Detection of Mycobacterium tuberculosis based on H₃₇R_v binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis

Abstract

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl–Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10³ colony forming units (CFU)/mL H₃₇R_v was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10³ CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.

Keywords:

• Mycobacterium tuberculosis
• phage display
• binding peptides
• magnetic microspheres
• quantum dots
• detection

Acknowledgments

The authors would like to thank Dr Malcolm Duthie, from Infectious Disease Research Institute, Seattle, USA for helpful revisions and comments regarding this manuscript.

The work was supported by the grant from the National Key Project for Infectious Disease (No 2012ZX10003002-008), the Science and Technology Commission of Shanghai Municipality, Shanghai, People’s Republic of China (No 11nm0506100, 124119a1500), the National Natural Science Foundation of China (No 51173135, 81301391), and the Fundamental Research Funds for the Central Universities (No 1511219013).

Disclosure

The authors have no conflicts of interest to disclose.