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Biosensor System for Rapid Diagnosis of Infection with Mycobacterium Tuberculosis - Essay Example

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This essay "Biosensor System for Rapid Diagnosis of Infection with Mycobacterium Tuberculosis" will look at biosensors that have the advantage of being simple and low-cost instruments, fast response time, require less sample pre-treatment, and display high sample throughput…
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Biosensor System for Rapid Diagnosis of Infection with Mycobacterium Tuberculosis
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Proposal to develop biosensor system for rapid diagnosis of infection with drug-resistant mycobacterium tuberculosis (TB) in accident and ER in hospital Aim: Proposal for developing a biosensor system for the rapid diagnosis of infection with drug-resistant Mycobacterium tuberculosis (TB) in hospital Accident and Emergency Departments. Introduction: Realizing the serious outcomes of Tuberculosis, Mr. Camilleri, said “TB is one of the greatest threats to global health and the terrifying ease with which it is transmitted means that national boundaries and global demographics are irrelevant in preventing its spread” (News release). It is therefore imperative to identify and commence the treatment at the earliest as it is the most valuable means of controlling TB infection rates. Various drugs have been designed to effectively combat tuberculosis but still it remains one of the world’s most dreaded diseases and is a serious health problem. It potentially kills more adults than any other infectious agent. Detection of infectious pulmonary cases at early stages of infection is imperative to gain control over the bacilli and also its spread. The most prevalent method of screening Mycobacterium tuberculosis is Mantoux TB test, where an injection is given under the patient’s skin and localised reaction is observed for up to 72 hrs. The test is slow, clumsy, requires specialist laboratory facilities and is only effective for detecting advanced disease, it is influenced by multiple factors and delivers false positive or false negative results and requires further testing and interpretations (News release). Research is going on constantly to develop rapid and accurate methodologies and techniques to detect the causal organism associated with tuberculosis. In the past two decades research is being carried out for the development of biosensors and biochips to understand the biological and medical fields and to quantify biomolecules (Vo-Dinh, 2000). It is manifested that organisms have some kind of bioreceptors; biosensors are developed as a means of chemical analysis with high selectivity (Vo-Dinh, 2000). Biosensors are chemical sensors that exploit the high selectivity and sensitivity of a biologically active material (Kumar, 2000). A biosensor can be defined as a device that consists of a biological recognition system called a bioreceptors and a transducer. When the analyte interacts with the bioreceptor (a molecular species e.g. an antibody, an enzyme, a protein, or a nucleic acid or cell, tissue or whole organism), it is measured by transducer as it converts information into an electrical signal (Vo-Dinh, 2000). Biosensors have the advantage of being simple and low-cost instruments, fast response time, require less sample pre-treatment and display high sample throughput. With the advances in research in this area, novel treatments are required to provide new and better analytical techniques to provide new and improved biosensors (Turner, 1989, Hahn, 1988, Guilbault, 1988, Ho, 1984). Biosensors are acquiring tremendous popularity in almost all areas of life, agricultural, horticultural and veterinary analysis; pollution, water and microbial contamination analysis; clinical diagnosis and biomedical applications; fermentation analysis and control; industrial gases and liquids; mining and toxic gases; explosives and military arena; and flavours, essences and pheromones (Dewa, 1994, Diamond, 1998, Lowe, 1985, Rogers, 1995). A biosensor comprises of two components, one is receptor and other is detector. Receptor is responsible. The receptor selects the sensor, like enzymes, antibodies and lipid bilayers while the detector/ transducer is