Preparation of Au-polydopamine functionalized carbon encapsulated Fe3O4 magnetic nanocomposites and their application for ultrasensitive detection of carcino-embryonic antigen

A novel carbon encapsulated Fe3O4 nanoparticles embedded in two-dimensional (2D) porous graphitic carbon nanocomposites (Fe3O4@C@PGC nanocomposites) were synthesized by situ synthesis strategy, which provided a sensor platform owing to a large aspect ratio and porous structure. Polydopamine (PDA) were modified on the surface of Fe3O4@C@PGC nanocomposites through self-polymerization of dopamine, acting as both the reductant and template for one-step synthesis of gold nanoparticles. The prepared Au/PDA/Fe3O4@C@PGC nanocomposites show ferromagnetic features, extremely excellent electron transfer, large specific surface area and excellent dispersing property. These are conducive to the electrochemical signal output and the immobilization of antibody. In this work, a highly label-free sensitive magnetic immunosensor was developed based on Au/PDA/Fe3O4@C@PGC nanocomposites for the detection of carcino-embryonic antigen (CEA). The magnetic glassy carbon electrode was used to fix the Au/PDA/Fe3O4@C@PGC nanocomposites with the help of magnetic force. Under the optimal conditions, the immunosensor exhibited a wide linear range (0.001 ng/mL–20.0 ng/mL), a low detection limit (0.33 pg/mL), good reproducibility, selectivity and acceptable stability. The proposed sensing strategy may provide a potential application in the detection of other cancer biomarkers.

Scientific RepoRts | 6:21017 | DOI: 10.1038/srep21017 potential as substrate materials for electrochemical immunosensor. The presence of thin onion-like carbon shells avoids the direct exposure of Fe 3 O 4 to the substrate solution and keeps the stability of the performance of Fe 3 O 4 structure 9 .
Dopamine (DA) is a naturally occurring compound with catechol and amine moieties, which owns extremely strong adhesive properties. Therefore, DA is selected as binding agents for forming thin, surface-adherent films onto the surface of inorganic and organic materials by secondary reactions [10][11][12][13] . In this work, polydopamine/Fe 3 O 4 @C@PGC nanocomposites (PDA/Fe 3 O 4 @C@PGC nanocomposites) were obtained through self-polymerization of DA in the solution, and the thickness of the polydopamine shell was controllable. Then, gold nanoparticles were one-step synthesized by as-prepared PDA/Fe 3 O 4 @C@PGC nanocomposites which acted as both the reductant and template. The obtained Au/PDA/Fe 3 O 4 @C@PGC nanocomposites show good electrical conductivity, large specific surface area and good dispersion.
In this paper, a novel label-free magnetic electrochemical immunosensor was developed based on Au/PDA/ Fe 3 O 4 @C@PGC nanocomposites modified magnetic glassy carbon electrode (MGCE). Using CEA as model analyte, the sensitive detection of CEA was demonstrated based on the peak current change of potassium ferricyanide (K 3 [Fe(CN) 6 ]) before and after the antigen-antibody reaction. The detection sensitivity of the fabricated immunosensor was further increased due to the large specific surface area and good electrical conductivity of the Au/PDA/Fe 3 O 4 @C@PGC nanocomposites. The simple and easy immunoassay has great prospect in the ultrasensitive detection of other cancer biomarkers.

Experimental
Apparatus and reagents. CEA antibody (Ab) and CEA are obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd. (China). Bovine serum albumin (BSA) is purchased from Sigma-Aldrich (Beijing, China). All other chemical reagents are analytical reagents grade and directly used without further purification.
All electrochemical measurements are achieved on a CHI 760D electrochemical workstation (Shanghai Chenhua Instrument Co. Ltd., China). Transmission electron microscope (TEM) images are recorded by a JEM-100C X II microscope (JEOL, Japan). Scanning electron microscope (SEM) is obtained from JSM-6700F micro-scope (JEOL, Japan). X-Ray Powder Diffraction (XRD) is performed with D8 advance X-ray diffractometer (Bruker AXS, Germany). Once cooled down to room temperature, the obtained solids are washed with ultrapure water until no chloride ion is detected. Finally, the resulting solids are dried at 50 °C.  Fabrication of the immunosensor. Figure 1 displayed the fabrication procedure of the immunosensors.

