This work has explored the development of impedimetric immunosensors based on magnetic iron nanoparticles (IrNP) functionalized with streptavidin to which a biotinylated FAB part of the antibody has been bound using a biotin-streptavidin interaction. SPR analysis shows a deviation on the measured (angle) during antigen-antibody recognition whereas label free detection using by EIS allows us to monitor variation of polarization resistance. Before detection, layers were analyzed by FTIR and AFM. Compared to immobilization of antibody on bare gold surface using aminodecanethiol SAM, antibody immobilization on nanoparticles permitted to reach lower detection limit: 500 pg/ml instead of 1 ng/ml to in the case of EIS and 300 ng/ml instead of 4.5
Immunosensors have arisen great interest with expectation of providing fast and highly sensitive immunological response. Immunosensors have widespread applications in clinical diagnostics, food safety and quality control, biological analysis, and environmental monitoring [
In the recent years, nanomaterials have been widely used in bioanalytical chemistry, bioseparations, and bioimaging for their unique and particular properties [
A new approach allows to attach these magnetic particles by covalent binding on the substrate previously functionalized with self-assembled monolayer (SAM). The tailoring of the physical chemistry of surfaces has led to an increasing interest in using SAMs of thiolor sulfur compounds as insulator [
In this work, we report the development of an electrochemical Immunosensor based on iron oxide nanoparticles functionalized with the streptavidine. The Biotine tag anti-D-dimer reduced antibody was immobilized on the surface of iron oxide nanoparticles linked to a previously functionalized gold electrode using 11-Amino-1-undecanethiol and glutaraldehyde as coupling agent.
For the purpose of developing this Immunosensors, surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) techniques were selected. In first time, surface plasmon resonance (SPR) was used as optical characterization technique [
Surface plasmons are generally excited in the Kretchmann configuration by directing p-polarized light to a glass prism and reflecting from a gold film [
Therefore, the information of biological binding (recognition) events on the gold film can be obtained by carefully monitoring the SPR coupling characteristics. In most SPR systems, the information of the biomolecular interactions is obtained from measurements of the angular or the spectral characteristics of light reflected under SPR.
In second time, electrochemical impedance spectroscopy (EIS) was used as transduction technique for the label free detection. This technique is an effective tool used on the one hand, for the qualitative and quantitative characterization of electrochemical processes occurring in the conducting polymer films [
While comparing with the development of an immunosensor-based on antibody coupled on a SAM, we attempt to show that the use of iron oxide nanoparticles allows to amplify the response signal and leads to the improvement of immunosensor characteristics.
The magnetic coated streptavidin nanoparticles that display a diameter of 200 nm and an iron oxide content of about 70% (cf. Figure
AFM images of the iron oxide nanoparticles.
The buffer solution used for the electrochemical impedance experiments was Phosphate Buffer Saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 0.01 M
Surface plasmon resonance spectrometer “Biosuplar 3” (
In the present study, the SPR spectrometer flow cell (
Cyclic Voltammetry and Electrochemical Impedance Spectroscopy (EIS) measurements were performed using a Voltalab 80 impedance analyzer. A conventional three electrode cell was used, including a saturated calomel electrode (SCE) as reference electrode, a platinum wire as counter electrode (0.54
Atomic Force Microscopy (AFM) experiments were performed in air, using a Pico Plus molecular imaging microscope with a 300 nm scanning head. The images were registered in tapping mode using silicon pyramidal
NICOLET 710 FT-IR spectrometer equipped with an MIR (Middle Infrared) source an MCT/A detector was used to obtain the FTIR spectra. All the spectra are collected during 128 scans for the reference and sample. Our strategy consisted of recording in reflexion mode the spectrum of the cleaned gold substrate and then of the gold substrate modified with the SAM. The spectrum of the cleaned electrode served as a reference. The difference between two spectra gave the spectrum of SAM.
Two systems were prepared, one based on the use of a thiol SAM (system 1) and the other based on the use of functionalized magnetic iron nanoparticles with the streptavidin (system 2).
