An ultrasensitive label-free electrochemical immunosensor based on signal amplification strategy of multifunctional magnetic graphene loaded with cadmium ions

Herein, a novel and ultrasensitive label-free electrochemical immunosensor was proposed for quantitative detection of human Immunoglobulin G (IgG). The amino functionalized magnetic graphenes nanocomposites (NH2-GS-Fe3O4) were prepared to bond gold and silver core-shell nanoparticles (Au@Ag NPs) by constructing stable Au-N and Ag-N bond between Au@Ag NPs and -NH2. Subsequently, the Au@Ag/GS-Fe3O4 was applied to absorb cadmium ion (Cd2+) due to the large surface area, high conductivity and exceptional adsorption capability. The functional nanocomposites of gold and silver core-shell magnetic graphene loaded with cadmium ion (Au@Ag/GS-Fe3O4/Cd2+) can not only increase the electrocatalytic activity towards hydrogen peroxide (H2O2) but also improve the effective immobilization of antibodies because of synergistic effect presented in Au@Ag/GS-Fe3O4/Cd2+, which greatly extended the scope of detection. Under the optimal conditions, the proposed immunosensor was used for the detection of IgG with good linear relation in the range from 5 fg/mL to 50 ng/mL with a low detection limit of 2 fg/mL (S/N = 3). Furthermore, the proposed immunosensor showed high sensitivity, special selectivity and long-term stability, which had promising application in bioassay analysis.

Human Immunoglobulin G (IgG) is an important component in the immune system, which plays a crucial role in recognizing bacteria and viruses 1,2 . In addition, it has also been administered therapeutically from exogenous pooled donor sources 3,4 . The concentration of IgG in blood and other body fluid is direct correlation to the standard of humoral immunity, which abnormal IgG concentrations often indict the risk of disease or vulnerability to infection 3,5 . Therefore, it is necessary to develop method for quantitative detection IgG for immunological diagnosis.
In recent years, various detection assays have been developed for the detection of IgG, such as chemiluminescence 6 , colorimetric sensors 7,8 , fluorescence 9,10 and electrochemical immunosensors 11,12 . In comparisons, electrochemical immunoassay may be an excellent candidate for the detection of tumor markers because of fast analytical time, low detection limits, high sensitivity, simple pretreatment procedure and inexpensive instrumentation 13,14 . As one important branch of electrochemical immunosensors, the label-free immunosensors have became an attractive and promising approach to direct detection of tumor makers because simple procedure, ease of use and rapidity of the assay and without the use of secondary antibody compared with sandwich-type immunosensors 15,16 .
Over the past decade, a wide variety of nanomaterials have been designed as transducing materials to fabricate label-free electrochemical immunosensors, such as, graphene sheets (GS) 17,18 , metal oxides 19,20 and noble metal nanoparticles 21,22 . As a kind of popular transducing materials, graphene oxide (GO) possess many oxygen-containing functional groups which make the water-solubility better and make it easy to form a stable chemical bond with various materials, such as, magnetic nanostructures, metallic or catalytic 23,24 . To date, Fe 3 O 4 NPs have attracted a considerable interest due to its good biocompatibility 25 and great auxiliary catalytic activity toward the reduction of hydrogen peroxide (H 2 O 2 ) 20,26 . Therefore, the graphene was introduced to combine with Fe 3 O 4 NPs by chemical reaction. Among the noble metal nanoparticles, gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs) have attracted a considerable interest because of its good biocompatibility 27 and superior auxiliary catalytic activity towards the reduction of H 2 O 2 28,29 . Further, Au and Ag NPs can facilitate the electron transfer because of its superior electrochemical properties. So, Au NPs are widely used in fixing antibody because of its superior biocompatibility and chemical stability 30,31 . Compared with Au NPs, Ag NPs exhibit excellent electro catalytic activity towards the reduction of H 2 O 2 32,33 . Compared with single metal NPs, bimetallic NPs with a core-shell structure show distinctly unique characteristics than their monometallic counterparts 34 . In addition, the Au@Ag NPs could also enable the facile conjugation of capture antibodies because of the stable Au-N 35,36 and Ag-N 37 bond between Au@Ag NPs and -NH 2 on antibodies. Simultaneously, the amino functionalized magnetic graphenes nanocomposites (NH 2 -GS-Fe 3 O 4 ) was applied to absorb Cd 2+ 38,39 due to the large surface area, high conductivity and exceptional adsorption capability. The adsorbed Cd 2+ , can further promote the redox of H 2 O 2 , which was applied to signal amplification. The signal amplification strategy, using the synergetic effect present in functional nanocomposites of gold and silver core-shell magnetic graphene loaded with cadmium ion (Au@Ag/ GS-Fe 3 O 4 /Cd 2+ ), can further increase electron transfer efficiency on electrode surface and the reaction efficiency of the nanocomposite toward H 2 O 2 reduction to improve the detection sensitivity of the immunosensor.
