BIOSENSOR

SUBMITTED BY:-

JYOTI

10093004

MSc nano 4th sem

BIOSENSORS

A sensor that integrates a biological element with a physiochemical transducer to produce an electrical signal proportional to a single analyte which is then conveyed to a detector.

COMPONENTS OF BIOSENSOR:

1. Physical components

2. Biological component

3. Analyte

Physical component:

a. Transduc er:- A transducer convert the biochemical signal to electronic signal. The signal produced may be in form of electrochemical [change in current or potential] , optical [change in colour} , piezoelectrical [mass change], calorimetric.

b. Amplifier: - The electronic signal produced by transducer is very small and is amplified by an amplifier.

c. Microprocessor: - The amplified signal is fed in to microprocessor. The signal is processed and is displayed in suitable unit.

Biological component:

a. Enzyme:- Enzyme may be used in purified form or may be present in micro organism or in slice of intact tissue. They can bind themselves to specific substrate and are biocatalyst for particular reaction. This catalytic action is made use of in biosensor.

b. Nucleic acid:- operate selectively because of their base pair characteristic.

c. Antibody :- Antibody binds specifically with corresponding antigen. They have no catalytic activity. Despite this , they are capable of developing ultra high sensitivity in biosensor.

Analyte : - An analyte is a compound whose concentration is to be determined.

Biosensor –a sensor incorporating a biological element that is an analytical device, which employs a biological material to interact with an analyte, this interaction produce some detectable physical changes that is measured and converted into an electrical signal by transducer. Finally electrical signal is amplified, interpreted and displayed as analyte concentration in the sample.

HISTORY OF BIOSENSOR:-

1916 First report on immobilization of proteins: adsorption of invertase on activated charcoal

1922 First glass pH electrode

1956 Clark published his definitive paper on the oxygen electrode

1962 First description of a biosensor:amperometric enzyme electrode for glucose (clark)

1969 Guilbault & Montalvo – First potentiometric biosensor :urease immobilized on an ammonia electrode to detect urea.

1970 Bergveld ion selective field effect transistor(ISFET)

1975 First commercial biosensor ( Yellow Spring Instruments glucose biosensor)

1975 First microbe based biobensor , first immunosensor

1976 First bedside artificial pancreas (Miles)

1980 First fibre optic pH sensor for in vivo blood gases(Peterson)

1983 First surface Plasmon resonance ( SPR) immunosensor

1992 Hand held blood biosensor by i-STAT

1996 Launching of glucocard

Advantages of biosensor :-

Fast response time

Simple to use

Miniaturized form

Portable

Disposibility

High selectivity

High specificity & selectivity

Disadvantages:-

Limited availability

Instability

Limited shelf life

Classification:-

Based on level of integration -

a. First generation:- The receptor is entrapped between or bound to membrane and the combination is fixed on surface of an appropriate transducer.

b. Second generation:-The receptor is bound covalently to the transducer surface , thereby eliminating the need for a semi permeable membrane.

c. Third generation:- The receptor is bound to an electronic device that transduces and amplifies the signal utilizing the technique of microelectronic & micromechanical.

Based on transducer :-

Electrochemical biosensor :- In this sensor ,the biological signal can be used to generate current or charge or may change conductivity between 2 electrode and therefore the corresponding transduction device has been described as- Amperometric ,potentiometric ,conductrometric.

i. Amperometric Biosensor :-involve the measurement of current generated through electro oxidation/reduction catalyzed by their enzymes, or by their involvement in a bioaffinity reaction at the surface of the working electrode.

ii. Potentiometric biosensor:-It consist of a perm-selective outer layer and a bioactive membrane. The reaction between the analyte and the bioactive material generates or consumes a charged species , which accumulates at an electrode surface.Change in distribution of charge is detected using ion –selective electrode.

iii. Conductimetric biosensor:- In this, the change of conductivity of a solution in correlation with the concentration of the analyte is determined.

Optical sensor :-This are based on the measurement of the light absorbed or emitted as the consequence of biochemical reaction .In such type of biosensor, the light waves are guided by optical fiber to suitable detector.This type of biosensor have been used for detection of oxygen ,co.

Optical sensor includes the following-

Colorimetric for color change

Photometric for change in light intensity

Calorimetric biosensor :- The basic principle of such biosensor is that biochemical reaction involve a change in enthalpy such a change in enthalpy is detected by calorimetric biosensor. For detection, thermal signal generated by redox reaction are measured as opposed to measuring the electrochemical signal. If the enzyme catalyzed reaction is exothermic, two thermistors may be used to measure the difference in resistance between reactant and product and, hencenthe analyte concentration.

