Selective Electrode For Microbial Biosensors example essay topic

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 physicochemical ly. 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 plasmon's 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, chloro phenols, bio available 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 - 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 hydrogen bonding or hydrophobic interactions. Entrapments hold the cell in close proximity of the transducer using dialysis or filer membrane or biological polymers or gels. Microorganisms used in biosensors can also be classified biologically, viable or non-viable forms.

Viable cells produce products like ammonia, carbon dioxide, and acids which can be monitored using a variety of transducers. These cells are mainly used when the overall substrate assimilation capacity of microorganisms is taken into account. Non-viable forms of cells are often used as a substitute for enzymes. A major limitation to using microorganisms is the diffusion of substrate and products through the cell wall resulting in slow responses.

One way to overcome this problem is to permeabilize the cells using physical (freezing), chemical (solvents), and enzymatic (lysozyme). The most common method is to use chemical solvents (toluene, chloroform, ethanol etc) to create pores by removing some of the lipids from the cell membranes, allowing for the free diffusion of small molecules across the membrane and retaining of the large enzymes within the cell. Permeabilization will make the cell non-viable but are important in certain situations nonetheless. Another limitation of using whole cells is the low specificity due to unwanted side reactions catalyzed by other enzymes within the cell. Permeabilization, heat or chemical treatments can be used to minimize such side reactions. We can also block unwanted metabolic pathways using inhibitors.

1 Microbial biosensors can be classified into several categories, with the two major categories being electrochemical and chemically based. The three types of electrochemical microbial biosensors are amperometric, potentiometric and conductimetric. The major application of electrochemical microbial biosensors is in the environmental field. 3 Amperometric biosensors involve detecting the current generated by the redox of specifies at the surface of the electrode, in comparison to a reference electrode. These sensors have been widely developed for determining the biochemical oxygen demand (BOD) for measuring biodegradable pollutants in solution.

BOD measures the microorganism's oxygen consumption / respiration levels over a set number of days; in other words, it's a value related to total content of organic materials in wastewater. Cell strains that are used under this system include Torpulopis candida, Pseudomonas put ida, and Bascillus subtilis. Sometimes, mixtures of two or more microorganisms are used to broaden the substrate because one strain has a narrow substrate spectrum. The first commercial BOD biosensor was produced by a Japanese company Nis shin Denk i in 1983, and many more sensors have now been commercialized by companies like DKK Corporation, Auto team F mbH, and Bioscience Inc. Significant efforts have been made toward the development of a disposable BOD sensor, and of a portable BOD system. 1 Amperometric microbial biosensors have also been applied to other chemicals.

Ethanol is the most important sensor next to BOD. Microorganisms that metabolize ethanol are immobilized on oxygen electrode to fabricate ethanol biosensors. These biosensors however, often have poor selectivity. Recent research has been geared mainly at increasing the selectivity of microbial ethanol biosensor. Sensors for sugars are also highly desired because sugars are important ingredients of different media. Finally, phenols have also grabbed much attention due to their high toxicity to living organisms.

One such biosensor for phenols is Rhodococcus erthropolis modified Clark oxygen electrode for 2, 4-dinitrophenol. Neurotoxic organophosphate compounds are also becoming more important in applications such as agriculture pesticide and chemical warfare. Potentiometric microbial biosensors consist of an ion-selective electrode (pH, ammonium, chloride etc) or gas-sensing electrode coated with an immobilized microbe layer. Microbes that consume analyte generate a change in potential resulting from ion accumulation or depletion. Transducers measure the difference between the highly active but stable reference electrode, and the signal is correlated to the analyte concentration. pH electrodes are the most widely applied ion selective electrode for microbial biosensors; others can also be used. An ammonium ion selective electrode coupled with unease yielding Bascillus sp. monitors the presence of urea in milk.

