Development of Genesensors for Pathogenic Bacteria based on ssDNA probes

Abstract

1 Electrochemical devices have played a major role in development of biosensors. Such devices are capable for simple, inexpensive, accurate and sensitive sensing in diagnostics field. Recently with advent of functional polymers and nanotechnology, we see use of functional conducting polymers and nanomaterials like nano metals, metal oxides and nanocomposites in modification of sensing probes and in enhancement of catalytic activity of the transducers. Conducting polymers got much more attention due to their conductivity as well as excellent matrix for immobilization of biomolecules. Similarly, metal nanomaterials are emphasized and used; due to its large surface energy provides the absorption of large number of small biomolecules, ease of functionalization, greater sensitivity and catalysis in bio-reactions with fast electron transfer. Metal nanoparticles are used as probe in detection and imaging of biomolecules. In certain cases nanoparticles are functionalized and made biocompatible to specifically and efficiently bind to a target analyte. Biocompatible nanomaterials are basically composite of metal nanoparticles and biopolymers like chitin, chitosan providing functionality for attaching the biomolecule. In the biosensors, stability, sensitivity and reproducibility are key factors and mainly depends on immobilization. Therefore, we focused on our research work on development of a stable sensing platform and second immobilization based on chemical binding with conducting polymer as well as nanoparticles dispersed polymer matrixes for water & food borne pathogens based on DNA as sensing species. Further, in view of need of portable, low cost sensors for onsite measurements, we made efforts for development of screenprinted electrodes using the same conditions as 2 developed for the conventional sensor probes. We demonstrated sensing for pathogens responsible for toxicity in food and water. We focused on sensing of food and water borne pathogens for food safety reasons. Food borne pathogens which cause serious health illness after infection includes Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Clostridium perfringens, Yersinia enterocolitica and Escherichia coli etc. Prior to treatment their diagnosis is necessary at early stage. A genosensor based on conducting polymer poly-5-carboxy indole (5C Pin) was developed for the detection of Listeria 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). Electrochemical impedance spectroscopic study was carried out to determine the extent of DNA hybridization over the probe. 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. Further, we developed of a simple and cost-effective genosensor probe based on a glassy carbon electrode modified with platinum nanomaterials dispersed in a chitosan matrix. This probe was explored for the label-free detection of Listeria monocytogenes obtained from milk samples. DNA-based interfacial interaction between target DNA and platinum nanomaterials (PtNPs) immobilized with 24 mer ssDNA was investigated using impedance spectroscopy. This user-friendly and simple platform was used for the detection of target DNA and shows excellent response and specificity (even for 1-bp mismatch of target DNA). Also, this sensing platform was utilized for the detection of Listeria monocytogenes in real samples (milk beverage) and had a 3 wide range of detection from 1 X10-12 M to 1 X 10 -4 M. Based on our stable nanomaterials sensing platforms, further E-coli sensors was developed based on ss-DNA of Escherichia coli St gene. Sensor is developed based on glassy carbon and conducting carbon ink screen printed electrodes decorated with platinum nanoparticles @ chitosan (PtNPs@CS) and immobilized with a specific oligonucleotides (ssDNA) for convenient and sensitive determination of Escherichia coli (E. coli) DNA. The relative change of RCT is used as sensing parameter for detection of E. coli DNA. Limit of detection for E. coli DNA is calculated as 3.2 X 10-14 M. The combination of our stable ssDNA probe with EIS provides a label-free, specific and user-friendly biosensing system. We successfully developed novel platforms for genosensors based on conducting polymer chemically coupled with ssDNA and nanoparticles dispersed in chitosan polymer physically immobilized with ssDNA. Both the platforms were found stable and demonstrated for sensing of two different pathogens. Further, the platforms were miniaturized by using screen printed electrode for low cost and on site detection in real samples. Our sensing platform and detection techniques showed enormous potential for development of sensitive, low cost portable sensors.