title: 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 10(3) 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 10(3) 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.
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Lianhua
authors.initials: L
authors.lastname: Qin
authors.affiliation: Shanghai East Hospital, The Institute for Biomedical Engineering and Nano Science, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Yilong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Shanghai East Hospital, The Institute for Biomedical Engineering and Nano Science, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Bingbo
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Ma
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Junmei
authors.initials: J
authors.lastname: Lu
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Xiaochen
authors.initials: X
authors.lastname: Huang
authors.affiliation: Shanghai East Hospital, The Institute for Biomedical Engineering and Nano Science, Tongji University School of Medicine, Shanghai, People's Republic of China ; The Materials Science and Engineering Program, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, OH, USA.
authors.forename: Donglu
authors.initials: D
authors.lastname: Shi
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
authors.forename: Zhongyi
authors.initials: Z
authors.lastname: Hu
mesh_terms: D002352:Carrier Proteins; D015169:Colony Count, Microbial; D006801:Humans; D006867:Hydrolases / Q000302:isolation & purification*; D057230:Limit of Detection; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D008863:Microspheres; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification*; D045663:Quantum Dots; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis*
chemical_list: D002352:Carrier Proteins; D058185:Magnetite Nanoparticles; D006867:Hydrolases; C527647:HsaD protein, Mycobacterium tuberculosis
keywords: Mycobacterium tuberculosis; binding peptides; detection; magnetic microspheres; phage display; quantum dots