The objective of this work is to elaborate an immunosensing system which will detect and quantify
Biosensor technologies play an increasingly important role in the detection of pathogenic bacteria because they present the great potential of satisfying the practical need for rapid, wearable, and low-cost detection [
The originality reported in this present study is the development of an immunosensor based on a diazonium electrografted gold electrode for the detection of
In this work, anti-
Polyclonal antibodies (developed in rabbit) against
Electrochemical measurements were performed using a Voltalab 40 potentiostat-galvanostat with a standard three-electrode configuration. The measurement set-up consisted of a three-electrode system, with a gold electrode (surface area
All electrochemical measurements were carried out in a measuring chamber of volume
Two-electrochemical techniques were used in this work as follows. Cyclic voltammetry was performed in 5 mM ferro/ferricyanide PBS solution (8 mM) at a scan rate of 100 mV/s. Faradaic EIS was used by applying a small sinusoidal signal (amplitude 10 mV; frequency range 100 mHz to 100 kHz) to the system at open circuit potential in 5 mM ferro/ferricyanide PBS solution (8 mM). The
The gold surface was cleaned in acetone, in an ultrasonic bath for 10 min, dried under a nitrogen flow, and then dipped for 2 min into a piranha solution 4 : 1 (v/v) [98% H2SO4/30% H2O2], rinsed with ultrapure water and dried under a nitrogen flow. The diazonium cations were synthesized
The electrochemical grafting on golf electrode was immediately performed in the mixture above described, by cyclic voltammetry (three cycles from 0.6 to −0.4 V/SCE at a scan rate of 100 mV/s). The consecutive cyclic voltammograms (CVs) of p-nitrophenyl diazonium at the gold electrode are presented in Figure
(a) Three cyclic voltammograms (numbered) for
The CVs are characterized by the first cycle exhibiting a well-defined, reproducible, and irreversible reduction peak located at 0.4 V/SCE. This feature corresponds to the typical electro-reduction reaction of the diazonium function, leading to the elimination of a nitrogen molecule and the production of highly reactive radicals. This radical was previously shown to attack the surface and to form a covalent bond between the aryl group and the Au electrode [
Very low currents were observed during the second and third voltammetric cycles, evidencing that surface saturation of grafted molecules has been achieved. Although Brooksby and Downard have pointed out that the electrochemical determination of surface concentration must be interpreted with caution [
The surface concentration,
A surface concentration (mole coverage) of p-NP:
The electrode was then washed and transferred to 0.1 M KCl solution and subjected to five potential scans between 0.4 and −1.25 V/SCE at 100 mV/s in order to reduce the nitro group and obtain a modified film of 4-aminophenyl on the electrode surface. Figure
Once the p-aminophenyl layer had been formed, the electrochemical behavior of the modified gold electrode surface was investigated by cyclic voltammetry in the presence of the Fe2+/Fe3+ redox couple. Figure
(a) Cyclic voltammograms of 5 mM Fe
Electrochemical impedance spectroscopy measurements were also performed to further characterize the modified surfaces. Impedance
Impedance
An electrical equivalent circuit for both a bare and a modified electrode, shown in Figure
It is worth noting that the CPE reflects the nonideality of the double-layer at the functionalized gold electrode/electrolyte interface due to the roughness and porosity of the interfacial film.
The CPE is defined as
A specific electrochemical element of diffusion, the Warburg element (
Here,
Following deposition of the p-aminophenyl (p-AP) film on the gold electrode, the terminal amine was activated by incubation for 60 min with glutaraldehyde (25%) at pH 4, at room temperature [
After rinsing with PBS, the electrode was incubated for 1 hour in a 0.2 mg/mL solution of anti-
Finally, after rinsing with PBS, the electrodes were incubated for 20 min in BSA (1%) to block the unreacted aldehyde groups. Cyclic voltammetry (CV) and EIS were employed to characterize the p-AP gold electrode (a), p-AP-GA gold electrode (b) and p-AP-GA-Ab gold.
Figure
(a) Cyclic voltammograms of (A) p-AP-Au electrode, (B) p-AP+GA-Au electrode, and (C) antibody modified Au-p-AP+GA-Au electrode, recorded in the presence of Fe
The Faradaic EIS measurements are in good agreement with CV measurements; the current density decreases (Figure
In order to calculate the number of binding sites we calculate the antibody surface coverage
55% of the surface is found to be covered with antibodies. Considering the surface density for a dense antibody layer determined in [
Having developed our sensor, we studied its response towards different concentrations of the
(a) Nyquist plots of impedance spectra obtained for increasing concentrations of
The percentage of surface coverage by bacteria
Assuming that the surface area of one
The reaction flow between immobilized antibody and bacteria is as follows:
Mass action applied to the equilibrium gives
If
The expression of
The measured relative variation of
The measured relative variation of
The charge transfer resistance increases gradually as the bacteria concentration increases after consecutive incubations from 10 up to 107 CFU per mL. The impedance immunosensor shows a linear relationship between the relative variation of charge transfer
The reproducibility of the immunosensor was investigated by repeating the experiments with three different immunosensors prepared in the same conditions. The response for three different immunosensors has an average standard deviation of 20%, which verifies the reproducibility of the system for the detection of
To ensure that the response of our immunosensor was not due to the adsorption of bacteria on the Au surface modified by p-aminophenyl, different electrodes were elaborated in the same conditions, without the antibody incubation step. The results show that there is no significant variation of the relative variation of the charge transfer resistance
The specificity of the impedimetric immunosensor system was determined for different bacteria strains at 106 CFU per mL. The relation variation of charge transfer resistance
A comparison of the analytical characteristics of the immunosensor developed in this work with relevant immunosensors for
Type of transducer | Quantification limit |
Dynamic range |
Reference |
---|---|---|---|
Impedance | 10 | 10–106 | [ |
102 | 102–107 | [ | |
10 | 10–106 | [ | |
10 | 10–107 | This work |
An impedance-based immunosensor for the detection of
The authors would like to thank EGIDE for its support through UTIQUE Program no. 09G 1128 and through PHC Maghreb no. 12 MAG 088. Amina Chrouda is grateful to the Tunisian Ministry of Higher Education and Technology for the “Mobility Grant”.