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An acceptable definition for biosensors should encompass all types of sensors and sensing configurations but that is not an easy task. Definitions should distinguish sensors from ordinary instrumental detectors, which do not necessarily exhibit biochemical selectivity, and from treshhold monitors, which function only as alarm devices. Furthermore, there has been an effort to set apart biosensors as a particular type of chemical sensor, distinguishable from those used for non-bioanalytical applications such as monitoring automobile exhausts or manufactured chemicals unrelated to biochemical processes.
Some scientists, scientific journals and IUPAC have been offered several definitions for biosensors over the decades. The last definition, which is involved in Goldbook of IUPAC:
Biosensor is a device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues,organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals.
Biosensors are roughly composed of five main parts(Figure1), these are;
Figure1: Schematic diagram showing the main components of a biosensor.
a)The biocatalyst(biological component) converts the substrate to the product.
b)The transducer determines the reaction and converts it to an electrical signal.
c)Amplifier intensifies output coming from transducer.
d)Processor converts the electrical signal to a significant data.
e)Monitor displayes the data.
Biosensors represent a rapidly expanding field, at the present time, with an estimated 60% annual growth rate; the major impetus coming from the health-care industry (e.g. 6% of the western world are diabetic and would benefit from the availability of a rapid, accurate and simple biosensor for glucose) but with some pressure from other areas, such as food quality appraisal and environmental monitoring. Since, there is clearly a vast market expansion potential, research and development in this field is wide and multidisciplinary, spanning biochemistry, bioreactor science, physical chemistry, electrochemistry, electronics and software engineering. New materials and technologies are providing a new generation of very sophisticated analytical devices which should be invaluable for biochemical analysis. Integration of components and miniaturization are significant characteristics of this new generation of instruments. In addition to this, a successful biosensor must possess at least some of the following beneficial features:
The biocatalyst must be highly specific for the purpose of the analyses, be stable under normal storage conditions and show good stability over a large number of assay.
The reaction should be as independent of such physical parameters as stirring, pH and temperature as is manageable. This would allow the analysis of samples with minimal pre-treatment. If the reaction involves cofactors or coenzymes these should, preferably, also be co-immobilised with the enzyme.
The response should be accurate, precise, reproducible and linear over the useful analytical range, without dilution or concentration. It should also be free from electrical noise.
If the biosensor is to be used for invasive monitoring in clinical situations, the probe must be tiny and biocompatible, having no toxic or antigenic effects. If it is to be used in fermenters it should be sterilisable. This is preferably performed by autoclaving but no biosensor enzymes can presently withstand such drastic wet-heat treatment. In either case, the biosensor should not be prone to fouling or proteolysis.
The complete biosensor should be cheap, small, portable and capable of being used by semi-skilled operators.
There should be a market for the biosensor. There is clearly little purpose developing a biosensor if other factors (e.g. government subsidies, the continued employment of skilled analysts, or poor customer perception) encourage the use of traditional methods and discourage the decentralisation of laboratory testing.
Biological elements provide the major selective element in biosensors. They must be substances that can attach themselves to one particular substrate but not to others. Some of them are examined fundamentally here with the advantages and disadvantages.
An enzyme is a large, complex macromolecule, consisting largely of protein, usually containing a prosthetic group, which often includes one or more metal atoms. In many enzymes, especially in those used in biosensors, the mode of action involves oxidation or reduction which can be detected electrochemically.
They bind to the substrate
They are highly selective
They have catalytic activity, thus improving sensitivity
They are fairly fast acting
They are the most commonly used biological component.
They are expensive. The cost of extracting, isolating and purifying enzymes is very high, and sometimes the cost of the source of the enzyme may be high. However, a very wide range of enzymes are available commercially, usually with well defined and assayed characteristics.
There is often a loss of activity when they are immobilized on a transducer.
They tend to lose activity, owing to deactivation, after a relatively short period of time.
Plant and animal tissues may be used directly with minimal preparation. Generally tissues contain a multiplicity of enzymes and thus may not be as selective as purified enzymes. However, the enzymes exist in their natural environment so they may be more stable to inhibition by solutes, pH and temperature changes.
The enzyme is maintained in its natural environment.
The enzyme activity is stabilized.
They sometimes work when purified enzymes fail.
They are much less expensive than purified enzymes.