Piezoelectric biosensor :- Piezoelectric devices use gold to detect the specific angle at which electron waves are emitted when the substance is exposed to laser light or crystals, such as quartz, which vibrate under the influence of an electric field.These biosensor operate on the principle of electic dipoles on subjecting anisotropic natural crystal to mechanical stress.Such type of biosensor are used for measurement of ammonia.,methane ,CO ,and other organophosphorus compound.This is also called as acoustic wave biosensor.

Thermal biosensor :- Thermometric devices predominantly measure the changes in temperature of the circulating fluid following the reaction of a suitable substrate with the immobilized enzyme molecule.

Catalytic biosensor :- Enzyme & microbial cell based biosensor come in this category. The immobilized sensing biomolecule act as a catalyst & catalyze the biochemical reaction. Various examples of catalytic biosensor are glucose biosensor (Glucose oxidase based), urea biosensor (urease based) , lactate biosensor (lactate oxidase & lactate dehydrogenase )etc.

Advances in several area have led to development of biosensor:-

-Technique for stabilizing enzyme /cell/antibody on to the surface of solid support while retaining their biological activity. (immobilization technology)

-development of tailor made membrane (Membrane technology)

-Integration of biomolecules & microelectronic/information technology

Immobilization :- The receptor must have intimate contact with the transducing system so that transmittance is proportional to the reaction rate of biocatalyst with the measured analyst for a high range of linearity. A number of methods have been developed for the immobilization of biological component.

1) Adsorption :- The simplest method is adsorption onto a surface. Adsorbed material is susceptible to changes in pH,temperature ,ionic strength and the substrate .However, the method is satisfactory for short- term investigations.

2) Microencapsulation :- This was the method used in early biosensor.The biomaterial is held in place behind a membrane, giving close contact between the biomaterial & transducer. It is adaptable It does not interfere with reliability of enzyme.It is stable towards changes in temperature ,pH ,ionic strength & composition.

3) Entrapment :- The biomaterial is mixed with monomer solution, which is then polymerized to a gel , trapping the biomaterial.The most common polymer used is polyacrylamide. Large barrier are created ,inhibiting the diffusion of substrate, which slows the reaction and hence the response time of the sensor.

4) Cross-linking :- A bio-functional agent is used to chemically bind the transducer and the biological material.

5) Covalent attachment :- In this technique , chemical groups on the active biological material which are not essential for activity (e.g., non-essential amino acids of enzymes) are attached to chemically activated supports in a wide variety of materials such as synthetic and natural polymers ,glass & metals. The linking molecules may form a bridge or be expelled after coupling.

Performance Factor:-

a. Selectivity:-This is the most important characteristic of biosensors- the ability to discriminate between different substrates.It is established by the nature of the biological material.A sensor may be so specific that only one compound in one stereoisomeric structure cause any response.This sounds ideal,but it can raise its own problems.Alternatively, a broad range of similar chemical types may cause a response to some extent or other. This is due to the nature of a very specific enzyme or it may be that the biological preparation actually contains a mixture of enzymes of similar specificity.

b. Sensitivity range:- This usually needs to be sub –millimollar , but in special cases can go down to the femtomolar range.

c. Accuracy:- This is usually around +- 5%

d. Reproducibility:- This is very important factor with any analytical technique, but especially so with biosensors , where it is impossible to reproduce the quality of biological preparation as well as with ordinary chemical substances.With biosensor the expected reproducibility between replicate determinations should be at least_+5-10%.

e. Times:-

The Response time is usually much longer than with chemical sensor. It may be 30s or longer.

The recovery time is the time before a biosensor is ready to analyse the next sample. It must not be too long –not more than a few minutes.

The working lifetime is usually determined by the instability of biological material. It can vary from a few days to a few months. The Exactech glucose biosensor is usable for over 1 year.

Immunosensor:

The most widely studied form of bioaffinity sensor is the immunosensor of which several types exist. The high specificity and sensitivity that typifies an antigen-antibody reaction has been used in a vast range of laboratory –based tests incorporating antibody components (immunoassays). The analytes detected and measured include many medical diagnostic molecules, such as harmones (pregnancy related steroids), clinical disease markers , drugs (therapeutic and abused ), bacteria, and such environmental pollutants like pesticides.Immunosensors can be of two types:-

Direct immunosensor:

The advantage of these sensor is that measurement of the antigen –antibody interaction can be accomplished immediately without the need for additional antibodies or markers (enzymes , fluorescent labels etc.).

Indirect immunosensor:-

They are based (like homogeneous and heterogeneous immunoassays) on labeling of one of the partners in an immune interaction. One class of indirect immunosensors derived mainly from enzyme immunoassays, encompasses the enzyme immunosensors .These combines the highly selectivity of an immunoreactions with the high sensitivity characterization of the enzymatic amplication effect.