A chloride ion selective electrode can be used to monitor in batch and continuous modes in wastewater. 1 Optical microbial biosensors involve measuring the UV absorption, reflectance and fluorescence that occurs with the interaction of the biocatalyst with the analyte. Optical based sensors are compact, flexible and resistant to electrical noise. Bioluminescence occurs when microorganisms emit light and it plays an important role in real-time process monitoring. The bacterial luminescence lux gene has been engineered in an inducible or constitutive manner. In the inducible manger, the lux gene is fused to a promoter that regulates the concentration of compound of interest.

The concentration of the compound can then be quantitatively analyzed by detecting the bioluminescence intensity. In the constitutive manner, the lux gene is fused to promoters that are always expressed as long as the organism is alive. This method is evaluates the total toxicity of contaminant. 1 Bio luminescent microbial biosensors have been extensively researched to monitor bioavailability metals such as copper, phosphorus, mercury and naphthalene. This method is especially important to environmental problems caused by industrial and agricultural pollution. These biosensors respond to pollution quickly and enable a rapid toxicity test.

Fluorescence spectroscopy is a sensitive chemistry technique that can detect low concentrations of analyte. Fluorescence emission intensity is directly proportional to the concentration. Fluorescent materials and green fluorescent protein (GFP) have been used in the construction of fluorescent biosensors. GFP microbial biosensors are useful in assessing heterogeneity of iron bioavailability on plants with the fusion between an ion-regulated promoter and gfp.

GFP has also been developed to measure water availability in a microbial habitat, monitoring cell populations and so on. This type of biosensor is becoming increasing powerful with the development of DNA recombinant technologies and other biotechnology advances. Other forms of fluorescence biosensors include oxygen-sensitive fluorescent material-based biosensor that measures oxygen in seawater microorganisms, and colorimetric biosensor, which identifies virulence activity associated with certain bacterial pathogens that change color change when infected. 1 Other types of microbial biosensors include sensors detecting pressure changes in microorganisms on baroxymeter, and sensors based on infrared analyzer for detecting microbial respiration of carbon dioxide. This is especially useful for measuring the extent of organic pollution in wastewater both in the lab and in wastewater treatment plants. 1 The biosensor concept has greatly inflated out of Clark and Lyon's first biosensor.

Microorganisms are now widely employed as the bio sensing element in the making of biosensors due to their low cost, long lifetime and wide range of suitable pH and temperature ranges. However in comparison to purified enzymes, microorganisms still lag in the long response time, low sensitivity and poor selectivity of analytes. As we venture ahead in the 21st century in cultivating this technology, and using learnings gained from genetic information of microbes and recombinant DNA technologies, we can synthesize faster responses and highly sensitive microbial biosensors. One such way is using microbes to serve as an enzymes's up port matrix, where the surface expressed enzymes can directly react with substrates without the entry of substrates into the microbes. We also need to design, select and screen for microorganisms with specific activity for chemicals, since non-specific cellular response to substrates can limit biosensor selectivity. To achieve this, we must combine classical microbiology with methodologies in genetic engineering to create metabolic pathways that cater to our needs.

One possible avenue for growth is Micro-Electro-Mechanical Systems (MEMS), which is an integration of sensors, electronics, and mechanical elements on a single silicon substrate through micro-fabrication technology. Bio chips and arrays for detecting a range of analytes can be made out of MEMS, making it feasible for simultaneous detection of multiple chemicals. Another trend in biosensors is developing biosensors for application under extreme conditions (highly acidic, alkaline, saline, temperature). Most microorganisms cannot survive under these extreme environments; thus, we need to select cells which not only survive, but also maintain high enzyme activity. Thus, populations of microbes like thermophiles, alkalophiles, halophiles, psychrophiles, and metallophiles etc. will gain importance in this industry.

2 These requirements are important for growing the biosensor industry because of the need for low cost, sensitive, selective and fast-response biosensors in the market. It seems that with the current advances in biosensors and modern biotechnology and the possibilities in the field of biosensors, it is imminent that many analytical applications will be replaced. It seems like microbial biosensors is on its way towards a promising future. Appendix Table 1.