Enzyme Technology

A biosensor is an analytical device consisting of an immobilized layer of biological material (e.g. enzyme, antibody, organelle, hormones, nucleic acids or whole cells) in the intimate contact with a transducer i.e. sensor (a physical component) which analyses the biological signals and converts into an electrical signal (Gronow, 1984). A sensor can be anything, a single carbon electrode, an ion-sensitive electrode, oxygen-electrode, a photocell or a thermistor.

The principle of the biosensor is quite simple (Fig. 17.6). The biological material is immobilized as described earlier on the immobilization support, the permeable membrane, in the direct vicinity of a sensor. The substances to be measured pass through the membrane and interact with the immobilized material and yield the product. A product (i.e. the monitored substrate) may be heat, gas (oxygen), electrons, hydrogen ions or the product of ammonium ions. The product passes through another membrane to the transducer. The transducer converts product into an electric signal which is amplified. The signal processing equipment converts the amplified signals into a display most commonly the electric signal which can be read out and recorded.

  Schematic outline of biosensor  

Fig. 17.6. Schematic outline of biosensor


A glucose-electrode is shown in Fig. 17.7. It can be built up by immobilizing glucose oxidase in polyacrylamide gel around a platinum oxygen-elec­trode separated by a teflon membrane. KCl solution is placed around the platinum-oxygen electrode. From upper surface glucose oxidase is intimately covered by a cellulose acetate membrane.

When glucose solution is brought into the contact of membrane, glucose and oxygen pass through membrane into the enzyme layer and, as a result of oxidation-reduction reactions, converted into gluconic acid and hydrogen peroxide in the presence of water, oxygen and glucose oxidase. Consequently, oxygen concentration in the gel around the electrode is lowered down. Hydrogen peroxide brings about a change in current i.e. measurable signal. Electrode records the rate of reactions. The rate of diminition of oxygen concentration is proportional to glucose concentration of the sample. It responds linearly to glucose concentrations over a range of 10-1-10-5mol dm-3 with a response time of 1 minute.
» Microorganisms
» Properties of enzymes

» Presence of species specificity

» Variation in activity and stability

» Substrate specificity

» Activation and inhibition
» Methods of enzyme production

» Isolation of microorganisms, strain development and preparation of inoculum 

» Medium formulation and preparation

» Sterilization and inoculation of medium, maintenance of culture and fluid filtration

» Purification of enzymes
» Immobilization of enzymes

» Advantages of using immobilized enzymes

» Methods of enzyme immobilization

» Adsorption

» Covalent bonding (Ionic bonding)

» Entrapping

» Cross linking

» Encapsulation

» Effects of enzyme immobilization on enzyme stability
» Enzyme engineering
» Application of enzymes

» Therapeutic uses

» Analytical uses

» Manipulative uses

» Industrial uses

» In dairy industry

» In detergent industry

» In starch industry

» In brewing industry

» In wine industry

» In pharmaceutical industry
» Biosensor

» Types of biosensor

» Applications of biosensor
» Biochips

» Principles of Biochips

» Application of Biochips
In 1987, for the first time Yellow Springs Instruments Co., USA developed a biosensor for diagnostic purposes for measuring glucose in blood plasma. It is a hand - held machine which measures six components of blood plasma for example, glucose, urea, nitrogen, sodium, potassium and chloride.

An indigenous glucose sensor has been developed by the scientists at Central Electro­chemical Research Institute (CECRI), Karaikudi. It gives electrical signal for a glucose concentration as low as 0-15 millimoles.