There may be interfering processes, i.e. there is some loss of selectivity.
Microorganisms play an important part in many biotechnological processes in industry, in fields such as brewing, pharmaceutical synthesis, food manufacture, waste water treatment and energy production. Many biosensors based on microorganisms immobilized on a transducer have been developed to assist with the monitoring of these processes and others. Microorganisms can assimilate organic compounds, resulting in change in respiration activity, and can produce electroactive metabolities.
They are cheaper source of enzymes than isolated enzymes.
They are less sensitive to inhibition by solutes and more tolerant of pH changes and temperature changes.
They have longer lifetimes.
They sometimes have longer response times.
They have longer recovery times.
Like tissues, they often contain many enzymes and so may have less selectivity.
Organisms develop antibodies which are proteins that can bind with an invading antigen and remove it from harm. Antibodies have long been used in immunoassays are biochemical tests that measure the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. They bind even more powerfully and specifically to the corresponding antigen than enzymes do to their substrates. In fact, they can be too selective, they lack the catalytic activity of enzymes.
They are very selective.
They are ultra- sensitive.
They bind very powerfully.
There is no catalytic effect.
There are also some biological substances are used as a molecular recognition elements.These are mitochondria, nucleic acids, receptors, etc.
3.IMMOBILIZATION TECHNIQUES OF BIOLOGICAL COMPONENT
In order to make a viable biosensor, the biological component has to be properly attached to the transducer. This process is known as immobilization. There are five regular methods of doing this as follows.
Adsorption is the simplest method and involves minimal preparation. However, the bonding is weak and this method is only suitable for exploratory work over a short time-span.
This was the method used in the early biosensors. The biomaterial is held in place behind a membrane, giving close contact between the biomaterial and the transducer. It is adaptable and does not interfere with the reliability of the enzyme. It limits contamination and biodegradation. It is stable towards changes in temperature, pH, ionic strength and chemical composition. It can be permeable to some materials such as small molecules, gas molecules and electrons.
The biomaterial is mixed with monomer solution, which is the polymerized to a gel, trapping the biomaterial. Unfortunately, this can cause barriers to the diffusion of substrate, thus slowing the reaction. It can also result in loss of bioactivity through pores in gel. This can be counteracted by cross-linking. The most commonly used gel is polyacrylamide, although starch gels, nylon and silastic gels have been used. Conducting polymers such as polypyrroles are particularly useful with electrodes.
In this method, the biomaterial is chemically bonded to solid supports or to another supporting material such as a gel. Bifunctional reagents such as gluteraldehyde are used. Again there is some limitation and there can be damage to the biomaterial. Also, the mechanical strength is poor. It is a useful method to stabilize adsorbed biomaterials.
Covalent bonding is the most strongest immobilizaiton technique thus, the enzyme will never be lost. An example is illustrated in figure2, showing the binding process of enzyme to a transducer in the presence of carbodiimide.
Figure 2 : Covalent bonding of an enzyme to a transducer via a carbodiimide.
Overall, the lifetime of the biosensor is greatly enhanced by proper immobilization. Typical lifetimes for the same biosensor, in which different methods of mobilization are used, are as follows:
Membrane entrapment: 1 week
Physical entrapment: 3-4 weeks
Covalent entrapment: 4- 14 months
Voltammetric and amperometric techniques are characterized by applying a potential to a working (or indicator) electrode versus a reference electrode and measuring the current. The current is a result of electrolysis by means of an electrochemical reduction or oxidation at the working electrode. The electrolysis current is limited by the mass transport rate of molecules to the electrode. The term voltammetry is used for those techniques in which the potential is scanned over a set potential range. The current response is usually a peak or a plateau that is proportional to the concentration of analyte. In amperometry, changes in current generated by the electrochemical oxidation or reduction are monitored directly with time while a constant potential is maintained at the working electrode with respect to a reference electrode. It is the absence of a scanning potential that distinguishes amperometry from voltammetry. The technique is implemented by stepping the potential directly to the desired value and then measuring the current, or holding the potential at the desired value and flowing samples across the electrode as in flow injection analysis. Current is proportional to the concentration of the electroactive species in the sample.
If the composition of the sample solution or medium changes in the course of a chemical reaction, it will result in a change in the electrical conductivity which has been monitored by conductometer. Conductometric biosensors often include enzymes whose charged products result in ionic strength changes, and thus increased conductivity. Conductometry has been used as the detection mode in biosensors for environmental monitoring and clinical analysis.
