biosensors Past, Present, and
  Future. 1996. http//www.cranfield.ac.uk/biotech/c
  nmap.htm (accessed Nov 2002).
• DBGET. http//www.genome.ad.jp.dbget-bin/www_bget?
  ec1.14.18.1 (Accessed Nov 2002).
• Wang, J. Chen, L. Anal. Chem. 1995. 67, 3824-27.
• Reiger, P.H Electrochemistry 2nd ed. 1994.
  Chapman and Hall London, 179-181.
• McArdle, F.A. Persaud, K.C. Analyst 1993. 118,
  419-23.
• Everett, R.W. Rechnitz, G.A. Anal. Chem. 1998.
  70, 807-10.
• Cosnier, S. Innocent, C. J. Electroanal. Chem.
  1992. 328, 361-66.