A presentation titled: THE USE OF BIOSENSORS IN DETECTING ENVIRONMENTAL POLLUTANTS

BY SIMISOLA A. OGUNDUGBA

ABSTRACT
The purpose of this research is to shed light on the subject of biosensors and its use as a viable means of detecting various pollutants in the environment.

INTRODUCTION
There is a worldwide effort towards the development of bioanalytical devices that can be used for detection, quantification and monitoring of specific chemical species. Conventional analytical techniques (e.g. G.C., Ads. Spec. Ph. metry), although highly precise, suffer from the disadvantages of high cost, complex sample preparations, the need for trained personnel and the fact that they are mostly laboratory bound (cannot be done on-site).

Dioxins as an example:
Formed as a by-product of the combustion of chlorophenols (mostly biocides); recognized as one of the most poisonous synthetic chemicals. Its effects include alleged birth defects (teratogenicity), severe skin disorders, carcinogenicity, mutagenicity, immuneresponse retardation, and bio-accumulation in aquatic organisms and people. (Hileman, 1994; U.S. E.P.A. , 2005a).

WHAT THEN IS A BIOSENSOR ?

an analytical device containing an immobilized biological material (which could be an enzyme, or antibody, or nucleic acid, or hormone, or whole cell), which can specifically interact with an analyte and produce physical, chemical or electrical signals that can be measured.

Operating Principle of Biosensors

biological material in use (e.g. an enzyme) is immobilized by conventional methods like physical or membrane entrapment, noncovalent or covalent binding. A contact is made between the immobilized biological material and a transducer. The analyte binds to the biological material to form a bound analyte which in turn produces the electronic response that can be measured. OR the analyte is converted to a product which could be associated with the release of heat, gas (oxygen), electrons or hydrogen ions. The transducer then converts the product-linked changes into electrical signals which can be amplified and measured.

SCHEMATIC DIAGRAM OF A BIOSENSOR

A typical biosensor comprises: Immobilized biologically-sensitive element, a transducer, amplifier and signal processor.

Classification of biosensors
According to the transduction (sensing) method as electrochemical, optical, piezoelectric and thermal sensors ; OR According to the bio-recognition principle as immunochemical, enzymatic, whole-cell and DNA biosensors

A CASE STUDY OF ENZYME INHIBITION-BASED BIOSENSORS.

This class of biosensors is based on the fact that specific toxic analytes (pollutants) inhibit the activity of specific enzymes. Relies on a quantitative measurement of the enzyme activity before and after exposure to a target analyte. It is chosen because of its wide application for detection of various pesticides, insecticides, heavy metals, glycoalkaloids, etc.

Inhibitors distribution in enzymatic biosensors investigations.

DEFINITION OF KEY TERMS

Enzyme: organic catalysts produced by microbes and used for speeding up biochemical reactions. Inhibitor: chemical agents which reduce the activity of enzymes (pollutants). Immobilization: process in which a soluble fraction (Bio-element) is rendered insoluble usually by attachment to a solid phase to make it stable. Transducers: devices which convert one form of energy to another form (chemical-electrical). Analyte: A substance or chemical constituent that is undergoing analysis.

PROCESS STEPS
STEP I
The determination of initial enzymatic activity (A) using optimum conditions (pH, temp., subst. conc.)

STEP II

The application of the biosensor to a substrate that contains an inhibitor (pollutant)

STEP III

The measurement of the residual activity (A*) i.e. activity after exposure of the biosensor to the inhibitor).

DESIGN CONSIDERATIONS
1. An extensive background knowledge of the enzyme characteristics:  taking into account the specificity of the enzyme Determining the optimum conditions for the enzyme (pH, temperature, etc.)

2. Immobilization technique
The enzyme is attached to the transducer surface (graphite, gold film) for stability and to put it in close proximity with the transducer.
(A) covalent binding; (B) adsorption; (C) gel entrapment; (D)cross-linking; (E)microencapsulation; (F) adsorption-cross-linking.