Synthesis of PDA/Fe
A MGCE with 4-mm diameter was polished with alumina powder (1.0, 0.3 and 0.05 μ m) and then thoroughly cleaned before use. To capture Ab, 6 μ L of Au/PDA/Fe 3 O 4 @C@PGC solution (1.0 mg/mL) was fixed on the surface of MGCE by the magnetism. Then 6 μ L of Ab was anchored on the Au/PDA/Fe 3 O 4 @C@PGC by the physical absorption and the chemical bonding of Au-N 14 or Au-S 15-17 bonds. After 1 h of incubation, 3 μ L of BSA (1 wt%) was modified onto the MGCE for blocking the nonspecific binding sites. Finally, 6 μ L of CEA was added onto the electrode surface and incubated for 1 h. After washing carefully with PBS, the electrode was ready for measurement.

Results and Discussion
Characterization of the Au/PDA/Fe 3 O 4 @C@PGC nanocomposites. The typical low-magnification SEM images and EDX spectrums of PDA/Fe 3 O 4 @C@PGC nanocomposites and Au/PDA/Fe 3 O 4 @C@PGC nanocomposites were showed in the Fig. 2. SEM image of the synthesized PDA/Fe 3 O 4 @C@PGC nanocomposites presented multi-pore structure with relative smooth surface (Fig. 2(A)). In addition, the PDA/Fe 3 O 4 @C@PGC nanocomposites with a large aspect ratio were well-dispersed in the sample 9 . After modified by Au nanoparticles, Au/PDA/Fe 3 O 4 @C@PGC nanocomposites kept original structure of PDA/Fe 3 O 4 @C@PGC nanocomposites ( Fig. 2(B)). The inset of the Fig. 2(B) showed the Au nanoparticles were immobilized on the surface of the PDA/ Fe 3 O 4 @C@PGC nanocomposites. The EDX spectrum of PDA/Fe 3 O 4 @C@PGC nanocomposites ( Fig. 2(C)) and The Au/PDA/Fe 3 O 4 @C@PGC nanocomposites were further characterized by the TEM (Fig. 3(A)). The result showed these Fe 3 O 4 nanoparticles were entirely encapsulated by thin onion-like carbon layers and Au nanoparticles were distributed on the surface of PDA/Fe 3 O 4 @C@PGC nanocomposites. In addition, Fig. 3 (Fig. 3(D)), which illustrated that Au/PDA/Fe 3 O 4 @C@PGC nanocomposites have great magnetism.
Characterization of the immunosensor. The performance of the immunosensor was also studied under continuous scans as shown in Fig. 4(A). The electrode was scanned successfully for 20 cycles in K 3 [Fe(CN) 6 ] solution (5 mmol/L) at 100 mV/s and no observable change of peak current and position was found, illustrating that the reaction between the electrode surface and the substances are not detached and diffusing into the solution during the experiment.
Electrochemical impedance spectroscopy (EIS) was used to research the impedance changes of the electrode surface during the electrode modification process. As shown in Fig. 4(B), bare MGCE showed a very small semicircle diameter (curve a), suggesting a diffusion limiting step of the electrochemical process. Owing to high conductivity and electronic transmission capacity of Au/PDA/Fe 3 O 4 @C@PGC nanocomposites, the semicircle diameter decreased slightly after modified Au/PDA/Fe 3 O 4 @C@PGC nanocomposites (curve b). The charge-transferring resistance (Ret) values increased with addition of Ab 1 (curve c), indicating that the protein    Figure 4(C) showed the current response of the cyclic voltammetry with the scan rate range from 10 mV/s to 1000 mV/s. It is found that the magnitude of both anodic and cathodic current response linearly dependent on the square root of scan rate in Fig. 4(D). The observed linear relation and well-defined stable redox peaks suggest that it was a diffusion-controlled electrochemical and quasi-reversible process 18 .
