“Prospect and application of Biosensor in plant disease management”

 Humankind has been performing bioanalysis since the dawn

of time, using the sensory nerve cells of the nose to detect the

scents or the enzymatic reactions in the tongue to taste food

Biological organisms are some of the most efficient machines ever created, scientist have sought to apply and copy their efficiency for use in man made creations.

Using bioreceptors from biological organisms or receptors that have been patterned after biological systems,Scientist have developed a new means of

chemical analysis that often has the high selectivity of biological recognition systems

Biorecognition elements in combination with various transduction modes have helped to create the rapidly expanding fields of bioanalysis and related technologies .

A biosensor can be defined as an analytical device with a biological sensing element in close proximity or integrated with a signal transducer inorder to quantify a compound or conditions.In the past two decades, the biological and medical fields haveseen great advances in the development of biosensors and biochips capable of characterizing and quantifying biomolecules .

Sensors are the electronic device

which uses the physical changes of the reaction to produce an effect.

According to the nature of input signals they can be broadly classified as

1. Physical sensor2. Chemical sensor (Sultana Afrin, 2004).

Biosensor• A biosensor is an analytical device which converts a

biological signal into electrical signal.

• Leland C. Clark Jr. known as the father of the biosensor.

• It is an analytical device where immobilized layer of biological material is in contact with sensor which ……..

• Analyses the biological signal & convert it in to electrical signal (Gronow,1984).

Principle of Biosensor 1. Immobilization of biological materialThe biological components is immobilized on the transducer surface. Gluter aldehyde Enzymes for immobilization.

2. Surface treatment to transducerSurface may be treated with 3-aminopropy l –triethoxysilane.Method yield non reproducible results and• Often causes a large reduction in the activity.

3. Interaction of analyte with biological material. Produces a physical change close to the transducer surface.Change in the mass of biological components as a result of the reaction.

4. Conversion of biological signal.The transducer detects the signal and convert into the electrical signals.

5. Amplification of signal.• The signal is then processed and interpreted and is displayed into the suitable units.

A)Biological components 1. Enzymes 2. Antibody 3. Microorganisms 4. Cell etc

B) Physical components. 1. Transducer 2. Detector 3. Signal processing unit 4. Amplifier

Methods of detection1. Electrochemical method of detectionProduction of electrical potential due to change distribution of the electrons.

2. Amperometric method of detection•Movement of electrons due to redox reactions.3. Thermistor method of detection•Heat released or absorbed by the reactions.

4. Optical method of detection•Light produced or absorbed by the reaction5. Piezoelectric method of detection-Change in the mass of biological components .

Types of Biosensors1. Electro-chemical biosensor Based on enzymatic catalysis of a reaction • The sensor contains three electrodes-a) reference electrode,b) an active electrode and c)a sink electrode. •An auxiliary or counter electrode may also be present as an ion source.

2. Potentiometric biosensorBased on conjugated polymers immunoassays.They have only two electrodes and are extremely sensitive and robust.

Signal produced by electrochemical and physical changes in the conducting polymer layer due to changes occurring at the surface of the sensor.

Changes can be attributed to ionic strength, pH, hydration and redox reactions, the latter due to the enzyme label turning over a substrate.

3. Amperometric biosensorIt measure the reaction of analyte with enzyme and

generate electrons directly or through mediator. eg…..Glucose oxidase biosensor.

4.Thermistor containing biosensorUse to record temperature changes during biochemical reaction using enzymes like cholesterol oxidase, invertase , tyrosinase.Also use to study antigen-antibody with very high sensitivity in ELISA.

5. Whole cell biosensorIn this biosensor whole cell or organelles use as a biological component. The cells are cheaper, have longer active lifetime, and are less sensitive to inhibition, pH, temperature variations than enzymes. In this biosensor whole cell of micro-organisms is use for the study.

6. Colorimetric test stripsThe most simplest form of biosensor. Cellulose is use which coated with appropriate enzyme and suitable reagents which gives colour change in the reaction. Eg ...To detect glucose in blood .

7. Optical biosensorIt detect how much light is produced or absorbed during the reaction.Biosensor used for detection of bacteria in food and clinical samples.

Biosensors in Agriculture

Agriculture includes the production of crops and the rearing of livestock producing various products which are used in daily life.

These elements damage by pests and diseases causing a

loss in the profit.

Biosensors may play a major role for providing rapid and specific detection techniques.

A biosensor has been developed for the detection of the fungus Phakopsora pachyrhizi that causes Asian rust or Soybean rust.

Important for monitoring agricultural by-products.

Biosensor for the detection of aflatoxin in olive oil has been developed.

Aflatoxins produced by molds Aspergillus flavus and Aspergillus parasiticus are carcinogenic to humans.

Concentrations of herbicides, pesticides and heavy metals in agricultural lands is increasing.

Biosensors can be used to measure the levels of pesticides, herbicide and heavy metals in the soil and ground water

.Biosensors can also be used to forecast the possible

occurrence of soil disease.

Applications of Biosensors

1. Clinical diagnosis and biomedicine2. Farm, garden and veterinary analysis3. Process control: fermentation control and analysis

food and drink4. production and analysis5. Microbiology: bacterial and viral analysis6. Pharmaceutical and drug analysis7. Industrial effluent control8. Pollution control and monitoring o Mining, industrial

and toxic gases9. Military applications

Biosensors in microbiologyBacteria, fungi, viruses and other microorganisms are

found widely throughout nature and the environment.

Bacterial pathogens are distributed in soil, marine and estuarine waters, the intestinal tract of animals, or water contaminated with fecal matter.