3. Transducer Choice
Any of the transduction method could be chosen (electrochemical, optical, piezo-electric, thermal). Electrochemical transducers are common since it can directly measure biochemical reaction properties in terms of current (I), voltage (v), or conductance (G). Various configurations of EC transducers are amperometric (I), potentiometric (V), conductometric (G), etc.

4. Enzyme-Inhibitor System.
The long-term function of enzyme-based biosensors depends on this. The inhibition can be either reversible or result in an irreversible inactivation of the enzyme. Reversible inhibition can either be competitive (inhibitor competes with active site of enzyme) OR uncompetitive (no competition with active site but competes with ES complex ).

4ai. Reversible competitive inhibition

(constant Vmax , high Km therefore high subst. conc. is needed)
example: trichloroethane and methane during methane oxidation

4aii. Reversible Uncompetitive inhibition.

(Decreases Vmax, Km constant)

example: cyanide and iron co-factors

4b. Irreversible Inhibition.

Enzyme Inhibitor interaction results in the formation of a covalent bond between the enzyme active center and the inhibitor. decomposition of the Enzyme Inhibitor complex results in the destruction of enzyme.

5. LIMIT OF DETECTION (LOD)

Defined as the concentration of the pollutant being measured which gives a minimum detectable difference signal (reduction in activity). Any concentration above the LOD value will yield a reduction in enzyme activity. The LOD value generally corresponds to 90 80% of residual activity, that is 10 20% inhibition.

6. REGENERATION
Strong inhibition of the enzymes can present a serious problem for practical applications by limiting the reuse of biosensors. Some chemical substances called regenerators can be used to treat enzymes after each exposure to inhibitors. This reduces ageing of the enzyme. Examples are NaF, pyridine-2-aldoxime methyliodide (2-PAM), 4-formylpyridinium bromide dioxime (TMB4).

Simple Illustration of enzyme biosensor applying amperometric transduction

Transducer Equation

I = ZFkmC*
where Z, F, Km are constants, C* is the is the bulk concentration of the product species and I (amperes) is the value of the detected electric current. Typical current rating are usually in the 10-6 - 10 -9

Facts and figures [3] (detection of some insecticide compounds)

OPAA (organophosphorus acid anhydrolase); OPH (organophosphorus hydrolase); OP (organophosphorus)

SOME CHALLENGES IN BIOSENSOR TECHNOLOGY.

1. Multianalyte Determination: biosensors capable of
determining different analytes simultaneously .

2. Miniaturization: reducing bulkiness, and making biosensors

more portable and handy.

3. Mass Production: batch-fabrication, which leads to the

production of inexpensive biosensors.

4. Network systems to establish connected analytical

stations in different natural environments. This will have enormous impact on all field-based environmental measurements.

5. Validation.
Despite the practical advantages of biosensors, they should be comparable to conventional analytical systems in terms of reliability and sensitivity. Biosensor measurements have to be verified by comparing them with the results of conventional chemical analysis.

Correlation between results obtained by an immunosensor and those obtained by LC/MS

COMPARATIVE ADVANTAGES OF BIOSENSORS OVER CONVENTIONAL METHODS On-site applications; Minimal sample treatment; Fast response times (typically 5-10 mins.); Low cost; Portability; Ease of use

CONCLUSION
The field of biosensor technology holds many exciting analytical applications for environmental use. Biosensor applications is not aimed at eliminating conventional methods but to complement it.

Some References:
1. Sara Rodriguez-Mozaza, et al, Biosensors for Environmental Monitoring: A global perspective (2004). 2. Aziz Amine, et al, Enzyme inhibition-based biosensors for food safety and environmental monitoring, Biosensors and Bioelectronics 21 (2006) 1405 1423. 3. Gamal A. E. Mostafa, Electrochemical Biosensors for the Detection of Pesticides, The Open Electrochemistry Journal, (2010), 2, 22-42.