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Published on: May 28, 2012
Development of DNA aptamers towards detection of tuberculosis biomarker Ag85B in a fluorescence-based sensing
Satakshi Hazra1, Manish Gupta2, Rakesh Bhatnagar2
1Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
Background:
Timely diagnosis of tuberculosis (TB) remains a critical challenge, highlighting the need for better screening tools. Traditional antibody-based detection methods for TB are often costly and cumbersome. To address this, we developed a streamlined centrifugal SELEX approach using a 69-nucleotide DNA library and the recombinant TB biomarker Ag85B, towards fabrication of an aptasensing platform offering a simpler and faster alternative.
Results:
Two high affinity DNA aptamers were screened through 12 rounds of SELEX and verified with in silico docking, circular dichroism spectroscopy and electrophoretic shift assays for binding interactions with Ag85B. The aptamer with highest binding affinity (KD values 76.36 ± 10.76 nM in binding buffer and 86.62 ± 6.20 nM in spiked serum) was used for fabrication of a fluorescence based aptasensing platform using graphene oxide as a quencher. The aptamer demonstrated specificity towards Ag85B without interference from two other recombinant TB proteins MPT64 and ESAT6. The aptasensing platform offered limits of detection of 5.83 nM in binding buffer and 6.51 nM in spiked serum.
Significance:
This work developed a modified SELEX approach combining a centrifugal filter and streptavidin-biotin magnetic separation technique for isolation of DNA aptamers. We report for the first time, a DNA aptamer against Ag85B biomarker that holds high prospects for clinical applications in diagnosing TB.
