Electrochemical sensory detection of Sus scrofa mtDNA for food adulteration using hybrid ferrocenylnaphthalene diimide intercalator as a hybridization indicator

Abstract

In this study, an electrochemical DNA biosensor was developed based on the fabrication of silicon nanowires/platinum nanoparticles (SiNWs/PtNPs) on a screen-printed carbon electrode (SPCE) for the detection of Sus scrofa mitochondrial DNA (mtDNA) in food utilizing a new hybrid indicator, ferrocenylnaphthalene diimide (FND). The morphology and elemental composition of the SiNWs/PtNPs-modified SPCE was analyzed by field emission scanning electron microscopy (FESEM) combined with energy dispersive X-ray spectroscopy (EDX). Cyclic voltammetry (CV) was used to study the electrical contact between the PtNPs and the screen-printed working electrode through SiNWs, while electrochemical impedance spectroscopy (EIS) was used to measure the charge transfer resistance of the modified electrode. The results clearly showed that the SiNWs/PtNPs were successfully coated onto the electrode and the effective surface area for the SiNWs/PtNPs-modified SPCE was increased 16.8 times as compared with that of the bare SPCE. Differential pulse voltammetry used for the detection of porcine DNA with FND as an intercalator confirmed its specific binding to the double-stranded DNA (dsDNA) sequences. The developed biosensor showed a selective response towards complementary target DNA and was able to distinguish non-complementary and mismatched DNA oligonucleotides. The SiNWs/PtNPs-modified SPCE that was fortified with DNA hybridization demonstrated good linearity in the range of 3 × 10^-9 M to 3 × 10^-5 M (R ² = 0.96) with a detection limit of 2.4 × 10^-9 M. A cross-reactivity study against various types of meat and processed food showed good reliability for porcine samples.

Fig. 1. (a) Ferrocenylnaphthalene diimide (FND) structure. (b) Schematic illustration of DNA hybridization with immobilized probe DNA and intercalation of ferrocenylnaphthalene diimide (FND) in between dsDNA.

Fig. 2. Schematic illustration of the fabrication and DNA hybridization process of SiNWs/PtNPs modified SPCE for electrochemical biosensor.

Fig. 3. (a) HRTEM of PtNPs. FESEM images for the surface of (b) bare SPCE, (c) SiNWs/SPCE, (d) SiNWs-PtNPs/SPCE with magnification of 50k. FESEM images for the cross-section of (e) bare SPCE and (f) SiNWs-PtNPs/SPCE with magnification of 2.5k, and (g) EDX of SiNWs/PtNPs-modified SPCE.

Fig. 4. The relationship between the peak potential and square root of scan rate for bare and SiNWs/PtNPs-modified SPCE (n = 3).

Fig. 5. Electrochemical study of the surface modification of SPCE. (a) EIS of modified electrode (n = 3). (b) DPV curves of different SPCE modifications after incubation in FND.

Fig. 6. Electrochemical study for the optimization of the hybridization conditions. (a) Optimum hybridization time, (b) optimum hybridization temperature and (c) optimum pH for DNA hybridization (n = 3).

Fig. 7. (a) DPV voltammograms of the FND peak current using different types of DNA oligonucleotides in 50 mM Tris–HCl containing 20 mM NaCl (pH 7.6). (b) Histogram of the same conditions (n = 3).

Fig. 8. (a) Histogram of DPV peak current of different DNA concentrations. (b) Plot of peak current of FND against logarithmic value of different DNA concentrations (n = 3).

Fig. 9. (a) Cross-reactivity study of DNA biosensor against various types of meat samples (n = 3). (b) Agarose gel electrophoresis of the PCR products amplified from raw pork, pork sausage, canned pork, raw beef and raw lamb (S1, S2, S3, S4, S5) by the specific 12S rRNA primer. The amplification of 1 μg of sample DNA is shown. M, 100 bp DNA ladder size standard.