An analytical device used to change biological response into an electrical signal is called biosensor (figure 2.1). The term biosensor refers to sensor devices used for determining the concentration of substances and other biological parameters even where biological system is not directly involved. Biosensors use a transducer to couple a biological sensing element with a detector. The first scientifically planned and successfully commercialised biosensors were the electrochemical sensors useful for multiple analytes. The biosensor contains a biological sensing element ( e.g., tissues, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, natural products , etc.), a material obtained biologically ( e.g., recombinant antibodies, engineered proteins, aptamers , etc.) or agents that mimic biological system ( e.g., synthetic receptors, biomimetic catalysts, combinatorial ligands, imprinted polymers , etc.) either closely associated to or integrated in a transducer.
The preferred biological material is generally a specific enzyme that is immobilised using conventional methods ( e.g., physical or membrane entrapment, non- covalent or covalent binding) and brought in close contact with the transducer.
The analyte on binding to the biological material forms a bound analyte that produces a measurable electronic response. Sometimes due to the release of heat, gas (oxygen), electrons or hydrogen ions, the analyte converts into a product; and changes associated to this product is transformed by the transducer to electrical signals that are amplified and measured.
The electrical signal coming from the transducer is low and superimposed by high and noisy baseline (could be due to high frequency signal component of random nature, or electrical interference generated in transducer electronic components). A baseline signal derived from a similar transducer without any biocatalyst membrane is called a reference baseline signal.
In signal processing, this reference baseline signal is subtracted from the sample signal; the signal difference obtained is amplified and the unwanted noise signals
are electronically filtered (i.e. smoothened ). The biosensor response is slow and eases the electrical noise filtration. The analogue sig nal produced directly is the output; however, it is converted to a digital signal, passed to a microprocessor for processing and manipulating the data to desired units , and then the output is displayed or stored.
A biosensor is of the following different types based on the type of sensor devices and the biological materials:
1) Electrochemical Biosensor: It is a simple device used to measure electronic current, ionic or conductance changes carried by bio-electrodes.
2) Amperometric Biosensor: It determines the movement of electrons or electronic curre nt due to a redox reaction catalys ed by enzyme. Usually , a normal contact voltage moves along the electrodes to be analysed. In the enzyme-catalysed reaction, the substrate or product obtained can transfer the electrons with the surface of electrodes to be reduced; h ence, an alternate current flow is measurable.
3) Blood Glucose Biosensor: It is employed extensively for diabetic patients. It contains a watch pen -shaped disposable electrode for single use. This
electrode has glucose oxide and deri vatives of a mediator (Ferrocen e). The electrodes are converted using hydrophilic mesh.
4) Potentiometric Biosensor: It measures the changes in the concentration of ionic species with the help of ion-selective electrodes present in it. It generally employs pH electrodes, thus in the release of hydrogen ions a large amount of enzymatic reactions are involved.
5) Conductometric Biosensor: Many reactions occurring in the biological system bring about a change in the ionic species . This change is helpful in measuring the electronic conductivity. Urea biosensor which utilises the immobilised areas is an example of conductometric biosensor.
6) Thermometric Biosensor: Several biological reactions involve production of heat and form the basis of thermometric biosensors. The diagram
representing a thermal biosensor consists of a heat insulated box fitted with a heat exchanger.
7) Optical Biosensor: It works on the principle of optical measurements , like fluorescence, absorbance , etc., and is utilis ed in fibre optics and optoelectronic transducers. Optical biosensor can even be safely used for non-electrical remote sensing of materials. It is involved in enzymes and antibodies in the transducer elements. This biosensor generally do es notrequire any reference sensor, and sampling sensor is used for generating comparative signals.
8) Fibre Optic Lactate Bio sensor: It measures the change in oxygen concentration at molecular level by identifying the effects of oxygen in fluorescent dye.
