Development of Ultrasensitive Electrochemical Aptasensor for Diagnosis of Latent Tuberculosis

Abstract

This thesis describes generation of ssDNA aptamer targeted to early secreted antigenic target-6kDa (ESAT6), culture filtrate protein-10kDa (CFP10) and Rv2654c (TB7.7). And development of novel diagnosis methods for latent tuberculosis through electrochemical aptasensor is also described. Tuberculosis is caused by an infection of Mycobacterium tuberculosis (M. tb)that is spread through the inhalation of aerosolized water droplets. Tuberculosis is a serious global health problem with about 10.4 million cases of new tuberculosis every year and 1.8 million deaths per year. Furthermore, it is estimated that two billion people live with latent M. tb infection and serve as a possible elements of future active tuberculosis. Tuberculosis is often difficult to diagnose because it has various clinical features. Although the diagnosis of tuberculosis can be made by the detection of mycobacteria from mycobacterial specimens obtained from suspected tuberculosis lesions, the tuberculosis can not be detected in many cases, and the tuberculosis culture test is time consuming. In addition, clinical, radiological findings and various inspection methods are integrated and diagnosed. The RD1 region, which is highly specific for M. tuberculosis complex and deficient in M. bovis BCG, is 9455 bp long and encodes ESAT6, CFP10 and TB7.7. After growth on a synthetic Sauton medium, ESAT6 and CFP10 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis. Both proteins have recently been shown to induce specific Th1 cell responses in memory immune model mouse after infection with Mycobacterium tuberculosis. Especially, ESAT6 can causes strong antibody responses and delayed type hypersensitivity skin reactions. Aptamers are single-stranded DNA (ssDNA) or RNA which have high affinity to their target molecules. Aptamers can be generated by repetitive selection process known as Systematic Evolution of Ligands by Exponential enrichment (SELEX) methods. Because of unique tertiary structure of aptamers, that can bind specifically to each their targets through hydrogen bonding, hydrophobic and electrostatic interactions, van der Waals forces. Aptamers have many advantages compared with antibody, now that they are oligonucleotides. For examples, aptamers can be synthesized easily at harsh condition, low cost and also have low immune-response in mammals. Electrochemical impedance spectroscopy (EIS) analysis is widely used for high sensitivity, speed, simplicity and low cost. Aptamer immobilized electrode surface can detect tuberculosis antigen within 1 hour and it takes maximum 10 minutes to confirm the results. In this study, we optimized the expression of the M. tb-specific protein ESAT6, CFP10 and TB7.7 in Escherichia coli, followed by purification using fast protein liquid chromatography (FPLC). Aptamers possessing high affinity towards the purified protein were discovered using SELEX. We proposed an aptamer-based electrochemical detection system for tuberculosis by using gold (Au) electrodes, which can become the basis of a POCT for tuberculosis.