In this research, a novel and ultrasensitive label-free immunosensor for the quantitative detection the IgG was prepared using Au@Ag/GS-Fe 3 O 4 /Cd 2+ as a signal amplification platform. The synergic effect between functionalized magnetic graphene nanocomposites (GS-Fe 3 O 4 ), Au@Ag NPs and Cd 2+ can not only increase the electrocatalytic activity towards hydrogen peroxide (H 2 O 2 ) but also improve the effective immobilization of antibodies, which greatly extend the scope of detection. The proposed immunosensor provides a useful technology for the quantitative detection of IgG in human serum, shows high sensitivity, good selectivity and stability for the quantitative detection of IgG, holding a great potential in clinical and diagnostic applications.

Experimental
Apparatus and reagents. Scanning electron microscopy (SEM) images and energy-dispersive X-ray spectroscopy (EDS) analysis were collected using a FEI QUANTA FEG250 coupled with INCA Energy X-MAX-50. Fourier transform infrared spectroscopy (FTIR) spectrum was collected using VERTEX 70 spectrometer (Bruker, Germany). All electrochemistry measurements were performed on a CHI760E electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd, China) by using a conventional three-electrode system consisted of a glassy carbon electrode (GCE, 4 mm in diameter) as working electrode, a saturated calomel electrode (SCE) as the reference electrode, and the platinum wire electrode as the counter electrode.

Preparation of the NH 2 -GS−Fe 3 O 4 . Graphene oxide (GO) was synthesized according an improved
Hummer's method 40 . In brief, graphite flakes (0.6 g) and KMnO 4 (3.6 g) were dispersed in a mixture of concentrated H 2 SO 4 (72 mL) and H 3 PO 4 (8 mL). Subsequently, the reaction was heated to 50 °C and maintained at this temperature for 12 h with stirred. The mixture was cooled to room temperature after reaction and poured onto ice (80 mL) with 30% H 2 O 2 (0.6 mL). Then, the mixture was centrifuged and removed the supernatant. After that, the remaining solid material was thoroughly washed with water, 0.2 mol/L HCl (60 mL), ethanol and ether. Finally, the obtained solid material was dried in vacuum overnight. GS− Fe 3 O 4 was synthesized according to a protocol described previously 39 . FeCl 3 ·6H 2 O (0.5 g) was dissolved in ethylene glycol (10 mL) to form a clear solution, then, NaAc (1.5 g), ethanediamine (5 mL) and GO (0.5 g) was added into the mixture orderly and dissolved under stirred vigorously for 30 min. Subsequently, the mixture was transferred into the teflon-lined stainless steel autoclave. The autoclave was heated to and maintained at 200 °C for 8 h and cooled down to room temperature after reaction. The prepared compound sample was thoroughly washed to remove the impurities and separated via a strong magnet. The resulting GS− Fe 3 O 4 was dried under high vacuum overnight. It should be noted that the GO was translated into the graphene sheet (GS) in the process of reaction.
The amino-functionalized GS- Preparation of Au@Ag/GS-Fe 3 O 4 /Cd 2+ . Au@Ag/GS-Fe 3 O 4 (10 mg) was dispersed into cadmium sulphate solution (10 mL, 2 mg/mL). The solution had been oscillated for 24 h to ensure that Cd 2+ could be fully absorbed onto the Au@Ag/GS-Fe 3 O 4 . The Au@Ag/GS-Fe 3 O 4 /Cd 2+ was obtained for further use after magnetic separation. Figure 1A shows the preparation process of the Au@Ag/GS-Fe 3 O 4 /Cd 2+ .
