Ultrasensitive Label-free Electrochemical Immunosensor based on Multifunctionalized Graphene Nanocomposites for the Detection of Alpha Fetoprotein

In this work, a novel label-free electrochemical immunosensor was developed for the quantitative detection of alpha fetoprotein (AFP). Multifunctionalized graphene nanocomposites (TB-Au-Fe3O4-rGO) were applied to modify the electrode to achieve the amplification of electrochemical signal. TB-Au-Fe3O4-rGO includes the advantages of graphene, ferroferric oxide nanoparticles (Fe3O4 NPs), gold nanoparticles (Au NPs) and toluidine blue (TB). As a kind of redox probe, TB can produce the electrochemical signal. Graphene owns large specific surface area, high electrical conductivity and good adsorption property to load a large number of TB. Fe3O4 NPs have good electrocatalytic performance towards the redox of TB. Au NPs have good biocompatibility to capture the antibodies. Due to the good electrochemical performance of TB-Au-Fe3O4-rGO, the effective and sensitive detection of AFP was achieved by the designed electrochemical immunosensor. Under optimal conditions, the designed immunosensor exhibited a wide linear range from 1.0 × 10−5 ng/mL to 10.0 ng/mL with a low detection limit of 2.7 fg/mL for AFP. It also displayed good electrochemical performance including good reproducibility, selectivity and stability, which would provide potential applications in the clinical diagnosis of other tumor markers.

According to our previous reports [21][22][23][24] , reduced graphene oxide functionalized with ferroferric oxide (Fe 3 O 4 -rGO) can be used in adsorbing a variety of heavy metals. In this case, a label-free electrochemical immunosensor based on multifunctionalized graphene nanocomposites (TB-Au-Fe 3 O 4 -rGO) was designed for the quantitative detection of AFP. First of all, gold nanoparticles (Au NPs) which could immobilize the antibodies through the good biocompatibility were applied to functionalize the Fe 3 O 4 -rGO to obtain the Au-Fe 3 O 4 -rGO [25][26][27][28][29][30][31][32][33][34][35] . Subsequently, taking advantage of the good adsorption property, Au-Fe 3 O 4 -rGO was used to adsorb toluidine blue (TB) to obtain the TB-Au-Fe 3 O 4 -rGO. TB is a kind of organic dye and widely used as an electron transfer mediator for providing electrochemical signal in the electrochemical immunosensor 36 . As is known to all, ferroferric oxide nanoparticles (Fe 3 O 4 NPs) have a good electrocatalytic performance [37][38][39][40] , which can promote the redox of TB. In addition, the graphene can provide large specific surface area for the immobilization of antibodies and high electrical conductivity for the electron transfer. Therefore, the usage of the TB-Au-Fe 3 O 4 -rGO modified electrode to fabricate the electrochemical immunosensor can achieve the effective and sensitive detection of AFP. Experimental Apparatus and reagents. All electrochemical measurements were performed on a CHI760E electrochemical workstation (Huakeputian Technology Beijing Co., Ltd., China). A conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire electrode as the counter electrode. Scanning electron microscope (SEM) images were obtained by using Quanta FEG250 field emission environmental SEM (FEI, United States) operated at 4 KV.
Human AFP and antibody to human AFP (anti-AFP) were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd., China. Bovine serum albumin (BSA) and TB were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. Sodium acetate trihydrate (NaAc·3H 2 O), chloroauric acid (HAuCl 4 ·4H 2 O) and trisodium citrate were purchased from Shanghai Aladdin Chemistry Co., Ltd, China. Ethanol, ethylene glycol (EG) and ethanediamine (EDA) were purchased from Tianjin Fuyu Fine Chemical Co., Ltd., China. Ferric chloride hexahydrate (FeCl 3 ·6H 2 O) was purchased from Tianjin Damao Chemical Reagent Co., Ltd., China. Phosphate buffered saline (PBS 1/15 M Na 2 HPO 4 and KH 2 PO 4 ) was used as an electrolyte for all electrochemical measurements, which was purged with nitrogen gas for 20 min to remove the dissolved oxygen. All other reagents were of analytical grade and ultrapure water was used throughout the study. Figure 1 shows the synthesis procedure of the TB-Au-Fe 3 O 4 -rGO.

