Quorum sensing (QS), a communication system involved in virulence of pathogenic bacteria like Pseudomonas aeruginosa is a promising target to combat multiple drug resistance. In vitro studies using clove bud oil (CBO) in P. aeruginosa revealed a concentration dependent attenuation of a variety of virulence factors including motility, extracellular DNA, exopolysaccharides and pigment production. Furthermore, treatment with CBO demonstrated a distinct dose-dependent reduction in biofilm formation as well as promoting dispersion of already formed biofilm, observations that were also supported by porcine skin ex vivo studies. Expression studies of genes involved in signalling systems of P. aeruginosa indicated a specific decrease in transcription of pqsA, but not in the lasI or rhlI levels. Additionally, the expression of vfr and gacA genes, involved in regulation, was also not affected by CBO treatment. CBO also influenced the PQS signalling pathway by decreasing the levels of kynurenine, an effect which was reversed by the addition of exogenous kynurenine. Though the synthesis of the signalling molecules of the Las and Rhl pathways was not affected by CBO, their activity was significantly affected, as observed by decrease in levels of their various effectors. Molecular modelling studies demonstrated that eugenol, the major component of CBO, favourably binds to the QS receptor by hydrophobic interactions as well as by hydrogen bonding with Arg61 and Tyr41 which are key amino acid residues of the LasR receptor. These results thus elucidate the molecular mechanism underlying the action of CBO and provide the basis for the identification of an attractive QS inhibitor.
Sandwich type voltammetric immunosensors were developed for the quantitative determination of cancer biomarkers, HER2 (human epidermal growth factor receptor 2), and CA125 (cancer antigen 125). Specific antibodies were immobilized on the gold electrodes by using a self-assembled monolayer of mercaptoundecanoic acid (MUA) activated by EDC-NHS ((1-ethyl-3-(3-dimethylamino propyl) carbodiimide and N-hydroxysuccinimide). Antibody conjugated gold nanoparticles decorated reduced graphene oxide (anti-HER2-Au/rGO and anti-CA125-Au/rGO) were used as enzyme-free nano labels for signal amplification. The sensors were characterized thoroughly by spectroscopic, microscopic, and electrochemical methods. Differential pulse voltammetric (DPV) studies in the presence of thionine revealed that the current signal produced is directly related to the biomarker concentration. A linear range of 0.2 pg mL-1 to 100 ng mL-1 was obtained, which indicated the immunosensors to be highly sensitive in lower concentrations of cancer biomarkers. Finally, the fabricated immunosensors were tested with serum samples spiked with HER2 and CA125. The results obtained from the test confirmed the data collected from clinical labs.
This study describes the development and testing of a simple and novel enzyme-free nanolabel for the detection and signal amplification in a sandwich immunoassay. Gold nanoparticles decorated reduced graphene oxide (rGOAu) was used as the nanolabel for the quantitative detection of human immunoglobulin G (HIgG). The rGOAu nanolabel was synthesised by one pot chemical reduction of graphene oxide and chloroauric acid using sodium borohydride. The pseudo-peroxidase behaviour of rGOAu makes the nanolabel unique from other existing labels. The immunosensing platform was fabricated using selfassembled monolayers of 11-mercaptoundecanoic acid (11-MUDA) on a gold disc electrode. The covalent immobilisation of antibody was achieved through the bonding of the carboxyl group of 11-MUDA and the amino group of the antibody using chemical linkers [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and N-hydroxysuccinimide. The fabricated immunosensor exhibited a linear range that included HIgG concentrations of 62.5-500 ng ml −1. The sensor was also used for the testing of HIgG in the blood sample.
An electrochemical immunosensing platform based on dual signal amplification strategy has been developed using gold nanoparticles. Human Immunoglobulin G was used as a model analyte in order to establish the immunosensing platform. The platform was fabricated using of 1,6-hexanedithiol self assembled on a gold disc electrode and was further modified by citrate capped gold nanoparticles. The direct immobilization of antibody was achieved through electrostatic interaction between negatively charged citrate capped gold nanoparticles and positively charged amino group of antibody. Each step of modification was analyzed using electroanalytical techniques like cyclic voltammetry and electrochemical impedance spectroscopy. The horseradish peroxidase (HRP)-labeled secondary antibodies conjugated on gold nanoparticles (AuNP-Ab2) acted as nanolabels. Thus the sandwich immunocomplex formed on the electrode surface produced an electrocatalytic response through the reduction of hydrogen peroxide by HRP in the presence of thionine. Electrochemical studies were carried out to understand the role of citrate capped AuNP and AuNP-Ab2 in dual signal amplification. The fabricated sensing platform can be used for the sensitive determination of various protein biomarkers by immobilizing specific antibody.
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