Rapid detection of Salmonella enterica in raw milk samples using Stn gene-based biosensor

Abstract

In this study, a DNA-based nanosensor using specific NH₂ labeled single standard probe was developed against stn gene of Salmonella enterica in milk samples. The single-stranded DNA probe was immobilized on carboxylated multiwalled carbon nanotube and gold nanoparticle (c-MWCNT/AuNP) electrode using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC): N-hydroxy succinimide-based cross-linking chemistry. Electrochemical characterization was performed using cyclic voltammetry (CV) and Differential Pulse Voltammetry (DPV) techniques. The electrode surface at each step of fabrication was characterized using scanning electron microscopy. The sensitivity and lower limit of detection were found to be 728.42 (μA/cm²)/ng and 1.8 pg/6 μl (0.3 pg/ml), respectively, with regression coefficient (R ²) of 0.843 using DPV. The sensor was further validated using raw and artificial milk samples, and results were compared with conventional methods of detection. The developed sensor was found to be highly sensitive and stable up to 6 months, with only 10% loss of initial peak current in CV analysis on storage at 4 °C.

Keywords: Electrochemical biosensor; Foodborne illness; Salmonella enterica; stn gene.

Scheme 1

Fabrication of electrochemical sensor using immobilization of 5′-NH₂ labeled ss-DNA probe of stn gene on c-MWCNT and hybridization with ssG-DNA of S. enterica

Fig. 1

CV of (a) ss-DNA (probe) (b–l) hybridization with 0.0976–100 ng/6 μl of S. enterica ssG-DNA at 50 mVs-1 using 1 mM MB in 50 mM PBS, pH 7.4. The inset-A shows a hyperbolic curve from 0 to 100 ng/6 μl with linear peak current (Ip) 0–0.19 ng/6 μl of ssG-DNA of S. enterica. Inset-B shows the linear plot from 0 to 0.19 ng/6 μl ssG-DNA for the calculation of sensitivity and LOD

Fig. 2

DPV of (a) ss-DNA (probe) (b–k) hybridization with 0.05–30 ng/6 μl of S. enterica ssG-DNA at 50 mVs-1 using 1 mM MB in 50 mM PBS, pH 7.4. The inset-A shows a hyperbolic curve from 0 to 30 ng/6 μl with linear peak current (Ip) 0.05–0.11 ng/6 μl of ssG-DNA of S. enterica. Inset-B shows the linear plot from 0.05–0.11 ng/6 μl ssG-DNA for the calculation of sensitivity and LOD

Fig. 3

Surface characterization of electrodes used in electrochemical biosensor fabrication for S. enterica. SEM micrograph of a c-MWCNT/AuNP b ss-DNA/c-MWCNT/AuNP and c dsDNA/c-MWCNT/AuNP after hybridization with 100 ng/6 μl ss-GDNA of S. enterica

Fig. 4

Specificity of the fabricated c-MWCNT/AuNP sensor for S. enterica and other foodborne pathogens. The relative Ip value of CV (concerning immobilized probe) and after hybridization with 1.0 ng/6 μl ssG-DNA of S. enterica and other foodborne pathogens

Fig. 5

Stability of c-MWCNT/AuNP electrochemical sensor measured as % relative peak current (for probe as zero using CV) after a regular interval of 30 days up to 6 months on storage at 4 °C

Fig. 6

Validation studies of c-MWCNT/AuNP electrochemical sensor by measuring relative peak current using CV after hybridization with ss-GDNA isolated microorganisms in raw milk samples