non-selective and interprets the physical and chemical change by recognizing the analyte and passes the signal as an electrical impulse. The detector encompasses pH-electrode, an oxygen electrode or piezoelectric crystal. Thus a biosensor incorporates a biological sensing element with a transducer and selectively distinguishes biological molecule through a reaction, specific adsorption or other physical or chemical process (Dewa, 1994, Diamond, 1998, Lowe, 1985, Rogers, 1995). WHO reports each year around 2 million deaths occur out of 8.8 million active cases of tuberculosis being diagnosed (News release). It was presumed that medical science is gaining control over the disease but the problem erupted in the form of virulent and drug-resistant strains of tuberculosis bacilli. This is a grave issue that must be taken seriously as the death ratio can increase because of the late diagnosis. It is observed that 80% of the reported cases are from developing countries. This could be due to huge population, closed and congested living where transmission is effortless (News release). The objective of this proposal is to develop a sensing device (a biosensor) for rapid diagnosis of tuberculosis and other infections caused by Mycobacterium. The proposal emphasize on the use of antibody-based piezoelectric crystal which accomplishes detection of mycobacterial antigens in diluted/ attenuated cultures of M. Tuberculosis. The reaction is highly immune-specific. Detection of antigen occurs in liquid or vapour phase (Kumar, 2000). Background: Recognition of biomolecules could either be bio-affinity recognition or bio-metabolic recognition. These processes involve chemical binding of the complementary structures. It involves shape-specific binding. In the process of bio-affinity recognition, strong binding occurs and the transducer detects the presence of this bonding (receptor-ligand ad antibody-antigen binding). In the process of bio-metabolic recognition, the analyte and other co-reactants are chemically transformed resulting in the product formation. It is manifested that any kind of biological molecule can be utilized for the development of biosensor. The interaction between antigen and antibody attempts to develop antibody-based chemical biosensors called as immunosensors. If antibody could be raised against a particular analyte, in the next step the immunosensor can be possible developed to recognize the analyte. This reaction should be sufficient to induce the electrical signal and therefore piezoelectric effect of crystalline substances is exploited for the detection (Luong, 1991, Guilbault, 1992, Minunni, 1994, Nakanishi, 1996, Rogers, 1997). The piezoelectric immunosensor is the most sensitive analytical instrument capable of detecting pictogram range. It can detect antigen in the gas and also in liquid phase. A piezoelectric sensor detects the mycobacterial antigen in biological fluids. It provides the results within couple of hours. It is very practical and can be used anywhere. The diagnosis of TB using this immunosensor is based on the detection of mycobacterial antigens in liquid and testing the degree of sensitivity and specificity caused by potentially interfering substances in biological fluids. Another proposed protocol is for designing a biosensor: A rapid analysis of the positive cases or suspected individuals is of high priority. For this a device is required which can sense the causal organism and can indicate its presence with rapid pace. Understanding the urgent need of this kind of analyser, on 24th March (the World TB day), Cambridge based Rapid Biosensor Systems (RBS) was launched with an intensive six-year development programme. It is a TB Breathalyser and is capable of providing reliable and cost-effective screening for early stage infection (News release). It is based on bio-optical sensor technology, delivers accuracy level of +95%. It provides the result within minutes, it has specificity of detecting active TB and not affected by the presence of HIV or other co-infections (News release). RBS is user friendly and does not require electricity or running water and therefore can be used in remote places. It is reported that RBS Breathalyser test is exclusive, and does not require the collection of sputum samples and can be used at the health centres. This test is an exhilarating