In the first step of the electrode modification, the 11-Amino-1-undecanethiol monolayer is chemisorbed on the gold electrode surface and exposed an array of amino groups towards the solution. In the second step, the electrode surface is activated with aldehyde groups and left the second carbonyl group of glutaraldehyde free on the top of the surface. Formation of bridge structures due to the reaction of both aldehyde groups of glutaraldehyde with the amino groups may be prevented by the application of a high concentration of the bifunctional reagent, that is, GA [
(a) System 1: schematic representation of the fabrication process of the multilayer immunosensor based on SAM. (b) System 2: schematic representation of the fabrication process of the multilayer immunosensor based on iron oxide nanoparticles. (c) Fab fragment biotinylated antibody.
Equivalent circuit model for complex impedance plot.
Two types of gold electrodes are used as substrate for the biolayer fabrication. Evaporated gold (~ 300 nm thickness) deposited on silicon using a titanium layer (30 nm thickness) used as substrate for the electrochemical experiments. These gold electrodes were provided by LAAS, CNRS Toulouse. Before modification, the gold surface was cleaned in an ultrasonic bath for 10 minutes in acetone, dried under a dry
The aminothiol monolayer was prepared by soaking a clean gold electrode (Au) in 0.1 mM 11-Amino-1-undecanethiol (AUT) in ethanol solution for 24 hours at room temperature in darkness, washing the electrode thoroughly with ethanol to remove physically adsorbed 11-Amino-1-undecanethiol, immersing the electrode in PBS (pH 7.4) containing 5% (v/v) glutaraldehyde (GA) solution for 1 hour 30 minutes, and rinsing with PBS. Afterwards, for system 1, the electrode was immersed into
The modified gold electrode with the self-assembled monolayer 11-Amino-1-undecanethiol was followed by several technique of characterizations.
Cyclic voltammetry is an effective and convenient method for probing the feature of the modified electrode surface. The step of the SAMs deposition was controlled by this technique. As is shown in Figure
Cyclic voltammetric measurement with the presence of the 5 mM redox-probe 3[Fe
Electrochemical Impedance Spectroscopy (EIS) is an effective tool for probing the feature surface-modified electrode while controlling its electrical properties [
Nyquist diagram (−Im(
Optical characterization of the SAM layer was performed by SPR technique. Figure
Optical parameters obtained by fitting the SPR curves for system 1 based on SAM.
Layer | Thickness (nm) | kapa | |||
---|---|---|---|---|---|
Bare gold | 50.72 | 0.17 | 3.988 | 1.355 | |
Thiol | 2.16 | 1.34 | 0 | 1.791 | 0 |
GA | 0.1 | 1.45 | 0 | 2.10 | 0 |
Nev. | 0.8 | 1.45 | 0 | 2.10 | 0 |
Ab | 0.4 | 1.45 | 0, | 2.10 | 0 |
As
A typical kinetics of step-by-step formation of the biofilm (system 1) is shown in Figure
Building-up kinetics of the biofilm based on SAM. SPR responses for the step-by-step formation of multilayer. All measurements were performed in the flow cell, in PBS 10 mM pH 7.4.
For each step of grafting of the layers constituting the immunosensor membrane, the spectra presenting the reflected intensity versus incidence angle, after flowing PBS buffer, were recorded (see Figure
In the same way, the building up of the membrane of immunosensor based on iron oxide nanoparticles was
Building-up kinetics of the biofilm based on iron oxide nanoparticles. SPR responses to the step-by-step formation of multilayer. All measurements were performed in the flow cell in PBS 10 mM pH 7.4.
For system 2, the covering rate of antibody reached
For each step of grafting of the layers constituting the immunosensor membrane, the spectra presenting the reflected intensity versus incidence angle, after rinsing by PBS buffer, were recorded (see Figure
Optical parameters obtained by fitting the SPR curves for system 2 based on iron oxide nanoparticles.
Layer | Thickness (nm) | kapa | |||
---|---|---|---|---|---|
Bare gold | 50.72 | 0.17 | 3.89 | 1.355 | |
Thiol | 2.16 | 1.34 | 0 | 1.791 | 0 |
GA | 0.1 | 1.45 | 0 | 2.10 | 0 |
Iron oxide nanoparticles | 301 | 0.28 | 2.98 | −8.802 | 1.6688 |
Ab | 1.2 | 1.45 | 0 | 2.10 | 0 |
Fitting parameters obtained from the proposed equivalent circuit for the different layers of the immunosensor based on SAM.