Types of biosensor

Biosensor are of different types based on the use of different biological material and sensor devices; a few of them are discussed below:
(i) Electro-chemical biosensor. This type of biosensor has been de­veloped by using electronic devices such as field effect transmitors or light emitting diode; the former measures charge accumulation on their surface and the later photoresponse generated in a silica based chip as an alternating current. Hence, the field effect transmitor measures a biochemical reaction at the surface and induce into current (Gronow et al, 1988). Moreover, the field effect transmitors can be modified to ion sensitive, enzyme sensitive or antibody sensitive ones by using selective ions, enzymes or antibodies respectively.
  A glucose electrode

Fig. 17.7. A glucose electrode


(ii) Amperometric biosensor. Amperometric biosensors are those which measure the reaction of anylate with enzyme and generate electrons directly or through a mediator. The amperometric biosensors contain either enzyme-electrode or without a mediator, or chemically modified electrodes. The oxygen and peroxide based biosensor and others (Table 17.4) discussed earlier are enzyme -electrode biosensor. Some advancement has been brought into this type of biosensor by using a mediator. In addition, more advanced types are the direct electron transfer systems. Principle of a mediated biosensor is shown in Fig. 17.8.

In this biosensor, a redox reaction catalyzed by an enzymes is directly coupled to an electrode where enzyme is presented with the oxidizable substrate. The electrons are transferred from the substrate to the electrode via enzyme and redox mediator. In this biosensor the oxidase replaces the oxygen requirement of the enzymes.

  Mediated biosensor.

Fig. 17.8. Mediated biosensor.


Enzyme electrodes. Enzyme electrodes are a new type of biosensors which have been designed for the amperometric assay of potentiometric assay of substrates such as urea, amino acid, glucose, alcohol, and lactic acid. The electrode consists of a given electrochemical sensor in close contact with a thin permeable enzyme membrane capable of reacting with the given substrates. The enzyme is embedded in the membrane and produce O2, H+, NH4+, CO2 or other small molecules depending on enzymatic reactions. This is detected by the specific sensor. The magnitude of the response determines the concentration of substrates (Fig. 17.9).

(iii) Thermistor containing biosensor. Thermistor is used to record even a small temperature changes (between 0.1-0.001°C) during biochemical reactions. By immobilizing enzymes like cholesterol oxidase, glucose oxidase, invertase, tyrosinase, etc. thermistors have been developed (Gronow et al, 1988). Moreover, thermistors are also employed for the study of antigen- antibody with very high sensitivity (10-13 mol dm-3) in case of thermometric Enzyme Linked Immunoabsorbant Assay (ELISA).

(iv) Bioaffinity sensor : Bioaffinity sensors are developed recently. It measures the concentration of the determinants, i.e. substrates based on equilibrium binding. This shows a high degree of selectivity. These are of diverse nature because of the use of radiolabelled, enzyme labeled or fluorescence-labeled substance (Kumar and Kumar, 1992). Principle of bioaffinity sensor is given in Fig. 17.10.
  A simple biosensor combining on electrochemical electrode and an enzyme immobilized on to a semipermeable membrane.

Fig. 17.9. A simple biosensor combining on electrochemical electrode and an enzyme immobilized on to a semipermeable membrane.

In this biosensor, a receptor is radiolabelled and allowed to bind with determinant analogue immobilized onto the surface of a transducer. When concentrations of a determinant are increased, the labeled receptor forms an intimately bound complex with determinant.
  Principle of bioaffinity sensor.

Fig. 17.10. Principle of bioaffinity sensor.

Table 17.3. Typical enzymes based biosensors.

Amines (for meat
Monoamine oxidase
4 min
50-200 m mol dm-3
Cholesterol oxidase
2 min
10-2 - 3x10-5 mol dm-3
Carbon monoxide
CO : acceptor
15 sec
0-65 m mol dm-3
Glucose oxidase
20 sec
2 x 10-3 -3x10-6 mol dm-3
25 sec
1-10 m mol dm-3
6 min
10-2 -2x10-3 mol dm-3
Uric acid
30 min
5x10-3-5x10-5 mol dm-3
Source : Gronow et al. (1988)

Finally, radiolabelled receptor-determinant complex is removed from the immobilized determinant analogue resulting in the increased concentrations of labeled receptor. This is measured by a reduction in signal of the labeled receptor. Gronow et al (1988) have discussed that the possibilities of this type of biosensors are the use of lectin receptors for saccharide estimation, hormone receptors for hormone, drug receptors for drug, antibodies receptors for antigens and nucleic acid (as gene probe) for inherited diseases and finger-printing.

