Please use this identifier to cite or link to this item: http://hdl.handle.net/123456789/14516
Title: APTAMERS FOR ADULTERANT DETECTION AND NANOBASED STRATEGIES FOR PATHOGEN CONTROL
Authors: Ramulu, Lambadi Paramesh
Keywords: Increasing Population
Rapid Urbanization
Microbial Pathogens
Toxins Represents
Issue Date: Oct-2014
Publisher: Dept. of Biotechnology iit Roorkee
Abstract: With ever increasing population and rapid urbanization, the food handling system has changed enormously across the globe to meet the demand of the population. Outbreak of diseases due to contamination of food by chemicals, microbial pathogens, and toxins represents a serious threat to human health and imposes economic burden on individuals, families, industries and eventually countries. Therefore, screening of food items meant for human consumption for presence of undesirable species and adulterants are of immense significance in agriculture, industrial and healthcare sectors. Urea is amongst the most commonly used adulterant in preparation of synthetic milk. A chemical biology approach was adopted for generation of urea specific aptamer by performing Flu-Mag SELEX and an aptamer-AuNPs based assay was developed for urea detection. The fluorescence assay demonstrated highest binding affinity for U38 aptamer with a dissociation constant of 238 nM. Circular dichroism study of U38 aptamer showed significant structural changes and an increase in the melting temperature by 9°C with urea, thus confirming stronger binding interactions. Based on the observation of ligand induced structural changes, the U38 aptamer was taken further for the development of an aptamer-AuNP based optical detection assay for urea in milk. A simple, rapid and user friendly method for milk sample preparation by methanol precipitation was standardized. Regions of U38 aptamer interacting with urea were determined by carrying out truncation studies, which highlighted the role of hair-pin motif in the forward region to be responsible optimum performance for the aptamer-gold nanoparticles assay. The aptamer-AuNPs biosensor developed in this study was specific for urea as compared to structural analogs viz glycine, alanine, serine and tyrosine. A urea dependent fluorescence recovery of gold nanoparticle quenched FITC-U38 aptamer further confirmed the specificity of the assay. In presence of 100 mM urea 85.5% fluorescence was recovered, while no significant recovery was observed with 100 mM glycine, confirming the specificity of the U38 aptamer for urea. Further, the robustness of the developed assay for other adulterants added to milk was evaluated and it was found to be unaffected in presence of salt (500 μM), sodium bicarbonate (20 mM), glucose (600 μM) and tween-20 (1%). The aptasensor demonstrated a linear range of detection of 20-150 mM, with a lower visual detection limit of 50 mM. While the method offers advantages of being rapid, simple and specific for detecting high concentration of urea in milk, it can be further improved for increasing the sensitivity. This method can also be adapted for detecting urea in clinical samples since urea is a pathological marker in several renal associated diseases. II Apart from adulteration of food, diseases caused by foodborne pathogens are worldwide problem. E. coli O157:H7 is declared by Centre for Disease Control and Prevention (CDC) amongst the most dreadful foodborne pathogen, causing haemolytic uremic syndrome, characterized by bloody diarrhea. E. coli O157:H7 is known to produce type IV pilus (TFP) called as hemorrhagic pilus (HCP), which plays a central role in attachment to the mucosal epithelial cells. Amplification of hcpA locus from E. coli O157:H7 genomic DNA was performed and was cloned in pET28(a) vector. The expressed His-tagged protein His-HcpA was immobilized to the nickel NTA agarose beads for carrying out an affinity chromatography based SELEX for the selection of HcpA specific aptamers. An initial binding study of different selection round by electrophoretic mobility shift assay (EMSA) demonstrated binding in 10th round population. Further cloning and sequencing of the 10th round of population was done and sequences were grouped in to different categories. Based on the structural folding, eight putative aptamers were selected based on the stability in terms of free energy. Preliminary binding study for the putative aptamers by EMSA demonstrated binding of HcpA-38, 41, 64 and 75 aptamers with target HcpA protein. Further, the best binding aptamer sequences can be coupled to different transducing elements like gold nanoparticles, single walled carbon nanotubes for detecting E. coli O157:H7. Bacterial infections are not only limited to foodborne diseases, but also have been extensively associated with nosocomial infection due to surgical devices and medical implants. Excessive use of antibiotics in hospitals has led to the emergence of antibiotic resistance among different nosocomial bacteria. Since the pace of conventional drug discovery is slow, there is urgent need for devise new strategies to kill or curb the growth of the antibiotic resistance microbes. In the present study, silver nanoparticles were bio-functionalized with polymyxin B antibacterial peptide using a facile method. The bio-functionalized nanoparticles (PBSNPs) were assessed for antibacterial activity against multiple drug resistant clinical strain Vibrio fluvialis and Pseudomonas aeruginosa. The results of antibacterial assay revealed that PBSNPs had ~3 fold higher antibacterial effect than the citrate capped nanoparticles (CSNPs). Morphological damage to the cell membrane was studied by scanning electron microscopy, testifying PBSNPs to be more potent in controlling the bacterial growth as compared to CSNPs. Bactericidal effect of PBSNPs was further confirmed by Live/Dead staining assays. Apart from the antibacterial activity, the bio-functionalized nanoparticles were also found to resist biofilm formation. Electroplating of PBSNPs onto stainless steel surgical blades retained the antibacterial activity against Pseudomonas aeruginosa. Further, the affinity of polymyxin for endotoxin was exploited for its neutralization using PBSNPs. It was found that the prepared III nanoparticles removed 97% of the endotoxin from the solution. Such multifarious uses of metal nanoparticles can be explored as viable means of enhancing the potency of antimicrobial agents to control infections.
URI: http://hdl.handle.net/123456789/14516
Appears in Collections:DOCTORAL THESES (Bio.)

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