Biosensors for bacterial detection generally involve a biological recognition component such as receptors, nucleic acids, or antibodies in intimate contact with an appropriate transducer

Various biosensor for detecting microorganism A) Direct (label-free) detection of bacteria• Optical biosensors• Bioluminescence sensors• Piezoelectric biosensors

B) Indirect detection of bacteria• Fluorescence labeled biosensors• Microbial metabolism based biosensors• Electrochemical immunodetection of bacteria

C) Flow immunosensorsD) GenosensorsE) The electronic nose

The electronic noseElectronic nose’ systems have advanced rapidly

during the past 10 years.

majority of applications being within the food and drink industry.

Is the signal pattern from a sensor array, comprised usually of individual sensing elements with limited specificity, is collected by a computer, where a first pre-treatment of the data is carried out.

These data then further processed and displayed on specific data base software.

THE ELETRONIC NOSE FUNCTIONING…..

The electronic nose

The ideal electronic nose sensors should fulfil the following criteria

High sensitivity,

They must respond to different compounds,

High stability and reproducibility;

Short recovery time; Easy calibration; They must also be robust and portable.

First attempts to identify microorganisms with an electronic nose was made by Craven et al. (1994). An array of four different commercial metal oxide gas sensors was used to sample the head space of six pathogenic bacteria….

Electronic nose was able to classify correctly 62% of the pathogens.

More recently, an investigation into the use of an electronic nose to predict the class and growth phase of two pathogenic bacteria, E. coli and S. aureus, has been performed by Gardner et al. (1998).

Contained six commercial metal oxide odour sensors.

The sensors were chosen based on secondary metabolites of growing microorganisms are hydrocarbons, alcohols, aldehydes, acids, ammonia and so on.

The growth phase of the bacteria was correctly predicted for 81% of all unknown samples.

The species investigated in culture were respectively four aromatic (Micrococcus and Staphylococcus) and three pathogenic bacteria strains.

100% percent of the bacteria samples were classified correctly into their respective groups based on factorial discriminant analysis.

The feasibility of electronic noses for the following applications: monitoring lot-to-lot variation in bioprocess medium ingredients,

Detection and simultaneous identification of microorganisms,

Bacteria classification; and

Evaluating bioprocess performance during cultivation of microorganisms at inoculum and production stages.

Gibson et al.1997

Electronic noses for the following applications: Monitoring lot-to-lot variation .Detection and simultaneous identification of microorganisms.Bacteria classification; and evaluatingBioprocess performance of microorganisms at inoculum and

production stages.E. coli, Enterococcus sp., Proteus mirabilis, P.aeruginosa, and

Staphylococcus saprophytica were selected for this study.

A classification tree was most important features.Due to this features a classification rate of 76% was

obtained. The detection and simultaneous identification of

microorganisms by measuring the volatile compounds produced.

The overall classification rate for 12 different bacteria and one pathogenic yeast was 93.4%. Data for a sub-set of seven bacteria gave 100% classification.

The responses are analysed mathematically, using pattern recognition techniques, to differentiate between different odours with a high level of sensitivity.

A large amount of different gaseous components are released from substrates contaminated with spoilage organisms.

In microbiology the smell of a culture of bacteria often provides a clue to the identification of the organism Present.

E.nose withdrawn for bacterial analyses (aerobic bacteria, lactic acid bacteria) and analysis of the volatile compounds during the storage period.

Electronic nose containing a sensory array composed of 10 metal oxide semiconductor field-effect transistors,

four Taguchi type sensors and one CO2-sensitive sensor.

The mathematical models were validated after 6 months using a new set of samples.

The method currently used for analysis of the total bacterial count.

A drawback with the bacteriological method is the incubation

period of 1–2 days that is required for colony formation.

Advantages of biosensor

Biosensors are sophisticated tools for detection and monitoring.

Biosensors are more specific and provide more accurate readings.

It is very easy to use.

They can measure non-polar molecules that do not respond to most measurement devices.

No need of continuous monitoring.

Disadvantages of biosensor

Heat sterilization is not possible as this would denature the biological part of the biosensor.

Cost is high. Reproducibility- it is not possible the same type of biosensor gives the same result.

Its sensitivity sometimes may be a problem. Reusability- Some type of biosensor such as

colorimetric test strips has single use.

obstacle is the tendency to focus only on the scientific basis of the technology.

• There are a number of practical and technical issues in the development of bacterial biosensors and their commercialization.

Biosensor must be able to provide a detection limit.

Technical problems Methods of sensor calibration, The requirement for reliable and low maintenance

functioning over extended periods of time,Reproducible fabrication of numerous sensors,Ability to manufacture the biosensor at a competitive

cost,Disposable format,and a clearly identified market. Biosensors for bacteria should reduce human

participation toAvoid contamination, and hence,Automation must be an inherent attribute of the

biosensor.

Future prospectsThough a lot of research activity has been involved in

developing biosensors.

For various purposes the time has come to bring this technology to make it commercially available.

Efforts and funds need to be mobilized to manufacture biosensors on a large scale to benefit and use for the general public.

With exposure to the commercial market the applications of this technology would be greatly enhanced.

Real time monitoring help cleaner and hygienic environment

Conclusions

In India, this technology, which has already made some imprints in many areas of plant pathology.

Is the second craze after biotechnology for innovative research.

Is the device that detects, records, and transmits information regarding a physiological change .

Biosensors combine the selectivity of biological system with the processing power of modern microelectronics.

Biosensor have new analytical tools.

This form allows for a rapid response is ideal in the agri-food process control.

Pesticides, fertilizers, and heavy metals can be quickly detected in small quantities .

Transduction, electrochemical and optical methods provide better detection sensitivity.

do not require any expert knowledge once programmed.

There are several markets which could support biosensors.

Highly sensitive and accurate biosensor systems could have great application.

The several diagnostics field offers real opportunities for the exploitation of biosensors for bacterial detection.