(v) Whole cell biosensors — (Microbial Biosensors). In this device, either immobilized whole cell of microorganisms or their organelles are used. These react with a large number of substrates and show generally slow response (Corcoran and Rechnitz, 1985). Immobilized Azotobacter vinelandii coupled with ammonia electrode shows sensitivity range between 10-5 and 8 x 10 mol dm-3. It measures the concentration of nitrate within 5-10 min-2. Examples of microbial biosensors are given in Table 17.4.

Table 17.4. Microbial biosensors containing oxygen electrode.
Sensoring for
Assimilase sugars
Linear above 1 m mole dm-3
Bacillus subtilis
Mutagen screening
1-6m g cm-3
upto 6.6m mol dm-3
Linear upto 22.5 mg dm-3
T. brassicae
below 22.5 mg dm-3
Source : Kumar and Kumar (1992)

(vi) Opto-electronic biosensor. In these biosensors either enzymes or antibodies are immobilized on the surface of a membrane. For measuring color, biosensor with enzyme and dye is immobilized to a membrane. When a substrate is catalyzed to yield product, changes in pH of the medium occur. This results in changes in dye - membrane complex. These changes in color are measured by using a light emitting diode and a photodiode (Gronow et al, 1988).

Applications of biosensor

In the beginning biosensor was applied in the field of medicine and industry. But in recent years, biosensors are becoming popular in many areas due to the small size, rapid and easy handling, low cost, and greater sensitivity and selectivity. Application of biosensor in some of the areas is described as below:

(i) Uses in medicine and health. Biosensors have tremendous potential for its application in the field of medical science. In 1979, the first glucose analyzer using biomolecule for the detection of blood glucose was commercialized by Yellow Springs Instruments Co., USA. A device, a minipump filled with insulin, has been constructed to deliver insulin to diabetics based on glucose levels of blood. When biosensor provides informations, the device delivers accurate amount of insulin required by the diabetics. Mitomycin, an aflatoxin, causes cancer in inborne infants. Therefore, mutagenicity of such chemicals can be detected by using the biosensor. Similarly, any other abnormal toxic substance produced in body due to infectious disease can also be detected.

(ii) Uses in pollution control. Biosensors are very helpful in environmental minitoring and pollution control, since they can be miniaturized and automated. As far as quality control of drinking water is concerned, the minitoring biosensors are successful in monitoring of pesticides in water. In Japan, a biosensor coupled with oxygen electrode and immobilized Trichosporon cutaneum is used for measuring biological oxygen demand (BOD) (Gronow et al, 1988).
The whole cell biosensor developed by immobilizing Salmonella typhimurium and Bacillus subtilis in conjugation with oxygen electrode can be used to measure mutagenicity and carcinogenecity of several chemical compounds.

(iii) Uses in industry. Generally, spectrophotometer and autoanalyzer are used to estimate the substrates utilized and the products formed in the fermented broth. In addition, there are a lot of problems associated with these. So the biosensors can be designed to measure the fermentation products to improve the feed back control, to carry out rapid sampling and rejection of below standard raw materials to improve the efficiency of workers. Isaokarube and coworkers of Tokyo University Research Centre of Advance Science & Technology have recently developed an ion sensitive field effect transistor (ISFET). This device is highly sensitive to change the ion concentration. Using this biosensor, it is possible to measure the odor, freshness and taste of foods. In dertemining fish freshness either ATPase, aminoxidase or putrescine oxidase is used. ATPase detects, the presence of ATP in fish muscle. As ATP is not present in staled food, therefore, signals do not occur. Recently, a biosensor has been developed at Cransfield Institute of Technology, UK which measures cholesterol levels in butter. The enzyme cholesterol oxidase, when immobilized on the electrodes, reacts with cholesterol of food.

(iv) Biosensor in military. The darker side of biosensor application is to provide support to military with such a biosensor that can detect toxic gases including chemical warfare agents. Such biosensors have advantages over the traditional methods of sensing of chemicals.