Detection of Pathogenic Mycobacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation

Abstract

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 104 bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples.

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

“…In the LOD test, it was found that 10 3 CFU/mL MTB was detectable even in sputum matrices without biotin-streptavidin amplification, as was used in Liandris et al's research. 22 Specificity and sensitivity of MTB detection was found, in this research, to be dependent on the combinations of ligands and nanoparticles. The highest specificity and sensitivity was found for the combinations of MMS-Pab+QD-H8 and MMS-H8+QD-H8.…”

“…22 The major principle is based on the separation of bacteria using magnetic beads coupled with genus-specific Pab and mAb for heparin-binding hemagglutinin. These complexes were then sequentially tagged with anti-mouse biotinylated antibody and streptavidin conjugated QDs for detection of the fluorescent signal.…”

“…Earlier studies showed detection of unamplified mycobacterial DNA and pathogenic mycobacteria by using fluorescent QDs and magnetic beads, suggesting the possibilities of similar nanotechnologies in TB diagnosis. 21,22 Recently, Foddai et al reported an IMS method for Mycobacterium avium subspecies paratuberculosis. 23 They obtained maximal capture of M. avium subspecies paratuberculosis from the broth, bovine milk, and fecal specimens by using the two phage display derived peptide ligands, which was initially developed by Stratmann et al 24,25 They also evaluated a series of bead types against the antibodies and peptide ligands studied, and found that capture efficiencies are related to the bead-ligand combinations.…”

“…In Liandris et al's research, samples of bacteria were coupled with several biological molecules and nanoparticles, sequentially: the genus specific Pab conjugated MMSs; the heparin-binding hemagglutinin (HBHA) mAb; an anti-mouse biotinylated antibody; and finally the streptavidin-coated QDs. 22 Although the final signal was amplified four times with the biotin-streptavidin reaction, these four steps render it too complex for routine and expanded use. In this study, it was found feasible to conjugate the MMSs and QDs with the ligands that can bind MTB cells directly.…”

Immunomagnetic nanoparticle-based assays for detection of biomarkers

“…Specifically, the capture moieties detect the desired target (ie, protein biomarker, DNA, RNA) for subsequent separation from the remaining solution via a simple magnet, and consequently, for various methods of analyses. [2][3][4][5][6] The noninvasiveness and simple nature of this method continues to enable the widespread use of magnetic particles. Furthermore, the high surface-area-to-volume ratio of the particles confers a correspondingly high probability of interaction with target biomarkers, and essentially increases the efficiency of the system.…”