Synthesis, characterization and bio-functionalization of magnetic nanoparticles to improve the diagnosis of tuberculosis

Abstract

Mycobacterium tuberculosis is the cause of one of the diseases with the highest mortality and morbidity rate in the Americas and in the world. In developing countries, the diagnosis of tuberculosis (TB) is based on baciloscopy and bacteriological cultures. The first method has a low sensitivity, and the second can take several weeks to reach a confirmatory diagnosis. The lack of a rapid diagnosis compromises the efforts to control this disease and favors the transmission of tuberculosis to the susceptible population. In this work, we present the synthesis, amine-silanization, characterization and bio-functionalization of magnetic nanoparticles (MNPs) to develop a sandwich ELISA to detect and concentrate antigens from M. tuberculosis. For this purpose, a recombinant mycobacterial heat shock protein Hsp16.3, which contributes to the persistence of TB, was cloned and expressed in the E. coli system. Polyclonal antibodies anti-Hsp16.3 were produced in a rabbit and in mice. Magnetic nanoparticles were synthesized by co-precipitation, amine-functionalized and characterized by several physical-chemical methods. The XRD, Mossbauer spectroscopy, zeta potential, TEM, and FTIR all proved the successful preparation of the MNPs showing a diffraction crystal diameter of 10.48 ± 2.56 nm, superficial net charge of : +23.57 ± 2.87 mV, characteristic patterns of magnetite and a structure similar to a sphere. Additionally, it showed a magnetization saturation of 37.06 emu.g−1. For the functionalization of nanoparticle surfaces with anti-Hsp16.3, the active ester method was used for bond formation, and parameters such as time of incubation, coupling agents ratio (EDC/NHS) and concentration as well as surface saturation level of amine-silanized MNPs (MNP@Si@NH2) were standardized. Finally, bio-functionalized MNPs were used to detect, fix and concentrate the recombinant antigen Hsp16.3 from M. tuberculosis in a sandwich ELISA-MNP assay.

Assessment of Gold Bio-Functionalization for Wide-Interface Biosensing Platforms

“…The assessment of sensor surface modification is fundamental when developing a biolayer-based device since the properties of the biorecognition element regarding its orientation, surface density, and activity toward the binding analyte can dramatically influence the assay analytical performances [ 41 , 42 ]. In SPR direct assays, the typical explored analyte concentration returns limit of detections in the 1–10 nM range [ 43 , 44 ], with further improvement only by using nanostructures or sandwich assays [ 45 ].…”

Study of the Stability of Citrate Capped AgNPs in Several Environmental Water Matrices by Asymmetrical Flow Field Flow Fractionation

“…These characteristics, convert NPs, specifically AgNPs, into tools used as potential applications in a wide variety of fields [9][10][11][12][13][14][15]. Moreover, and despite the benefits that their use can generate for the diagnosis of diseases [15,16] or their treatment by acting as anticancer or antimicrobial agents [17], NPs also appear on the scene as potential emerging pollutants whose effects on human health [9,18,19] and the environment are not yet fully known [20][21][22].…”

Is interleukin-2 an optimal marker for diagnosing tuberculosis infection? A systematic review and meta-analysis

“…However, IGRA frequently has indeterminate results in children, especially in those less than 5 years old [18]. Clinically, positive Mtb culture is a gold standard for diagnosing ATB [19]. However, culture is time wasting, which takes around six weeks for truly negative detection [20].…”