Example: Scientists at the Hebrew University of Jerusalem ,Israel and Brookhaven National Lab.,US, have used gold nanoparticles to attach glucose oxidizing enzymes to electrodes. These enzyme-electrodes could ultimately be used as miniaturized biosensors inside the body that measure blood glucose .The nanoengineered electrodes could also have application in bionanoelectronic devices such as biofuel cells By fabrication of implanted biofuel cell devices , one will be able to use glucose in blood as the fuel source for the generation of electricity.Such implanted biofuel cells would generate electricity from body fluids and the electrical power could activate pacemakers, insulin pumps or prosthetic units.

Kin ExA TM Model Biosensor (An affinity biosensor)

Chemical Sensor :- The sensor chemically senses the biomolecules present in the sample. These are used for sensory analyses of foods. In the past, chromatography & mass spectroscopy have been used as sensing systems, although these are usually expensive and time consuming. Currently chemical sensors, in the field of sensory analysis, are replacing these techniques and allow the food & beverage industry to cover a large proportion of the sensory perception of the consumer These are two types:

1.Electronic tongue

2.Electronic noses

Electronic Tongue:- Its application includes

Bitterness measurement of new chemical entities

Bitterness masking efficiency

Shelf life & stability of formulation

Comparison with competitor product

Screen for best additives & taste agent

These have been developed for analysis of taste and non-volatile chemicals in food with an objective to compliment electronic nose. They measure typically non-volatile compounds responsible for sweetness , bitterness, saltiness and sourness. A group of researchers in Malasia have developed an electronic tongue for herbal samples.This system helps in standardization of herbal samples, both quantitatively and qualitatively.

Electronic Nose:-The US Department of Agriculture (USDA) is testing the zNose technology to see whether the sensor device can pick up bug infestation in plant. The zNose could have a wider use for food & beverage companies , such as for aroma chemistry testing or for the analyses of wine & beer .The zNose is unique in its ability to separate & quantify the chemistry of any fragnance or odour of chemical vapour with part per trillion sensitivity within 10sec

Electronic nose are made of two parts:-

Pattern Recognition system:- Each order leaves a characteristic pattern of certain compounds called fingerprint. Known odors can be used to build a database for the construction of a pattern recognition system and classify them with signature . This can be done by using artificial neural networks, which can detect more chemicals than the number of sensors , they are utilizing.

Sensing system:-These measure the voltage change from the normal or standard conditions .Due to presence of certain chemical in the odor, the oxygen concentration in the air over the sensor changes.This in turn changes the resistance across the sensor .The transducer converts this altered resistance into an electronic signal.

APPICATIONS OF BIOSENSORS:-

Application in Health Care & Medicine:-

Biosensor are used for diagnosis of metabolites , drugs , enzymes ,vitamins ,harmones ,allergies, infection ,diseases, cancer markers ,pregnancy etc.

Consumer self-testing ,especially self-monitoring of blood components ,is an important area of clinical medicine and healthcare which has been impacted by commercial biosensors. One of the successful examples is the commercialization of biosensor devices for monitoring of blood glucose level in diabetic patients. Monitoring systems for lactate,creatinine ,phenyalanine ,and histamine are also available.

Biosensor are being developed which can be wirelessly controlled and that can detect and treat an acute condition in body.

External biosensors are used in emergency rooms as point-of-care diagnostic units, which can reveal almost immediately whether a patient is in cardiac arrest by testing blood chemistry.

In America, Britain ,Sweden and Switzerland ,brain microdialysis has been deployed to follow changes in glucose ,lactate and glutamate.

Drugs such as phenytoin (antiepileptic ) and digoxin (heart drug) have been detected using amperometric immunosensors. Quantities in the range of 10-20ug/ml phenytoin and 0.5-2.5ug/ml digoxin were detected in pateins serum sample.

Biosensors have also paved way for in-vivo analysis like long term in vivo control of metabolites and drugs, control for artificial prosthetic organs , surface imaging of organs during implantation.

Biosensors offer potential of reusable self-monitoring systems by employing electrochemistry rather than color changes to help alleviate the problems of poorly sighted people.

Environmental monitoring: -

Because they can be miniaturized and automated , biosensors have many roles to play in the environmental and monitoring. They are used for pollution control, monitoring and screening of toxic compounds in water supplies, solid and liquid wastes, soil and air (e.g., pesticides, inorganic ions, explosives ,oils, PAHs, PCBs ,microorganisms, volatilevapors and gases.)

The detection of gaseous compounds has been done using gas sensors, which possess semiconductors to sense the substance concentration in terms of electric conductivity.

Microbial sensors have also been applied to analyze ammonia, methane, and nitrogen dioxide. The principle of microbial sensors indicates selective determination of compounds from the respiratory activities of immobilized microbial cells with the help of oxygen electrode.