new development in TB control. It will help us learn more about when patients become infectious and how long they remain a risk to other people. In a small study in Ethiopia we used a prototype of the test to identify TB patients within ten minutes. It is important that further studies are undertaken to ascertain the sensitivity and specificity of the test. If the TB Breathalyser proves sufficiently sensitive then it might have a major impact on the way we control tuberculosis.” (http://www.rapidbiosensor.com/exciting_new_development_in_TB_control.asp) Experimental Design: Electrode Fabrication Process: The study proposes the use of alpha quartz, the detector crystal, which is insoluble in water and resistant to high temperatures and can sustain the temperature as high as 579° C with no loss to piezoelectric properties. AT and BT-cut crystals are most helpful as piezoelectric detectors as these cuts consign to the orientation of the plate with respect to the crystal structure and are most stable, with a temperature coefficient of 1 ppm per degree centigrade over a temperature range of 10°C to 50°C and take the form of discs, squares, and rectangles. The crystals are mounted on the holder with stainless steel leads (figure 3, Appendix) and a silver composite to connect the electrode to the wire. Dimensions to be used are: Crystals: 14 mm in diameter and electrodes on both sides were 8 mm in diameter. These crystals will then be mounted on HC6/u holders (Figure 3, Appendix). The piezoelectric quartz crystal will be driven by a low-frequency transistor oscillator, which runs on a 1-3-V d.c. regulator power supply and will be set at 9V d.c. the frequency of the vibrating crystal will be monitored with Protek multifunction frequency counter. The crystal mounted on its holder will be connected to the oscillator circuit and frequency counter will be connected to the oscillator device. Then the coating process will be performed, first with metal depositions followed by the biomolecular-analytes. Then the frequency reading will be recorded (Hussain, 1998) (Figure 4, Appendix). Immunosensing: The crystal electrodes will then be modified with a 5 ml coating of protein A (a polypeptide isolated from S. aureus binds exclusively with IgG antibodies and so not interact with the antigen site) for better adhesion of antibodies on the surface of the transducer. A tertiary complex will be formed (protein A + Ab + Ag). The electrodes will be oxidized with 0.5M NaOH and will be cleaners in 0.5 M HCl and 0.5 m HCrO2. They will be dried in an incubator for 3 hrs and then coating of protein A followed by IgG coating will be performed again followed by drying process. At each step the frequency of the crystal will be recorded. In the process one control will be kept in the similar manner and coating in control will be of HBV- honey bee antibody, whereas the experimental sample will be coated with M. Tuberculosis. The difference in frequency change between the control and experimental crystals will be compared reflecting the immunologically specific binding (Figure 5, Appendix). Time table: the proposal requires the time frame of 6 months to 8 months: 2 months for electrode fabrication process. 2 months for immunosensing process 2 months to carryout clinical trials Marketing of the Product under MHRA/ PASA: The UK Medicines and Healthcare Product Regulatory Agency (MHRA) is the government relevant essential requirements of the appropriate Medical Devices Directives. The NHS is the largest single healthcare delivery organization in the world. NHS PASA is an executive agency of the Department of Health (DH) and makes the most effective use of resources by procuring NHS makes the most effective use of resources by getting the best possible value for money when purchasing goods and services. NHS constitutes a major opportunity for UK medical device companies, NHS is assisted by the NHS Purchasing and Supply (NHS PASA). The research and clinical trials of the biosensor for the diagnosis of TB will be as per the guidelines of GCP (Good Clinical Practice) Literature Review/ References: 1. Vo-Dinh, T., Cullum, B. 2000. Biosensors and biochips: advances in biological and medical diagnostics Fresenius J Anl Chem, 366, pp, 540-551. 