Tuberculosis (TB) is a major fatal infectious disease caused by Mycobacterium tuberculosis (MTB). The current World Health Organization (WHO) figures estimate a worldwide TB incidence of 10.4 million with 1 million with HIV per year and 1.7 million deaths annually (WHO 2018 report). The diagnostic approaches used today rely on tests that are slow, expensive and are lab-constrained lacking sometimes sensitivity and specificity. The only reliable diagnostic approach being is the GeneXpert™ technology, which though is specific and sensitive, is carried out on a huge lab-restricted equipment and need trained personnel to operate, hence cannot be outreached.
Current research focused on miniaturizing the whole laboratory setup for TB diagnostics on a microfluidic chip. The chip have three compartments, the first compartment has a short DNA sequence (aptamer) that detects the specific early proteins excreted from the TB bacteria. The second and third compartments have the proteins that will detect the Multi Drug resistance (MDR) and Extensive Drug resistance (XDR) respectively. These are the different types of TB disease based on resistance of the bacteria to set of drugs used against the infection. The sample that is tested on the chip is sputum of the diseased individual. We have already developed electrochemical aptasensor for plain TB detection and tested it on clinical samples.
a.Working electrode 1 (W1): 5’-(NH3) IS6110, b.Working electrode 2 (W2): (Katg315-INH), c.Working electrode 3 (W3): inhA promoter INH, d.Working electrode 4 (W4): rpoB-RIF, e.Working electrode 5 (W5): embB-EMB, f.Working electrode 6 (W6): A1401G- RRS-SLDs, g.Working electrode 7 (W7): EIS-SLDs h.Working electrode 8 (W8): gyrA-FLQ, i.Counter electrode, j.Dielectric film, k.Reference electrode, l.Connectors, m. Base template
In this pursuit, another alternative, that is published in International journal of nanomedicine, we used graphene oxide-chitosan nanocomposite (GO-CHI), which is a biocompatible matrix, and has a large electroactive area with an overall positively charged surface. The obtained GO-CHI nanocomposite is then immobilized on the ITO surface to form a positively functionalized electrochemical sensor for the detection of Mtb. DNA probe, specific for the IS6110, was electrostatically anchored on a positively charged electrode surface and the resistance of charge transfer was investigated for the sensitive and specific (complementary vs non-complementary) detection of Mtb by cyclic voltammetry and differential pulse voltammetry techniques.
Gel electrophoresis of four Mtb DNA samples used in the study run in duplicate, confirming the presence of 1S6110.
A DNA genosensor was developed for the rapid detection of tuberculosis using indium tin oxide (ITO) slides as the electrode base. The ITO slides were coated with a graphene oxide-chitosan nanocomposite (GO-CHI) to enhance conductivity and impart an overall positive charge to the electrode surface. The GO-CHI nanocomposite was prepared by mixing graphene oxide derived from graphite flakes with chitosan. This nanocomposite was uniformly applied to the ITO surface, providing an effective platform for the binding and immobilization of single-stranded DNA (ssDNA) probes.
The DNA genosensor exhibited specificity in distinguishing between non-complementary DNA and complementary DNA (IS6110) specific to Mycobacterium tuberculosis. The immobilization of the ssDNA probe on the positively charged surface of the electrode facilitated the detection of target DNA associated with tuberculosis. This genosensor demonstrates a potential application for the rapid and specific detection of Mycobacterium tuberculosis, offering a promising tool for early diagnosis and monitoring of tuberculosis infections. The obtained results also suggested that the GO-CHI based electrochemical DNA genosensor could distinguish among the pathogenic strengths of raw sputum samples. Hence, it clearly indicates that GO-CHI based electrochemical biosensor can be regarded as a successful candidate for tuberculosis point-of-care medical diagnosis.
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