Biosensors for monitoring environmental emissions
By J.D. Newman, Institute of BioScience and Technology, Cranfield University Increasing concerns over the environment and the consequent political pressures to monitor consent levels, check for pollution and safeguard fragile ecosystems, have led to many initiatives to develop biosensors for environmental applications.
Since the description of the first biosensor in 1962, huge advances have been made in scientific research combining the extensive knowledge of electrochemistry, biochemistry, physics, electronics (particularly silicon technology), and software design. This has made possible the development of highly specific, sensitive and accurate sensors, based on the exquisite recognition properties of biological molecules. These devices offer many promising solutions to on-site, real time monitoring of target analytes in the environment. They have even been sent into space to look for extraterrestrial life.
Biosensors are most commonly used in the environment for monitoring the following potential classes of pollutants:
A good example of a biosensor system for environmental analysis, is the “RIANA” river analyser instrument, which was developed by a European consortium, led by the University of Tübingen. The instrument relies on specific immunoassays for analyte recognition, which is detected using Total Internal Reflection Fluorescence (TIRF).
This relies on the interaction of a light beam, which passes along a planar waveguide, with fluorescent labels, which attach themselves to the waveguide via very highly selective antibodies.
The RIANA platform is very flexible. By changing the antibodies used, a wide range of potential analytes can be monitored. Initial efforts concentrated on pesticides and some other common pollutants, including: atrazine, Irgarol 1051, Methylchlorophenoxy-propionic acid (MCPP), simazine, 2,4-dichlorophenoxyacetic acid (2,4-D), paraquat, isoproturon, pentachlorophenol (PCP) and alachlor. For the majority of substances, the detection limit is 0.1µg/l or lower.
Ultra-low detection limits are achievable with many affinity sensors and electrochemical detection may be readily integrated with chromatographic techniques to yield user-friendly devices. This approach overcomes the need for multiple sample manipulation steps, which was a major drawback of many early sensors of this type.
In an alternative approach, double-stranded DNA may be used as a receptor element. “Sandwich”-type biosensors based on liquid-crystalline dispersions formed from DNA-polycation complexes may find application in the determination of a range of compounds and physical factors that affect the ability of a given polycation molecule to maintain intermolecular crosslinks between neighbouring DNA molecules.
Broadly based biosensors can be produced, based on the principle of enzyme inhibition. Acetylcholine esterase (AChE) is the enzyme most commonly used for biosensors of this type. A variety of compounds, including pesticides and heavy metals, inhibit this enzyme. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, has been detected in this way. By using mutated enzyme, the sensitivity of detection was brought to below the nM level. The biggest drawback to devices like this is that they are unselective.
Biosensors utilising AChE operate by comparing a kinetic measurement of the initial velocity of the enzyme-catalysed reaction before and after exposure to the pollutant:
Acetylcholine + H2O ———–> Acetic acid + Choline
The kinetic measurement is often performed using electrochemical transducers. In many cases these are simple carbon electrodes, or pH probes, although more complex transducers, such as ion-selective field effect transistors (ISFETs), light addressable potentiometric sensors (LAPS), or conductivity cells can also be used.
Many other enzyme systems can be utilised in inhibition biosensors. Lactate dehydrogenase has been used, in this way, to measure pentafluorophenol. Detection limits of 20-40 µg/l have been reported.
Advances are not limited to the liquid phase. For example, a gas-phase micro biosensor for phenol, in which polyphenol oxidase was immobilised in a glycerol gel on an interdigitated microelectrode array has been demonstrated. Phenol vapour partitioned directly into the gel, where it was oxidised to quinone. Signal amplification was enhanced by redox recycling of the quinone/catechol couple, resulting in a sensor able to measure 30 ppb phenol. Detection limits of parts per trillion of volatile organic carbons are feasible with this type of approach.
It has long been realised that advanced fabrication techniques are a key to the successful development of commercially viable biosensors in many applications. Fortunately, many technologies have been developed in other industries (such as microelectronics) and are, therefore, available with much greater reliability and at a much lower cost than would otherwise be the case, although they obviously require certain modifications and considerable development.
Screen-printing is a thick-film process, which has been used for many years in artistic applications and, more recently, for the production of miniature, robust and cheap electronic circuits. Since the technique has been developed for mass production of biosensors, it has revolutionised the industry, particularly in the lucrative medical sector. The process has been one of the major reasons for the commercial success of many biosensors and is the process by which MediSense (now Abbott) produce over 1 billion biosensor strips annually. As well as low cost, there are other attractions from an environmental standpoint, since the technique makes it possible to produce very large numbers of reproducible, inexpensive devices at high speed.
The ability to handle small volumes of liquids with high precision is a key area in the development of novel, next generation, biosensors. As devices become smaller and more sophisticated, it becomes increasingly difficult to handle the analytical reagents involved in production. Some of the latest advances in transducer design, for example, make the production of 1 million measurement points on a 1 cm squared chip a possibility. The most difficult aspect of the production of these devices is, currently, incorporating the biological reagents onto the surface of such arrays.