Layer | CPE ( | ||||
---|---|---|---|---|---|
Bare gold | 51.63 | 8292 | 1.8847E-5 | 0.96414 | 0.0011 |
Thiol | 47.18 | 37120 | 4.0271E-6 | 0.9475 | 0.00058 |
GA | 49.26 | 62535 | 3.2506E-6 | 0.94902 | 0.0011 |
Neutv. | 43.22 | 48703 | 3.9494E-6 | 0.93945 | 0.0010 |
Ab | 44.63 | 39869 | 4.1848E-6 | 0.93169 | 0.0010 |
The comparison of the results of both systems proved that the use of the iron oxide nanoparticles increases the thickness of the layer of antibody from 0.4 to 1.2 nm, from where, the increase of the grafting density of antibody on the electrode.
We used EIS to control the building up of the biofilm in PBS (10 mM, pH 7) at potential −400 mV. Figures
Fitting parameters obtained from the proposed equivalent circuit for the different layers of the immunosensor based on iron oxide nanoparticles.
Layer | CPE ( | ||||
---|---|---|---|---|---|
Bare gold | 51.63 | 8292 | 1.8847E-5 | 0.96414 | 0.0011 |
Thiol | 47.18 | 47280 | 4.4323E-6 | 0.95475 | 0.00023 |
Iron oxide nanoparticles | 43.22 | 52673 | 3.0594E-6 | 0.92432 | 0.0011 |
Ab | 44.63 | 74200 | 4.1848E-6 | 0.92349 | 0.0012 |
Nyquist diagram (−Im(
Nyquist diagram (−Im(
In the case of system 1 (based on SAM) (Figure
In the same measuring conditions, the building up of the biofilm of immunosensor based on iron oxide nanoparticles was controlled by EIS (Figure
In order to study the comparison of the two immunosensors responses, the optical calibration curves corresponding to the variation of incidence angle
Optical calibration curves describing the variation of angle shift
Detection using system 1 based on SAM
Detection using system 2 based on iron oxide nanoparticles
Antigen—Antibody interactions were monitored by impedance spectroscopy in PBS solution (10 mM, pH 7) at −400 mV for system 1 and for system 2. Systematically, the electrodes modified with biotin-tagged antibody fragment were equilibrated with a range of concentrations of the specific antigen. The impedance spectra obtained after incubation with different concentrations of Antigen are shown in Figure
Nyquist diagram (−Im(
Detection using system 1 based on SAM
Detection using system 2 based on iron oxide nanoparticles
In order to study the comparison of both immunosensors responses, the electrical calibration curves corresponding to the variation of membrane resistance
Electrical calibration curves describing the variation of membrane resistance
Detection using system 1 based on SAM
Detection using system 2 based on iron oxide nanoparticles
In this study, we have developed an immunosensor based on functionalized iron oxide nanoparticles. By comparison with the development of an immunosensor based on SAM, we have demonstrated that the use of the functionalized iron oxide nanoparticles offers several advantages. The comparison of the results of both systems proved that the use of the iron oxide nanoparticles amplifies the response signal, so increases the thickness of the layer of antibody from 0.4 to 1.2 nm, from where, the increase of the grafting density of antibody on the electrode. In second time, we have demonstrated that by using electrochemical impedance spectroscopy combined with theoretical equivalent circuits models it is possible to determine the electrical properties. Furthermore, EIS allowed a thorough monitoring of the engineering of the biolayer membrane.
Table
Comparison of analytical parameters of immunosensors based on SAM and immunosensor based on iron oxide nanoparticles.
Analytical parameters | SAM system | IrNp System | |
---|---|---|---|
Optical characterization | detection limit | 4.5 | 300 ng/ml |
sensitivity | |||
Dynamic range | 4.5 to 15 | 0.3 to 1 | |
The time response | 45 minutes | 30 minutes | |
reproducibility | 6.3% | 5.7% | |
Electrical characterization | detection limit | 1 ng | 500 pg |
sensitivity | |||
Dynamic range | 1 to 50 ng | 0.5 to 50 ng | |
The time response | 45 minutes | 20 minutes | |
reproducibility | 8.7% | 7.5% |