For detection of ammonia, both the genera of nitrifying bacteria (nitrosomonas and nitrobacter) have been used. The nitrosomonas oxidizes ammonia, while Nitrobacter oxidizes nitrite. These reaction require oxygen, hence, oxygen consumed by the bacteria can be measured by an oxygen electrode coated with immobilized microbial cells.

Whole cell biosensors help in environmental water monitoring by determination of BOD. The determination time varies from 20 seconds to several minutes as compared to 5 days interval in the classical BOD test.

Application in Defence & Security:-

Biosensors are used for detection of chemical and biological warfare agents (e.g.,nerve gas ,anthrax viruses.).

Organophosphorous compounds have been detected in the air by immobilizing the enzyme acetyl-cholinesterase onto a crystal, a device which could be used to detect nerve gas.

Other gases like carbon monoxide, ammonia etc, can be measured by employing selective membrane coatings while immobilizing antibodies or special receptor molecules to the crystal may offer possibility of constructing selective electronic noses for applications like detecting illegal drugs.

Biosensor tags (RFID biosensor) are being developed, which are microscopic structures, coated with bacteriophages or viruses that can bind with anthrax and other biological and chemical agents. These tags will be attached to food packages. When an agent binds with the phage coating,the tag produces a signal for transmission to a receiver and can instantly alert the suppliers and consumers to anthrax or other toxins in the foods. In addition ,the food companies hope that the technology will protect them from lawsuits brought by victims of deliberately contaminated foods. Several food companies have already begun testing these RFID biosensors like Golden State Foods ,which is McDonald’s largest beef patty provider and its leading sauce supplier.

Fresh Alert system, from RFID chipmaker Infratab, combines RFID tags with temperature sensors and timers , to signal when perishables have become unsafe to eat.

Biosensor technologies can result in a field portable kit to provide rapid, real-time analysis of combat rations, fixed-field feeding sites, and military dining facilities ensuring the safety of the defense personnel.

Application of biosensor for analyses of food composition:-

For food composition analysis and detection of metabolites and contaminants ,various biosensors have been developed like for glucose, lactose, fructose ,biogenic amines, glutamate, malate, glycerol, lactate, lactulose.Sensors for pesticides(organochlorine and carbate),sensors for toxins as aflatoxins in milk,seafood toxins, and ocratoxins in wine can be used to analyze these compounds for food freshness and safety. A number of examples can be cited in this context:

YS 12700 from Yellow Spring, Ohio , can be used for food composition analysis to measure common food ingredients such as glucose ,sucrose ,falactose,.choline ,L-glutamate and starch.

Biacore Inc offer a SPR based biosensor for specific analysis e.g., Quantitative measurement of folic acid ,biotin, vitamin B12 and future development for protein quality measurement and detection of allergens.

Amperometric biosensors have been developed for the detection of biogenic amines in fish and fruit samples and lactulose in milk samples.

A Tetrathiafulvalene (TTF)-mediated, amperometric biosensor has been developed for the specific determination of L-lactate. Biosensors based on both whole cells(Acetobactor pasteurianus) and enzymes(lactate dehydrogenase,lactate monoxygenase,lactate oxidase) have been reported.

The emerging health issues related to food-induced allergenic reactions present an important challenge to food industry. The major food allergens that account for more than 90% of all food allergens are found in peanuts,soybeans,milk,eggs,fish,shellfish,tree nuts and wheat.A biosensor using surface surface Plasmon resonance(SPR) is a new

technology for rapid, cost effective , yet sensitive, on-site detection of allergens in fooda.The assay is based on total sample binding to the allergen specific antibodies on the sensor surface.

Biosensor for detection of pathogens in food:-

A flow –injection sensor has been developed for the detection of Staphylococcal enterotoxin B using serum antibodies. This sensor has also been used for the detection of Candida albicans ,and Vibrio cholera.

Piezoelectric sensor surfaces have been coated with immobilized antibodies specific to whole microbial cells, surface expressed antigens or even microbial toxin.

Georgia Research Institute has developed a new interferometric biosensor for the poultry industry. Laboratory tests indicate that this device can detect the presence of Salmonella and campylobacter in less than 30 minutes.

Fig. biosensor for detection of Salmonella.

Immuno-optical capillary based biosensor to detect E.coli and Salmonella typhimurium during poultry processing.

Reference:-

Rogers ,Kim and Mulchandani 2003 Affinity Biosensor-technology and protocols, Humanas press.

Anthony P.F Turner ,Isao Karube ,George S.Wilson 1987 Biosensor : Fundamental and application. Oxford university press.

Ashok and Zhou ,weichang 1995 Biosensor and chemical sensor technology: process monitoring and control ,oxford university press.

Gogosti, Y.G and Uvarova ,Irsna V 2003. Nanomaterial and sensor application ,Spinger.