Synthesis of the TB-Au-Fe
SEM image was performed to characterize the shape and size of the Au-Fe 3 O 4 -rGO (inset of Fig. 1). Graphene oxide (GO) was synthesized by an improved Hummers method 41 . In brief, a mixture of concentrated H 2 SO 4 (36 mL) and H 3 PO 4 (4 mL) was added into a mixture of graphite flakes (0.3 g) and KMnO 4 (1.8 g), producing a slight exotherm to 35~40 °C. The reaction was then heated to 50 °C and stirred for 12 h. After that, the reaction was cooled to room temperature and poured onto ice (40 mL) with 30% H 2 O 2 (0.3 mL), and the mixture was centrifuged and the supernatant was decanted away. For workup, the remaining solid material was washed in succession with water, 0.2 M HCl, ethanol and ether. The obtained solid was dried in vacuum overnight.
In a typical synthesis of Fe 3 O 4 -rGO 21 . FeCl 3 ·6H 2 O (0.5 g) was dissolved in EG (10 mL) to form a clear solution, followed by the addition of NaAc (1.5 g), EDA (5 mL) and GO (0.5 g). The mixture was stirred vigorously for 30 min and then sealed in a teflon-lined stainless steel autoclave. The autoclave was heated to 200 °C and maintained for 8 h, and then was cooled to room temperature. The resulting black powder was washed several times Fabrication of the immunosensor. Figure 2 shows the schematic diagram of the label-free electrochemical immunosensor fabricated on the GCE. A GCE was polished to a mirror-like finish with alumina powder (1.0, 0.3 and 0.05 μ m), and then it was thoroughly cleaned before use. First, an aqueous solution of TB-Au-Fe 3 O 4 -rGO (2 mg/mL, 6 μ L) was added onto the surface of bare GCE and then dried. After washed, anti-AFP dispersion (10 μ g/mL, 6 μ L) was added onto the electrode. According to the ref. 43, anti-AFP can be conjugated on the surface of TB-Au-Fe 3 O 4 -rGO due to the bonding between amino groups of antibodies and Au NPs. After incubated at 4 °C for 1 h and washed, BSA solution (10 mg/mL, 3 μ L) was added onto the electrode to eliminate nonspecific binding sites. After incubated for another 1 h at 4 °C, the electrode was washed and incubated with a varying concentration of AFP (1.0 × 10 −5 ~ 10.0 ng/mL, 6 μ L) for 1 h at room temperature, and then the electrode was washed extensively to remove unbounded AFP molecules for measurement. For Square Wave Voltammetry (SWV) to record the amperometric response in PBS at pH = 6.8, a detection potential from − 0.6 V to 0 V was selected. The response time of one electrode is 30 seconds.

