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Microbial Biosensors - Past, Present and Future

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Biosensors are analytical devices used to measure biological information that converts a bodily response into an electrical signal. Biosensors consist of three major parts, the sensitive biological element (tissue, microorganisms, enzymes etc.), the transducer, and the detector element which works physicochemically. The major component of a biosensor is the transducer, which uses the physical changes of a reaction to produce an effect. Such physical changes could be thermal output, electrical potential change, redox reaction, electromagnetic radiation etc. The triggered electrical output from the transducer can then be amplified, processed, displayed and analyzed. Biosensors are a rapidly expanding field of study with an estimated annual growth rate of 60%, with the majority of the growth coming from the health-care industry.

The biosensor concept can be traced back to Professor Leland C Clark Junior, who invented the oxygen electrode in 1956. He wanted to expand the range of analytes that we could measure in the body. His first experiment involved entrapping glucose oxidase enzyme in an oxygen electrode using a dialysis membrane. The observed decrease in oxygen concentration was proportional to glucose concentration. This is the first of many variations of the basic biosensor design that emerged out of Dr Clark's concept. Through the next several decades, the biosensor technology took off radically as a variety of new devices was discovered including enzymes, nucleic acids, and cell receptors. In looking at the historical development of this technology, 1980's was certainly the inventive decade, with commercialization being the theme in the 1990's.

Biosensors' primary functions in terms of research and commercial applications are identifying target molecules, identifying the availability of a suitable biological recognition element, and the potential for disposable detection systems to replace sensitive lab techniques. Examples of these functions include monitoring health related targets, detecting pesticides, detecting pathogens, and determining the level of toxicity in an organism or in the environment. The most widespread and commercialized example is the blood glucose biosensor, which uses an enzyme to break down blood glucose. Once broken, the biosensor transfers an electron to an electrode which is converted into a measure of blood glucose concentration. This process is especially important to diabetics to monitor glucose levels in their bodies. However, many biosensors are still not commercialized and most are still single analyte devices. In recent years, the development of biosensors for environmental and clinical applications has gained much interest from researchers, due to the need for fast and cost effective methods for analysis in both environmental and clinical situations. The advantages of using biosensors are analyte specificity, ease of operation, fast analysis time, and minimal sample preparation. The future of this technology will likely head in the direction of innovating the current processes, and finding ways to make this technology more commercially viable. Table 1 shows some of the most commonly used biological recognition components and transducers in biosensor research.

There are several main categories of biosensors. Piezoelectric biosensors and optical biosensors are based on the concept of surface plasmon resonance, where surface plasmons are released off the surface of some materials. Piezoelectric sensors use crystals which undergo transformation when an electrical current is applied. Electrochemical biosensors are based on enzymatic catalysis of a reaction that produces ions. Two other types of biosensors, thermometric and magnetic based biosensors are rarely used.

Microbial biosensors are another major class of biosensors. It uses whole cell microorganisms as the recognition element in biosensors and has been a popular area of research especially in food processing, fermentation processing and environmental applications. There has been an increasing demand for quick and specific analytical tools. To ensure the quality and assurance of foods, analysis is required to monitor nutritional parameters, food additives, contaminants, microbes etc. For the environment, microbial biosensors measure the amount of chemicals in nature such as nitrite, cyanide, chlorophenols, bioavailable carbon etc.

Microorganisms are preferred in these research areas as opposed to enzymes because cells are cheaper to grow than to purify enzymes, and enzyme activity is often enhanced in cells and is more stable due to the optimized cellular environment; this in turn would make it harder to measure the desired response. Whole cells also provide a multipurpose catalyst when processes require the participation of a number of sequential enzymes.2 Additionally, advances in recombinant DNA technologies has opened possibilities of tailoring microorganisms to improve the activity of existing enzymes or express foreign proteins in host cells. Dives discovered the first microbial biosensor in 1975 for determining ethanol. His biosensor consisted of Acetobacter xylimum bacterial cells immobilized onto an oxygen electrode.

A major disadvantage however of using cell-based electrodes is the poor selectivity because microbes contain other enzymes which may catalyze competing reactions. Another problem with these sensors is the long recovery time required. Microbes require anywhere from several minutes to hours to recover because many cell-based biosensor measuring principle is based on the cell's respiratory function. With this said however, cell-based biosensors are extremely useful for functional information Ð'- how a living organism is affected by a stimulus.4

Microbial biosensors require the close contact between the microorganism and the transducer. Thus, microorganisms must be immobilized on the transducer with close proximity. Both the technology and choice of immobilization technique is critical to the biosensor response and must be chosen carefully to obtain the desired measurements. Two buckets of immobilization techniques are available and fall under chemical methods and physical methods. Chemical methods of microbe immobilization include covalent binding and cross-linking. Covalent binding relies on forming a stable covalent bond between functional groups of the microorganisms' cell wall components and the transducer. Cross-linking involves bridging between functional groups on the outer membrane of the cells to form a network. On the other hand, physical methods involve adsorption and entrapments. These methods are preferred when viable cells are required because they do not significantly disturb the microorganism's natural state. Physical adsorption uses simple interactions such as ionic, polar or



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