Fabrication of the immunosensor. The schematic diagram of the stepwise self-assembly procedure of the proposed label-free immunosensor is shown in Fig. 1B. Generally, GCE was polished to a mirror-like, and washed thoroughly with ultrapure water. Firstly, the aqueous solution of Au@Ag/GS-Fe 3 O 4 /Cd 2+ (2 mg/mL, 6 μ L) was coated onto the surface of GCE and dried at room temperature. After drying for 1 h, the resultant Au@Ag/ GS-Fe 3 O 4 /Cd 2+ /GCE was incubated with anti-IgG (10 μ g/mL, 6 μ L) by the chemical bonding between Au@Ag NPs and available amine groups of anti-IgG. After incubated for another 1 h at 4 °C, BSA solution (1 wt%, 3 μ L) was added onto the electrode to eliminate nonspecific binding sites. After 1 h incubation, the BSA/anti-IgG/Au@ Ag/GS-Fe 3 O 4 /Cd 2+ /GCE was washed with ultrapure water and incubated with a varying concentration of IgG (5 fg/mL to 50 ng/mL, 6 mL) for 1 h at room temperature, and then the IgG/BSA/anti-IgG/Au@Ag/GS-Fe 3 O 4 / Cd 2+ /GCE was washed extensively to remove unbounded IgG molecules. Ultimately, the proposed immunosensor was stored at 4 °C for further usage.
Detection of IgG. Phosphate buffered saline (PBS, pH = 6.8) were prepared by mixing Na 2 HPO 4 and KH 2 PO 4 stock solution and used as the electrolyte in the process of electrochemical measurements. Amperometric i-t curve was used to record the amperometric response by scanning the potential at − 0.4 V. 5 mM H 2 O 2 was added into the PBS (10 mL) after the back ground current was stabilized. The cyclic voltammetry (CV) experiments were recorded in 5 mM K 3 [Fe(CN) 6 ] by scanning the potential from − 1 V to 1 V. For A.C. impedance measurements, a frequency range of 0.1 kHz to 100 Hz and AC voltage amplitude of 5 mV were used.

Results and Discussion
Characterization of Au/Ag@ GS-Fe 3 O 4 . As shown in (Fig. 2A), the surface of GO exhibits wrinkled, paper-like structure. After magnetization, GS was loaded with a lot of nearly monodisperse microspheres Fe 3 O 4 ( Fig. 2B). It was observed that these Fe 3 O 4 NPs own quasi-monodisperse size with an average grain diameter of 230 nm by the SEM. Obvious Fe, C, and O elements were observed which prove Fe 3 O 4 was loaded on the GS successfully (Fig. 2F). Furthermore, FT-IR spectra of GS-Fe 3 O 4 were recorded to prove that GS was loaded successfully with Fe 3 O 4 (Fig. 2H). As shown in Fig. 2G, the peak at 3420 cm When the Au@Ag NPs were coated on the surface of NH 2 -GS, the surface morphology was greatly alternated. Au@Ag NPs are well monodispersed and uniformly spherical in shape. As shown in (Fig. 2C), many small particles were loaded on the NH 2 -GS by constructing stable Au-N and Ag-N bond between Au@Ag NPs and -NH 2 . Subsequently, EDX spectrum of Au@Ag-GS was recorded which clearly confirmed the presence of Au@Ag NPs attached on the surface of the NH 2 -GS. As shown in (Fig. 2G), obvious Au, Ag, C, and O elements were observed, in which the signal of Au, Ag are assigned to the Au@Ag NPs, and that of C and O elements are belonged to the GS. Figure 2D,E shows the SEM image of Au@Ag/GS-Fe 3 O 4 in different scale respectively, which contains two kinds of size of particle. It is the combination of the Fig. 2B,C, suggesting the synthesized of Au@Ag/GS-Fe 3 O 4 successfully. Furthermore, obvious Au, Ag, C, and O and Fe elements were observed in Fig. 2I, which also suggest the synthesized of Au@Ag/GS-Fe 3 O 4 successfully. Figure 2J also proved that the magnetic field presents significant effect on the dispersion.
The mechanism of multiple signal amplification strategy. The sensitivity of the label-free immu-  20 . When GS-Fe 3 O 4 was applied as the signal amplification platform, the current response was significantly increased (curve c). Simultaneously, a much larger current response was observed when Au@Ag NPs was applied to modify the bare GCE due to the synergistic effect of Au NPs 28 and Ag NPs 45 towards the reduction of H 2 O 2 (curve d). Subsequently, the current response was further increased (curve e) when the electrode was modified with Au/Ag@GS-Fe 3 O 4 . As expected, using Au@Ag/GS-Fe 3 O 4 /Cd 2+ as signal amplification platform displayed the highest current change due to synergistic effect (curve f). The result indicted that Fe 3 O 4 NPs, Au@Ag NPs and Cd 2+ promote the multiple signal amplification toward the reduction of H 2 O 2 as an analytical signal. Hence, the resultant nanocomposites (Au@Ag/GS-Fe 3 O 4 /Cd 2+ ) was adopted as signal amplification platform due to excellent electrochemical performance to improve the sensitivity.
