PCR-assisted impedimetric biosensor for colibactin-encoding pks genomic island detection in E. coli samples

Abstract

A fast PCR-assisted impedimetric biosensor was developed for the selective detection of the clbN gene from the polyketide synthase (pks) genomic island in real Escherichia coli samples. This genomic island is responsible for the production of colibactin, a harmful genotoxin that has been associated with colorectal cancer. The experimental protocol consisted of immobilizing the designated forward primer onto an Au electrode surface to create the sensing probe, followed by PCR temperature cycling in blank, positive, and negative DNA controls. Target DNA identification was possible by monitoring changes in the system's charge transfer resistance values (R_ct) before and after PCR treatment through electrochemical impedance spectroscopy (EIS) analysis. Custom-made, flexible gold electrodes were fabricated using chemical etching optical lithography. A PCR cycle study determined the optimum conditions to be at 6 cycles providing fast results while maintaining a good sensitivity. EIS data for the DNA recognition process demonstrated the successful distinction between target interaction resulting in an increase in resistance to charge transfer (R_ct) percentage change of 176% for the positive DNA control vs. 21% and 20% for the negative and non-DNA-containing controls, respectively. Results showed effective fabrication of a fast, PCR-based electrochemical biosensor for the detection of pks genomic island with a calculated limit of detection of 17 ng/μL.

Keywords: DNA biosensor; Electrochemical impedance spectroscopy; Polyketide synthase genomic island; Polymerase chain reaction (PCR).

Fig. 1

Schematic representation of the custom miniature gold electrode and its dimensions

Fig. 2

Graphic scheme showing forward primer immobilization protocol and target DNA sensing mechanism, where DNA primers are colored red, and positive target DNA strand and amplicon are colored blue and green, respectively

Fig. 3

A Side and B top view of the electrochemical cell arrangement. C Size comparison and D representative clean cyclic voltammogram for a miniature gold electrode. CV parameters: in 0.5 M H₂SO₄, from 0.2 to 1.6 V vs. Ag/AgCl (3 M KCl) at 50 mV/s

Fig. 4

Nyquist plots following the surface probe immobilization and target DNA recognition after PCR treatment: Bare Au electrode (black), TGA-modified Au electrode (orange), TGA/primer-modified Au electrode (blue), and after DNA elongation through PCR treatment (pink)

Fig. 5

Graphical scheme showing the resistance to charge transfer (R_ct) percentage difference before and after PCR treatment (orange bar chart), and ΔR_ct/time ratio (blue scatter plot) at different PCR cycle numbers

Fig. 6

Resistance to charge transfer (R_ct) percentage change bar chart representing the selectivity of the sensor by exposure to non-containing blank (red), positive (green), and negative (blue) DNA controls at six (6) PCR cycles

Fig. 7

pks-containing positive DNA control calibration curve, R_ct percentage difference vs. positive control DNA target concentration, at six (6) PCR cycles