2. News Release, 10th February 2009, A Revolution in TB Screening http://www.babraham.co.uk/documents/RBS_PR0209.pdf 3. Dewa, A., S., Ko, W.H. 1994. Biosensors, Semiconductor Sensors, ed. S.M. Sze (New York: Wiley Interscience,p pp. 415. 4. Diamond, D. 1998. Principles of Chemical and Biological Sensors, vol. 150 (New York: John Wiley & Sons) 5. Lowe, C., R., 1985. An Introduction to the Concepts and Technology of Biosensors, Biosensors, 1, pp 4. 6. Rogers, K., R., 1995. Biosensors for Environmental Applications, Biosensors Bioelectronics, 10, pp, 533-541. 7. Turner, A., P.,F., 1989. Current Trends in Biosensor Research and Development, Sensors Actuators, 17, pp, 433-450. 8. Hahn, E., C., 1988. Piezoelectric Crystal Detectors and Their Applications, Ph.D. dissertation, University of New Orleans. 9. Guilbault, G., G., and Jordan, J., M., 1988. Analytical Uses of Piezoelectric Crystals: A Review, CRC Crit. Rev. Anal. Chem., 19 (1), pp, 1-28. 10. Ho, M. 1984. Applications of Piezoelectric Quartz Crystal Microbalances, ed. 11. Lu, C., and Czanderna, A., W., 1984. (Amsterdam: Elsevier/North Holland). 12. http://www.rapidbiosensor.com/exciting_new_development_in_TB_control.asp 13. Luong, J., T., H., and Guilbault, G., G. 1991. Analytical Applications of Piezoelectric Crystal Biosensors, Biosensor Principles and Applications, ed. L.J. Blum and P.R. Coulet (New York: Marcel Dekker, pp. 107-138. 14. Guilbault, G., G., Hock, B., Schmid,R. 1992. A Piezoelectric Immunobiosensor for Atrazine in Drinking Water, Biosensors Bioelectronics, 7 , pp. 411-420. 15. Minunni, M., Skladal, P., Mascini, M., 1994. A Piezoelectric Quartz Crystal Biosensor for Atrazine, Life Chemistry Reports, 11, pp, 391. 16. Nakanishi K. 1996. Detection of the Red Tide-Causing Plankton Alexandrium Affine by a Piezoelectric Immunosensor Using a Novel Method of Immobilizing Antibodies, Anal. Lett., 29, pp, 1247-1258. 17. Rogers, K., R., Koglin, E., R. 1997. Biosensors for Environmental Monitoring: An EPA Perspective, Biosensors for Direct Monitoring of Environmental Pollutants in Field, ed. D.P. Nikolelis et al. (Norwell, MA: Kluwer Academic Publishers), pp. 335-349. 18. Hussain, I. 1998. Development and Applications of Piezoelectric Biosensors, M.S. thesis, University of South Alabama. 19. Kumar, A. 2000, Biosensors Based on Piezoelectric Crystal Detectors: Theory and Application, JOM-e, 52 (10) http://www.tms.org/pubs/journals/JOM/0010/Kumar/Kumar-0010.html. 20. Kelley, K, Selling to NHS http://www.nic.nhs.uk/About/HowToGuides/Marketing%20and%20the%20NHS/Pages/Selling%20to%20the%20NHS.aspx Discussion: The newly invented biosensor-based tuberculosis serodiagnostic device detects antibodies to lipid antigens of the bacterial pathogen as surrogate markers of infection. This method is ideally suited to testing HIV-infected patients, as production of the antibodies to the lipid antigens are not affected by the HIV as much as with other antigens. Tuberculosis is a bacterial infection that has regained prominence in the world due to drug resistance and its association with AIDS. A large proportion of HIV-infected individuals succumb to tuberculosis. This is mainly due the abundance of the pathogen in dormant state, especially amongst people living in developing countries. HIV infection leads to a weakened immune resistance that is exploited by the tuberculosis pathogen to proliferate and infect. Although tuberculosis can be successfully treated with antibiotics, a cure is only affected after at least six months of combination therapy. Non-compliance by patients to this extended treatment regime is highly probable, and often results in a re-activation of the dormant mycobacteria in a drug-resistant mode, which is usually fatal. It also puts medical staff at risk. It is important that HIV-infected people who are hospitalized for various reasons are effectively screened for tuberculosis co-infection and are monitored for compliance to the treatment, to prevent hospitals becoming breeding sites for drug-resistant tuberculosis and places of unacceptable risk for health workers. Appendix Figure 1. Schematic diagram of a biosensor device. Figure 2. Schematic diagram of an immunosensor device. Figure 3. Quartz crystal and holder. Figure 4. Experimental apparatus for a piezoelectric sensor. Figure 5. Schematic diagram of the antibody-antigen binding. (Kumar, 2000) Read More
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