CV was applied to further verify that the Cd 2+ was adsorbed successfully onto the Au@Ag/GS-Fe 3 O 4 and have better electrocatalytic properties toward the reduction of H 2 O 2 (Fig. 3B). A reduction peak at − 0.7 V is obvious when a bare GCE was scanned in 0.1 mg/mL of Cd 2+ solution (curve a). Simultaneously, a reduction peak (curve b) was also observed at − 0.7 V when a bare GCE was scanned in PBS (pH = 6.8) using Au@Ag/GS-Fe 3 O 4 /Cd 2+ as signal amplification platform. Hence, it inferred that Cd 2+ was adsorbed successfully onto the Au@Ag/GS-Fe 3 O 4 . Furthermore, there was no current response (curve c) when the electrode was scanned in PBS (pH = 6.8) using

Electrochemical impedance spectroscopy (EIS) characterization of immunosensor.
Electrochemical impedance spectroscopy (EIS) was regarded as an effective method to characterize the fabrication process of the proposed immunosensor by monitoring the interfacial properties 46  − , the frequencies from 0.1 to 10 5 Hz and the potentiostatic at 0.188 V. As shown in Fig. 3, the bare GCE exhibited a smaller Ret (curve a). After the Au@Ag/GS-Fe 3 O 4 /Cd 2+ was modified on the electrode, the semicircle is much smaller (curve b) due to the high electrical transport properties of Au@Ag/GS-Fe 3 O 4 /Cd 2+ . After incubation with anti-IgG, the Ret (curve c) was significantly increased due to the anti-IgG is protein which can hinder electron transfer, which indicates anti-IgG was immobilized on the electrode successfully. Followed by blocking the nonspecific binding spots with BSA, the Ret (curve d) was further increased due to the blocking effect on electron transferring by the modified protein molecules on the surface of the electrode. Additionally, the Ret further increased with the addition of IgG (curve e), which indicates the successful capture of IgG and the formation of immunocomplex layer hinder the electron transfer. As a result, we can conclude that the proposed immunosensor was fabricated successfully.
Optimization of experimental conditions. In order to detect optimal electrocatalytic signal, it was necessary to optimize the experimental conditions including pH and the concentration of Au@Ag/GS-Fe 3 O 4 /Cd 2+ . The pH of the PBS was investigated with same concentration of Au@Ag/GS-Fe 3 O 4 /Cd 2+ (2.0 mg/mL). As shown in Fig. 4A, the current signal increases with the variation of pH from 5.6 to 6.8, and then decreases with the variation of pH from 6.8 to 8.1. The optimal amperometric response was obtained at pH = 6.8. It was inferred that the pH value obviously affected the electrocatalytic process of Au@Ag/GS-Fe 3 O 4 /Cd 2+ toward the reduction of H 2 O 2 . Simultaneously, the highly acidic or alkaline surroundings would influence the activity of the antigens, antibodies and damage the immobilized protein 48,49 . By contrast, the antigens and antibodies could keep their bioactivity in this approximate neutral conditions of pH 50 .
The concentration of Au@Ag/GS-Fe 3 O 4 /Cd 2+ would affect the amperometric response by accelerateing the electron transfer efficiency and immobilize of antibodies. As shown in Fig. 4B, with the increasing of the concentration from 0.5 mg/mL to 2.0 mg/mL, the current signal increased, but the current signal decreased with the concentration from 2.0 mg/mL to 3.5 mg/mL. The optimal amperometric response was obtained at 2.0 mg/mL. It was inferred that the increasing of Au@Ag/GS-Fe 3 O 4 /Cd 2+ film thickness might lead to an increase of interface electron transfer resistance, and the electron transfer become more difficult. Simultaneously, the higher or lower concentrations of Au@Ag/GS-Fe 3 O 4 /Cd 2+ influenced the catalytic performance for the reduction of H 2 O 2 51 . Therefore, the concentration of 2.0 mg/mL was used as the optimal concentration for this study.
In addition, the rest experiment conditions were also required strictly. For example, the concentration of antibodies was 10 μ g/mL, the incubation time of 1 h, which was enough to capture antigen (ng/mL) and achieve the specific recognition between antigens and antibodies 52 . Under the optimal conditions, the proposed immunosensor will obtain an optimal electrochemical response for quantitative detection of IgG.
