Label-free impedimetric detection of Listeria monocytogenes based on poly-5-carboxy indole modified ssDNA probe.
Listeria monocytogenes is a life threatening pathogenic bacteria concerned with human health. The accurate and rapid detection of L. monocytogenes is required for preventing of listeriosis. In this study, DNA sensing probe based on conducting polymer poly-5-carboxy indole (5C Pin) was developed for the detection of L. monocytogenes hlyA gene responsible for pathogenicity. The probe sequences (24 mer ssDNA) were covalently immobilized on 5C Pin via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS). The probe having ssDNA was further hybridized with the target DNA sequence. Electrochemical impedance spectroscopic study was carried out to determine the extent of DNA hybridization over the probe. Significant change was observed in the impedance spectra before and after hybridization of ssDNA immobilized over the probe with the target DNA. RCT (charge transfer resistance) was estimated from the Nyquist plot (impedance plot) for target DNA (hlyA gene) in the solution. RCT vs. logarithmic concentrations of the target (genomic) DNA plot showed a linear range (1 × 10(-4) to 1 × 10(-12)M) in case hybridization was performed under optimized conditions. The method proposed, is simple, free from any label, and highly sensitive for the detection of L. monocytogenes in environmental and clinical samples.
School of Materials Science and Technology, Indian Institute of Technology, Banaras Hindu University, Varanasi 221005, India.
Kashish
Centre of Advanced Study in Botany, Faculty of Science, Banaras Hindu University, Varanasi 221005, India.
Dharmendra Kumar
DK
Soni
Central Instrument Facility Centre, Dr. H.S.G. Central University, Sagar 470003, India.
Sunil Kumar
SK
Mishra
School of Materials Science and Technology, Indian Institute of Technology, Banaras Hindu University, Varanasi 221005, India.
Rajiv
R
Prakash
Centre of Advanced Study in Botany, Faculty of Science, Banaras Hindu University, Varanasi 221005, India. Electronic address: skdubey@bhu.ac.in.
Suresh Kumar
SK
Dubey
D001427:Bacterial Toxins / Q000235:genetics; D015374:Biosensing Techniques; D015342:DNA Probes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D058266:Dielectric Spectroscopy; D004566:Electrodes; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007211:Indoles; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification; D011108:Polymers; D015203:Reproducibility of Results; D013696:Temperature
D001427:Bacterial Toxins; D015342:DNA Probes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D007211:Indoles; D011108:Polymers; C514680:poly(indole-5-carboxylic acid); C038705:hlyA protein, Listeria monocytogenes
Conducting polymer; EIS; Genosensor; Listeria monocytogenes; hlyA gene