9) Optical Biosensor for Blood Glucose: In diabetic patients, the blood glucose level is important to be monitored. It is based on a simple technique
in which paper strips saturated with reagents, like glucose oxide, Horseradish Peroxidase and a chromogen are used. The intensity of the dye colour is measured using a portable reflectance meter. The calorimetric test strips of cellulose layered with suitable enzymes and reagents are also widely used for testing blood and urine parameters.
10) Piezoelectric Biosensor: It is also called acoustic biosensor as its principle relies on sound vibrations. It contains piezoelectric crystals and th e characteristic frequencies vibrate with the positive ly and negatively charged crystals. With the help of electronic devices, certain molecules on the crystal surface can be measured. The response frequencies can be changed by using these crystals with at tached inhibitors. For example , the b iosensor for cocaine ( in the gas phase) works by attaching the cocaine antibodies on
Applications in Pharmaceutical Industries
Biosensors are made up of a biological element and a physiochemical detector used for detecting analytes. These devices have a wide range of applications in fields ranging from clinical to environmental to agricultural and to food industries. Given below are some of the fields in which biosensor technology is used:
1) General healthcare monitoring,
2) Screening of diseases,
3) Clinical analysis and diagnosis of diseases,
4) Veterinary and agricultural applications,
5) Industrial processing and monitoring, and
6) Environmental pollution control.
Biosensors, Protein and Genetic Engineering
Biosensors can be used for quantitative determination of numerous biologically important substances in body fluids , e.g., glucose, cholesterol, urea. Glucose biosensor is widely used for regular monitoring of blood glucose in diabetic patients. Also biosensors are used fo r blood gas monitoring for pH, pCO 2 and pO2 during critical care and surgical monitoring of patients. Mutagenicity of a few chemicals can be determined by using biosensors. Presence of toxic compounds produced in the body can also be detected. Thus, b iosensors possess many applications
Genetic engineering involves deliberate DNA manipulation in organisms to alter their genes. Although the organisms whose genes are being altered may not be microbes, but the substances and techniques involved are obtained from microbes and adapted for use in more complex organisms.
The term genetic engineering initially was used for various techniques used for modifying or manipulating organisms through heredity and reproduction processes. Genetic engineering involves artificial selection and also all the interventions of biomedical techniques (artificial insem ination), in vitro fertilisation (e.g., test-tube babies), cloning, and gene manipulation. In the 20 th century, the term genetic engineering was used to indicate more specific methods of recombinant DNA technology (or gene cloning), in which DNA molecules obtained from two or more sources are combined in vivo (within cells) or in vitro (outside cells) and then inserted into the host organisms for propagation. The techniques of recombinant DNA technology were developed with the discovery of restriction enzymes by Werner Arber (a Swiss microbiologist) in 1968. The next year Hamilton O. Smith (an American microbiologist) purified type II restriction enzymes having the ability to cleave a specific site within the DNA (in contrast to type I restriction enzymes that cleave DNA at random sites), and thus essential to genetic engineering. Daniel Nathans (an Americanmolecular biologist) helped in modifying the DNA recombination technique in 1970-71 and demonstrated that type II enzymes could be useful in genetic studies. Genetic engineering based on recombination was p ioneered in 1973 by Stanley N. Cohen and Herbert W. Boyer (American biochemists) , who were the first to cut DNA into fragments, rejoin different fragments, and insert the new genes into E. coli bacteria, which then reproduced.
Gene cloning involves inserting a specific piece of ‘desired DNA’ into a host cell in such a manner that the inserted DNA is replicated and handed onto the daughter cells during cell division. The factors involved in gene cloning are:
1) Isolation of the gene to be cloned.
2) Insertion of the gene into a vector (piece of DNA) which allow it to be taken by bacteria and replicate within them as the cells grow and divide.
3) Transfer of the recombinant vector into bacterial cells by transformation or infection with viruses.
4) Selection of the cells containing the desired recombinant vectors.
5) Growth of the bacteria, that can be continued indefinitely, to give the required cloned DNA.
6) Expression of the gene to get the desired product.