Analytical performance of the immunosensor. Under the optimal experimental conditions, amperometric i-t curve was used to detect different concentrations of IgG (Fig. 5A) using Au@Ag/GS-Fe 3 O 4 /Cd 2+ as a signal amplification platform in pH 6.8 PBS. The amperometric response towards the reduction of 5 mmol/L  (Fig. 5B). The results demonstrate the proposed method have an acceptable quantitative performance for the IgG detection. The performance of the immunosensor was compared with previously reported methods for the detection of IgG. As shown in Supplementary Table S1, the proposed immunosensor has a wider linear range and lower detection limit than previously report. The low detection limit was attributed to several factors. Firstly, Au@Ag/GS-Fe 3 O 4 /Cd 2+ , a novel material, is a combination of Fe 3 O 4 -GS, Au@Ag NPs and Cd 2+ through covalent bonding, which has high electrocatalytic performance towards the reduction of H 2 O 2 leading to higher sensitivity. Secondly, Au@Ag NPs not only possess good biocompatibility which firmly conjugated with a relatively large amount of antibodies, but also in assisting the electron transfer process to amplify the signal. In addition, Cd 2+ possess good electrocatalytic activity towards the reduction of H 2 O 2 , which can be used to amplify the detection signal and leaded to the high sensitivity of the designed immunosensor. Consequently, the synergic effect between Au@Ag/GS-Fe 3 O 4 /Cd 2+ can not only increase the electrocatalytic activity towards H 2 O 2 but also improve the effective immobilization of antibodies, which could greatly improve the probability of antibody-antigen recognition and extended the scope of detection. Hence, high sensitivity is one of the characteristics of the proposed immunosensor.
Reproducibility, selectivity and stability. To evaluate the reproducibility of the immunosensor, a series of five different electrodes were prepared for the detection of 0.05 ng/mL of IgG (Fig. 6A). The relative standard deviation (RSD) of the measurements for the five electrodes was 3.6%, indicating the precision and reproducibility of the immunosensor is acceptable.
To investigate the selective of the proposed immunosensor, interferences study was performed using (alpha fetoprotein) AFP, BSA, carcinoembryonic antigen (CEA), and glucose. IgG solution (0.5 ng/mL) containing 50 ng/mL of interfering substances were measured by the proposed immunosensor (Fig. 6B). The electrocatalytic current response variation was 4.2%, less than 5% of that without interferences, suggesting the selectivity of the immunosensor is good.
The stability was investigated at 0.05 ng/mL of IgG. As shown in Fig. 6C, the current response of the immunosensor kept constant after 3 days, the current response of the immunosensor only had a minor change of 4.2% after 2 weeks. Subsequently, the current response retained 90.5% of their initial current after 3 weeks. It could be found that current responses to same concentration of IgG has no apparent change compared to the immunosensor freshly prepared which was used to directly detect the same concentration of IgG without being stored, suggesting the stability of the immunosensors was also acceptable. The better stability can be attributed to the good biocompatibility of transducing materials. The reproducibility, selectivity and stability were all acceptable, indicating that the proposed immunosensor was suitable for quantitative detection of IgG in real samples.

Real sample analysis.
In order to test the feasibility and precision of the proposed label-free immunosensor, standard addition method was used to detect the recoveries of different concentrations of IgG in human serum samples. As shown in Supplementary Table S2, the RSD was in the range from 1.40% to 3.01% and the recovery was in the range from 99.53% to 100.4%.The results imply that the proposed immunosensor could be effectively applied to the quantitative determination of IgG in human serums.

Conclusions
In this paper, a novel and ultrasensitive label-free electrochemical immunosensor for quantitative detection of IgG was prepared using Au@Ag/GS-Fe 3 O 4 /Cd 2+ as a signal amplification platform. To ensure a high-performance electrochemical immunosensor, Au@Ag/GS-Fe 3 O 4 /Cd 2+ was immobilized on the electrode, which can improve the electronic transmission rate and increase the surface area to capture a larger amount of antibodies. The proposed label-free immunosensor displayed wide linear range, low detection limit, high sensitivity, good reproducibility, long-term stability and acceptable selectivity. Herein, this proposed method will not only expand the application of Au@Ag/GS-Fe 3 O 4 /Cd 2+ , but also provide an attractive way to detect other biomolecules.