Genosensor for the diagnosis of rheumatic heart disease

Abstract

newline x newlineABSTRACT The present study was carried out for the development of nano composite based biosensor for the instant diagnosis of rheumatic heart disease caused by Streptococcus pyogenes with more accuracy and sensitivity. Streptococcus pyogenes infection in human causes initially pharyngitis due to inhalation of aerosols emitted by infected persons and develops rheumatic fever which leads to rheumatic heart disease causing permanent damage of heart valves. The available detection methods are bacterial culture, and#946;-hemolysis, bacitracin sensitivity, hippurate test, phadebact test, CRP (C-reactive protein), ESR and PCR. All these methods are either expensive or non-confirmatory and have some limitations. This study was performed by development of PCR based genetic marker, SPGE based biosensor, nanosensor and nano composite based biosensor for the detection of S. pyogenes. In first experimental set up the PCR based detection of S. pyogenes in human was evaluated using specific primers of speB gene. Streptococcus pyrogenic exotoxin gene (speB) is chromosomally encoded pyrogenic and cardiotoxic virulence factor of particular bacteria. The present reported PCR based genetic marker for the detection of S. pyogenes in human completes overall analysis in 80 min including electrophoresis which is the minimum time reported so far for the confirmation of this disease. Amplicon of 423bp of speB gene obtained in present study can be used as a specific genetic marker as it does not show homology with other organisms for early detection of rheumatic heart disease. In next objective further, speB genosensor was fabricated for early detection of S. pyogenes in human throat swab samples using screen printed modified gold electrode. The electrochemical changes before and after hybridization was measured using cyclic voltammetry (CV) in the presence of redox indicator. The sensitivity of the genosensor was observed to be 80 and#956;A/cm2/ng and lower limit of detection (LOD) was 0.10 ng/6and#956;l with the regression coefficient (R2) of 0.921 using CV. The sensor was highly specific, simple and takes only 30 min for detection of S. pyogenes infection. The sensor was further characterized by atomic force microscopy (AFM). Genosensor was found stable upto 6 months with only 10% loss in initial peak current in CV analysis on storage at 4oC. newlinexi newlineIn further study, for the fabrication of nanosensor a specific 5´ NH2 labeled speB gene based DNA probe was immobilized onto the gold nanoparticles/ carboxylated multi walled carbon nanotubes (Nano-Au/cMWCNT) screen printed electrode using EDC/NHS cross linking chemistry. This was followed by hybridization with 0.5-50 ng/6and#956;l of Streptococcus pyogenes single standard genomic DNA (ssG-DNA) from patient throat swab samples. The sensitivity of the sensor was found to be 104.7 and#956;A cm-2 ng-1 using CV with a regression coefficient (R2) of 0.907 and LOD was 0.01 ng/6and#956;l. The modified surface morphology was characterized by scanning electron microscopy (SEM). This modified electrode was found stable for 6 months at 4°C with only 6% loss in the initial current. This sensor is again gene specific and can detect the pathogen within 30 min. To further increase the sensitivity a nano-Au/-MWCNT/ATP/GQD composite based sensor was developed. The sensitivity (S) of the sensor was found to be 1095.2 and#956;A cm-2 ng-1 and the limit of detection (LOD) was 0.2pg/6 and#956;l. The sensitivity and LOD of this nanocomposite based biosensor is very high in comparison to the previous developed techniques. In summary, the developed nanocomposite based biosensor is the new innovative device for the diagnosis of Rheumatic Heart Disease and can detect the particular disease in 15 min. Keywords: Rheumatic heart disease, Streptococcus pyogenes, speB gene, speB genosensor, Screen printed gold electrode, Nano-Au/MWCNT, Nano-Au/MWCNT/ATP/GQD composite.