Potentiometric sensors are based on measuring the potential of an electrochemical cell while drawing negligible current. Common examples are the glass pH electrode and ion selective electrodes for ions such as K+, Ca2+, Na+, Cl-. The sensors use an electrochemical cell with two reference electrodes to measure the potential across a membrane that selectively reacts with the charged ion of interest. These chemical sensors can be turned into biosensors by coating them with a biological element such as an enzyme that catalyzes a reaction that forms the ion that the underlying electrode is designed to sense. For example, a sensor for penicillin can be made by coating a pH electrode with penicillinase, which catalyzes a reaction of penicillin that also generates H+. The pH electrode senses the change in pH at its surface, which is an indirect measure of penicillin.
There are two main areas of development in optical biosensors. These involve determining changes in light absorption between the reactants and products of a reaction, or measuring the light output by a luminescent process. The former usually involve the widely established, if rather low technology, use of colorimetric test strips. These are disposable single-use cellulose pads impregnated with enzyme and reagents. The most common use of this technology is for whole-blood monitoring in diabetes control. In this case, the strips include glucose oxidase, horseradish peroxidase and a chromogen. The hydrogen peroxide, produced by the aerobic oxidation of glucose, oxidising the weakly coloured chromogen to a highly coloured dye.
Piezo-electric crystals (e.g. quartz) vibrate under the influence of an electric field. The frequency of this oscillation (f) depends on their thickness and cut, each crystal having a characteristic resonant frequency. This resonant frequency changes as molecules adsorb or desorb from the surface of the crystal, obeying the relationship;
where Df is the change in resonant frequency (Hz), Dm is the change in mass of adsorbed material (g), K is a constant for the particular crystal dependent on such factors as its density and cut, and A is the adsorbing surface area (cm2). For any piezo-electric crystal, the change in frequency is proportional to the mass of absorbed material, up to about a 2% change. This frequency change is easily detected by relatively unsophisticated electronic circuits.
Figure 3: A Scheme of Transduction and Biosensor Types
5.APPLICATIONS OF BIOSENSOR
5.1.Summary of potential applications for biosensors
Clinical diagnosis and biomedicine
Farm, garden and veterinary analysis
Process control: fermentation control and analysis food and drink production and analysis
Microbiology: bacterial and viral analysis
Pharmaceutical and drug analysis
Industrial effluent control
Pollution control and monitoring of mining, industrial and toxic gases
5.2.1.Measurement of Metabolites: The initial impetus for advancing sensor technology came from health care area, where it is now generally recognized that measurements of blood gases, ions and metabolites are often essential and allow a better estimation of the metabolic state of a patient. In intensive care units for example, patients frequently show rapid variations in biochemical levels that require an urgent remedial action. Also, in less severe patient handling, more successful treatment can be achieved by obtaining instant assays. At present, the list of the most commonly required instant analyses is not extensive. In practice, these assays are performed by analytical laboratories, where discrete samples are analyzed, frequently using the more traditional analytical techniques.
5.2.2.Diabetes: The 'classic' and most widely explored example of closed-loop drugcontrol is probably to be found in the development of an artificial pancreas. Diabetic patients have a relative or absolute lack of insulin, a polypeptide hormone produced by the beta-cells of the pancreas, which is essential to the metabolism of a number of carbon sources. This deficiency causes various metabolic abnormalities, including higher than normal blood glucose levels. For such patients, insulin must be supplied externally. This has usually been achieved by subcutaneous injection, but fine control is difficult and hyperglycaemia cannot be totally avoided, or even hypoglycaemia is sometimes induced, causing impaired consciousness and the serious long-term complications to tissue associated with this intermittent low glucose condition.
5.2.3.Insulin Therapy: Better methods for the treatment of insulin-dependent diabetes havebeen sought and infusion systems for continuous insulin delivery have been developed. However, regardless of the method of insulin therapy, its induction must be made in response to information on the current blood glucose levels in the patient. Three schemes are possible , the first two dependent on discrete manual glucose measurement and the third a 'closed-loop' system, where insulin delivery is controlled by the output of a glucose sensor which is integrated with the insulin infuser. In the former case, glucose has been estimated on 'finger-prick' blood samples with a colorimetric test strip or more recently with an amperometric 'pen'-size biosensor device by the patient themselves. Obviously these diagnostic kits must be easily portable, very simple to use and require the minimum of expert interpretation. However, even with the ability to monitor current glucose levels, intensive conventional insulin therapy requires multiple daily injections and is unable to anticipate future states between each application, where diet and exercise may require modification of the insulin dose. For example, it was shown that administration of glucose by subcutaneous injection, 60 min before a meal provides the best glucose/insulin management.
