Abstract

Tuberculosis (TB) remains one of the top 10 causes of death worldwide. It is an infectious disease caused by the bacillus Mycobacterium tuberculosis (MTB). It typically affects the lungs (pulmonary TB) but can also affect other sites (extrapulmonary TB). It spreads when people who are ill with pTB expel MTB into the air by coughing, sneezing or spitting. In 2015, 10.4 million people fell ill due to TB and 1.8 million died. Many lives were saved through TB diagnosis and treatment, but gaps persist. There are two kinds of tests for detecting MTB infection: TB skin test (TST) and TB blood tests, but a positive TST or TB blood test does not tell whether the person has latent TB infection (LTBI) or progressed to TB disease. We hypothesized that developing a microfluidic device (MFD) for capturing MTB from sputum samples of people with pTB can differentiate people with pTB from those having LTBI. Here we developed an aptasensor (aptamer-based sensor) by making use of microfluidic technologies and bioconjugate techniques for capturing MTB efficiently for POC diagnosis of pTB. We used a single-stranded DNA (ssDNA) aptamer that recognizes the cell surface mannose-capped lipoarabinomannan (Man-LAM) antigen of MTB. We immobilized this aptamer on micropillars present in the MFD to develop it into an aptasensor. These micropillars act as passive mixers for creating turbulence flow inside the MFD due to which MTB comes in contact with the aptamer coated micropillars and gets captured. Validation of the aptasensor bio-functionalization is carried out with horseradish peroxidase (HRP) enzymatic reaction as well as acridine orange staining, and capturing of the 4',6-diamidino-2-phenylindole stained MTB H37Ra strain is validated by observing the MFD under a fluorescence microscope. In this way, the concept of microfluidics-based POC diagnosis of pTB by capturing MTB was achieved.