Detection of Mycobacterium tuberculosis based on H37Rv 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 H 37 R 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.

Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP

“…tuberculosis associated with magnetic beads has been employed, including sandwich detection using functionalized magnetic microspheres and quantum dots conjugated with specific antibodies (Yang et al . ) and direct detection using TB beads coated with polymeric ligand and auramine‐O fluorochrome staining (Wilson et al . ; Albert et al .…”

“…The detection of Myco. tuberculosis associated with magnetic beads has been employed, including sandwich detection using functionalized magnetic microspheres and quantum dots conjugated with specific antibodies (Yang et al 2015) and direct detection using TB beads coated with polymeric ligand and auramine-O fluorochrome staining (Wilson et al 2010;Albert et al 2011). Herein, anti-Ag85B mAb clone AM85B-8 specific to mycobacterial Ag85B protein coupled with magnetic beads were facilitated for selective separation of Mycobacterium spp.…”

Diagnosis of Tuberculosis: Nanodiagnostics Approaches

“…Due to these advantages, SPR has emerged as a powerful optical tool, which can provide valuable data in the analysis of biomedical and chemical analyses. The SPR-based CFP-10 antigen detection system was developed in clinical samples by Yang et al (2014), which showed reputable usefulness in TB diagnostics (Hsieh et al 2012;Yang et al 2014). Zhu et al (2017) developed AuNPs modified indium tin oxide (ITO) electrode for the direct detection of M. tuberculosis using genomic DNA (gDNA) isolated directly from clinical samples.…”

Advantages and limitations of nanoparticle labeling for early diagnosis of infection

“…[96][97][98] One particularly interesting example of the utility of QDs also took advantage of the separation/concentrating characteristics of MNPs to develop a method to detect Mycobacterium tuberculosis (Mtb) biomarkers in the sputum of infected patients. [99] The intent was to take advantage of the optical strengths of QDs -high quantum yields and low fluorescent NPs offer many sought-after advantages in expanding the fluorescent toolbox, such as robustness, versatility, and synthetic simplicity. However, like QDs, there are still questions regarding the toxicity of these NPs and the reagents used in their synthesis.…”