5.2.4.Artificial Pancreas: The introduction of a closed-loop system, where integrated glucose measurements provide feedback control on a pre-programmed insulin administration based on habitual requirement, would therefore relieve the patient of frequent assay requirements and perhaps more desirably frequent injections. Ultimately, the closed-loop system becomes an artificial pancreas, where the glycaemic control is achieved through an implantable glucose sensor. Obviously, the requirements for this sensor are very different to those for the discrete measurement kits.
5.3.Industrial Process Control
5.3.1.Bioreactor Control: Real-time monitoring of carbon sources, dissolved gases,. in fermentation processes could lead to optimization of the procedure giving increased yields at decreased materials cost. While real-time monitoring with feedback control involving automated systems does exist, currently only a few common variables are measured on-line (e.g. pH, temperature, CO2, O2)) which are often only indirectly related with the process under control.
5.4.1.Dip Stick Test: The requirement for rapid analysis can also be anticipated in military applications. The US army, for example, have looked at dipstick tests which are based on monoclonal antibodies. While these dipsticks are stable and highly specific (to Q-fever, nerve agents, yellow rain fungus, soman, etc.) they are frequently two-step analyses taking up to 20 min to run. Such a time lapse is not always suited to battlefield diagnostics.
A particularly promising approach to this unknown hazard detection seems to be via acetylcholine receptor systems. It has been calculated that with this biorecognition system, a matrix of 13-20 proteins are required to give 95% certainity of all toxin detection.
5.5.1.Air and Water Monitoring: Another assay situation which may involve a considerable degree of the unknown is that of environmental monitoring. The primary measurement media here will be water or air, but the variety of target analytes is vast. At sites of potential pollution, such as in factory effluent, it would be desirable to install on-line real-time monitoring and alarm, targeted at specific analytes, but in many cases random or discrete monitoring of both target species or general hazardous compounds would be sufficient. The possible analytes include biological oxygen demand (BOD) which provides a good indication of pollution, atmospheric acidity, and river water pH, detergent, herbicides, and fertilizers (organophosphates, nitrates, etc.). The survey of market potential has identified the increasing significance of this area and this is now substantiated by a strong interest from industry. The potential applications of biosensors are summarized in Table 1.4.
5.5.2.Tuning to Application: The potential for biosensor technology is enormous and is likely to revolutionize analysis and control of biological systems. It is possible therefore to identify very different analytical requirements and biosensor developments must be viewed under this constraint. It is often tempting to expect a single sensor targeted at a particular analyte, to be equally applicable to on-line closed-loop operation in a fermenter and pin-prick blood samples. In practice, however, the parallel development of several types of sensor, frequently employing very different measurement parameters is a more realistic.
Whatever the market, wherever the application, the development of the Sensor Device requires separate and linked investigation at various levels. Even without a particular final goal, our basic understanding of immunoassay, enzyme-linked assay, recognition proteins, catalytic active sites and their 'electronic transduction will continue to occupy the field, in addition to more 'downstream' considerations such as life-time levels of detection etc.; the list could be unending, these for example, are just some of the considerations:
nature of the analyte and identification of a specific recognition pathway
and transduction parameter.
identification of the physico-chemical method for transduction of that
parameter and its optimisation.
Optimization of the transducer technology.
Linking the recognition reaction with the transduction.
immobilization of the recognition species and optimisation of its
immobilization of any other 'transduction' species and their
assessment of levels and range of detection
assessment of interferents
consideration of needs of particular application:
quantitative or qualitative (alarm)?
operation in 'real' samples?
required working life?
required shelf life?
ease of fabrication
Many other considerations!
There appears to be no simple summary of areas which should be targeted for further investigation, or statement of what might be involved. Perhaps a suitable description might be that we are concerned with the matching of natural and synthetic materials and technologies to allow interference free communication between analyte and a data handling circuit.