Detection of Mycobacterium tuberculosis Hsp16.3 by visual biosensor based on aptamers

XIANG Hui; LI Duo; ZHAO Wei; YUAN Shishan

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Journal of Hunan Normal University(Medical Science) ›› 2025, Vol. 22 ›› Issue (4) : 172-177.

Laboratory Medicine

Detection of Mycobacterium tuberculosis Hsp16.3 by visual biosensor based on aptamers

XIANG Hui, LI Duo, ZHAO Wei, YUAN Shishan

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Abstract

Objective To construct two visual biosensors named as “competitive method” and “sandwich method” based on heat shock protein 16.3(Hsp16.3) aptamer and gold nanoclusters (AuNCs) for detection of Mycobacterium tuberculosis Hsp16.3 and provide reference for diagnosis of latent tuberculosis infection (LTBI). Methods Recombinant Hsp16.3 was induced to express in Escherichia coli BL21(DE3) containing recombinant expression plasmid pQE30-hsp16.3 and purified by metal chelate chromatography. AuNCs were prepared by glutathione reduction and coupled with the amino modified aptamer H66 and aptamer complementary strand H63 SL-2 M6. aptamer H63 SL-2 M6 was fixed on the microplate, and Hsp16.3 was used as the diagnostic antigen. The “competitive” biosensor was established by adding aptamer complementary chain H63 SL-2 M6 modified with AuNCs, and the “sandwich” biosensor was established by adding aptamer H66 modified with AuNCs. The reaction of trisodium citrate reduction method to prepare gold nanoparticles (AuNPs) was introduced as color development reaction. The color change in the preparation process of AuNPs caused by the concentration of hydrogen peroxide (H₂O₂) was used as the output signal. The absorbance value at 550 nm was measured by microplate detector, and the detection limit of the two biosensors was calculated to evaluate the detection performance. Results The conjugated AuNCs retained peroxidase-like activity. After reacting with Hsp16.3, the color reaction of the “competitive method” biosensor appears red; The color reaction of the sandwich biosensor is blue. The linear response range of the two visual biosensors was 100-700 ng/mL, and the detection limit for competition method and sandwich method was 87 ng/mL and 43 ng/mL respectively. Conclusion The two visual biosensors based on aptamers can effectively detect Hsp16.3.

Key words

Mycobacterium tuberculosis / aptamer / Au nanoclusters / heat shock protein 16.3

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XIANG Hui, LI Duo, ZHAO Wei, YUAN Shishan. Detection of Mycobacterium tuberculosis Hsp16.3 by visual biosensor based on aptamers[J]. Journal of Hunan Normal University(Medical Science). 2025, 22(4): 172-177

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