Optimization of detection conditions. In order to obtain the best analytical performance of the fabricated immunosensor, the concentration of Au/PDA/Fe 3 O 4 @C@PGC nanocomposites has been optimized in Fig. 5(A). Owing to the improvement of electron transfer ability, the current response increased with the increasing concentrations of Au/PDA/Fe 3 O 4 @C@PGC nanocomposites. However, when the concentration of Au/PDA/ Fe 3 O 4 @C@PGC nanocomposites was higher than the optimal concentration, the current response decreased with further increasing Au/PDA/Fe 3 O 4 @C@PGC thickness, which attenuated the electron transfer. Therefore, 1.0 mg/ mL was selected as the optimum concentration for the following experiments.
The concentration of K 3 [Fe(CN) 6 ] has a marked impact on the signal output of the immunosensor. Therefore, the different concentrations of K 3 [Fe(CN) 6 ] from 1 to 10 mM were used as basal solution. Figure 5(B) showed that the amperometric response increased with the increasing K 3 [Fe(CN) 6 ] concentration and tended to decrease after 8 mM. Thus, 8 mM was selected as the optimal concentration. Assay performance. Differential pulse voltammetry (DPV) was used to evaluate the performance of the fabricated immunosensor under optimized conditions in Fig. 6(A). As shown in Fig. 6(B), the calibration plots between the peak current and the concentration displayed good linear relationships in the range of 0.001 ng/mL to 20.0 ng/mL for CEA with a regression equation of Δ I = 1.23 + 1.75 c, R = 0.988. The detection limit was estimated to be 0.33 pg/mL. The obtained detection limit was lower than that in some previous reports, such as 3 pg/ mL 19 , 1 ng/mL 20 , 1.7 pg/mL 21 and 1.0 ± 0.04 pg/mL 22 . The results showed enough sensitivity for detection of CEA.
In addition, the properties of immunosensor based on Au/PDA/Fe 3 O 4 @C@PGC nanocomposites for CEA detection were compared other electrochemistry immunoassay in Table 1. The results showed the assay performance of immunosensor was accepted.
Selectivity and reproducibility of the fabricated immunosensor. In order to study the selectivity of the immunosensor, the squamous cell carcinoma antigen (SCCA), alpha fetal protein (AFP), vitamin C (Vc) and glucose found in the serum were selected as the interfering substances. In this paper, the 1 ng/mL CEA containing 20 ng/mL interfering substances was measured by the immunosensor. As indicated from Figure S1, when CEA coexisted with these sample interfering agents, no apparent signal change took place in comparison with that of only CEA. Meanwhile, significantly higher current response change was observed with the target CEA than with other biomarkers, which indicated that the proposed immunosensor revealed sufficiently selectivity for the detection of CEA. The relative standard deviation (RSD) of the measurements was 1.02%, which indicated that the selectivity of the immunosensor was acceptable.
In addition, the reproducibility also was an important parameter of the performance of the immunosensor. Therefore, five of identical batches of immunosensors were fabricated and measured for 1 ng/mL CEA in Figure  S2. The RSD of the measurements was less than 3%, illustrating that the fabricated immunosensor has acceptable fabrication reproducibility.
Analysis of real sample. In order to validate the feasible of the immunosensor, the recovery test with different concentrations of CEA in serum sample was carried out by standard addition method. The relative errors of the results were less than 5% for CEA detection in Table S1, and the recoveries were in the range of 98.4%-102.2%, suggesting good accuracy of the proposed method for real samples. Conclusions A novel label-free immunosensor based on Au/PDA/Fe 3 O 4 @C@PGC nanocomposites was fabricated for the high sensitivity detection of CEA. Moreover, the fabricated immunosensor shows a wide linear range, a low detection limit, good specificity and acceptable reproducibility for the detection of CEA. In addition, the method is also applied to determine the CEA in the serum and shows the great application prospect in diagnosis and treatment of cancer.