Results and Discussion
SEM and EDX characterization. Figure  Optimization of experimental conditions. In order to achieve an optimal electrochemical signal, optimizations of experimental conditions are necessary. The pH value of PBS and the concentration of TB-Au-Fe 3 O 4 -rGO influence the electrochemical current response. Figure 4A shows the different electrochemical current responses of the electrode modified with 2 mg/mL TB-Au-Fe 3 O 4 -rGO in different pH values of PBS for the detection of 10.0 ng/mL of AFP. Figure 4B shows the different electrochemical current responses of the electrode modified with different concentrations of TB-Au-Fe 3 O 4 -rGO in PBS at pH = 6.8 for the detection of 10.0 ng/mL of AFP. As shown in these figures, the optimal amperometric response was achieved at a pH of 6.8 and at a concentration of 2 mg/mL. Therefore, PBS at pH = 6.8 and 2 mg/mL TB-Au-Fe 3 O 4 -rGO were selected for the test throughout this study.
In addition, the other factors were controlled strictly, for example, the incubation time was 1 h, the incubation temperature was the room temperature, and the concentration of antibodies was 10 μ g/mL. All these above factors would make sure antibodies and antigens could effectively and specifically recognize with each other. SWV was also used to verify the successful fabrication of the immunosensor (Fig. 5B). It can be observed that the bare GCE (curve a) modified with TB exhibits an obvious electrochemical peak current at − 0.24 V (curve b). After modified with anti-AFP (curve c), BSA (curve d) and AFP (curve e), the decreasing electrochemical current response indicates biological active substances can hinder the efficiency of electron transfer. The slight shift of the electrochemical peak potential might be due to the modification of the biological active substance. In order to further characterize the fabrication process of the label-free electrochemical immunosensor, Nyquist plots of the A.C. impedance method was recorded from 1 to 10 5 Hz at 0.2 V in a solution containing 0.1 M KCl and 2.5 mM Fe(CN) 6 3− /Fe(CN) 6 4− . Nyquist plots are consisted of two portions. The linear portion at low frequencies is associated with electrochemical behavior limited by diffusion. The semicircle portion at high frequencies is associated with the electrochemical process subject to electron transfer, where the diameter corresponds to the resistance. Simply, resistance change could be judged by observing the diameter change of semicircle portion. Thus, A.C. impedance is a suitable method for monitoring the changes in the surface features during the fabrication process [45][46][47][48] . The inset in Fig. 5C shows the Randles model for the equivalent circuit [49][50][51]  : solution resistance (R s ), charge transfer resistance (R ct ), capacitance of double layer (C dl ), Warburg impedance (Z w ). As shown in the Fig. 5C, it can be observed that the bare GCE exhibits a very small resistance (curve a), which is characteristic of a diffusion-limiting step in the electrochemical process. After the modification of TB-Au-Fe 3 O 4 -rGO, the electrode shows a decreasing resistance (curve b). It implies that the successful immobilization of TB-Au-Fe 3 O 4 -rGO on the surface of bare GCE and TB-Au-Fe 3 O 4 -rGO can provide a sensitive interface to accelerate the electron transfer. The gradually increasing resistance of electrodes further modified with anti-AFP (curve c) and BSA (curve d) indicates the successful immobilization of the non-conductive bioactive substances. It also indicates that TB-Au-Fe 3 O 4 -rGO can offer a biocompatible surface for the capture of anti-AFP. After the modification of AFP, the electrode shows an increasing resistance (curve e), implying that the successful specific recognition between anti-AFP and AFP.
Characterization of the immunosensor. Under optimal conditions, the label-free electrochemical immunosensor was employed to detect different concentrations of AFP. Figure 6A shows the electrocatalytic current responses of the designed immunosensor for the detection of AFP covering the concentration range from 1.0 × 10 −5 ng/mL (curve a) to 10.0 ng/mL (curve g). Figure 6B   parallel blank measures, the x b1 = 19.50 and s b1 = 0.034 in this work). The low detection limit of 2.7 fg/mL was obtained, which was ascribed to the novel signal amplification strategy based on multifunctionalized graphene nanocomposites in the fabrication of the designed immunosensor. The linear range and detection limit of the proposed immunosensor were compared with other reported methods for the detection of AFP in Table S1. It could be found that the proposed immunosensor showed a satisfactory linear range and detection limit.
Reproducibility, selectivity and stability. To evaluate the reproducibility of the label-free electrochemical immunosensor, a series of five electrodes were prepared for the detection of 1.0 ng/mL AFP (Fig. 7A). The relative standard deviation (RSD) of the measurements for the five electrodes was less than 5%, suggesting the precision and reproducibility of the designed immunosensor was quite good.
To investigate the specificity of the label-free electrochemical immunosensor, interference study was performed by using CEA, prostate specific antigen (PSA), human immunoglobulin (IgG) and BSA. 1.0 ng/mL AFP with and without 100.0 ng/mL interfering substances solution was measured by the designed immunosensor (Fig. 7B). It can be observed that the electrochemical current response variation due to the interfering substances was less than 5% of that without interferences, indicating the selectivity of the designed immunosensor was excellent.
To test the stability of the fabricated electrochemical immunosensor, it was stored at 4 °C when not in use. After one month, the electrochemical current response change was less than 5% for the detection of 1.0 ng/mL AFP. The good stability of the designed immunosensor can be ascribed to the good biocompatibility of the multifunctionalized graphene nanocomposites. The reproducibility, selectivity and stability of the designed immunosensor were all acceptable, thus it was suitable for quantitative detection of AFP in real human samples.

Real sample analysis.
In order to validate the label-free electrochemical immunosensor, a comparative experiment with the commercialized available ELISA method for the detection of AFP in human serum sample was conducted (Table S2). The relative error between the two methods was in the range from − 4.9% to 4.0%. These data revealed a good agreement between the two analytical methods, indicating the feasibility of the designed immunosensor for clinical application. Conclusion A novel label-free electrochemical immunosensor for the quantitative detection of AFP has been developed in this work. Thanks to the good adsorption property and the good electrochemical performance of the multifunctionalized graphene nanocomposites, the proposed immunosensor showed a wide linear range, a low detection limit, good reproducibility, fine selectivity and acceptable stability. The designed immunosensor might provide wide potential applications for the detection of AFP in clinical diagnosis and also can be applied in clinical detection of other tumor markers.