Biotechnology Research at Amrita Vishwa Vidyapeetham

An electrochemical immunosensing platform capable of detecting Immunoglobulin G (IgG) concentration as low as femtograms was developed based on signal amplification strategy. The immunosensing platform was fabricated using self-assembled monolayers (SAM) of 11- mercaptoundecanoic acid (MUDA) on a gold disc electrode. The covalent immobilization of antibody was achieved through the bonding of a carboxyl group of MUDA and amino group of antibody using [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] (EDC) and N-hydroxysuccinimide (NHS) chemistry. The formation of SAM and antibody immobilization was analyzed using cyclic voltammetry and electrochemical impedance spectroscopy. The gold nanoparticles conjugated with horseradish peroxidase-labeled secondary antibodies were used as nanolabels, to increase the sensitivity and catalytic efficiency of the immunosensor. The sandwich immunocomplex formed on the electrode surface produced an electrocatalytic response through the reduction of hydrogen peroxide in the presence of thionin. The fabricated immunosensor exhibited two linear ranges that included IgG concentrations of 10 fg mL−1 to 0.1 ng mL−1 and 0.1 to 100 ng mL−1 respectively.

Nontuberculous mycobacterial (NTM) species present a major challenge for global health with serious clinical manifestations ranging from pulmonary to skin infections. Multiomics research and its applications toward clinical microbial proteogenomics offer veritable potentials in this context. For example, the Mycobacterium abscessus, a highly pathogenic NTM, causes bronchopulmonary infection and chronic pulmonary disease. The rough variant of the M. abscessus UC22 strain is extremely virulent and causes lung upper lobe fibrocavitary disease. Although several whole-genome next-generation sequencing studies have characterized the genes in the smooth variant of M. abscessus, a reference genome sequence for the rough variant was generated only recently and calls for further clinical applications. We carried out whole-genome sequencing and proteomic analysis for a clinical isolate of M. abscessus UC22 strain obtained from a pulmonary tuberculosis patient. We identified 5506 single-nucleotide variations (SNVs), 63 insertions, and 76 deletions compared with the reference genome. Using a high-resolution LC-MS/MS-based approach (liquid chromatography tandem mass spectrometry), we obtained protein coding evidence for 3601 proteins, representing 71% of the total predicted genes in this genome. Application of proteogenomic approach further revealed seven novel protein-coding genes and enabled refinement of six computationally derived gene models. We also identified 30 variant peptides corresponding to 16 SNVs known to be associated with drug resistance. These new observations offer promise for clinical applications of microbial proteogenomics and next-generation sequencing, and provide a resource for future global health applications for NTM species.