Highly Sensitive Immunosensing of Carcinoembryonic Antigen Based on Gold Nanoparticles Dotted PB@PANI Core-Shell Nanocubes as a Signal Probe

Herein, a method was developed for the sensitive monitoring of carcinoembryonic antigen (CEA) by gold nanoparticles dotted prussian blue@polyaniline core-shell nanocubes (Au NPs/PB@PANI). First, a facile low-temperature method was used to prepare the uniform PB@PANI core-shell nanocubes with the assistance of PVP, where PB acted as the electron transfer mediator to provide electrochemical signals, and the PANI with excellent conductivity and desirable chemical stability not only played the role of a protective layer to prevent etching of PB in basic media but also effectively improved electron transfer. Importantly, to further enhance the electrical conductivity and biocompatibility of PB@PANI and to further enhance the electrochemical signal and capture a large amount of Ab2, Au NPs were doped on the surface of PB@PANI to form Au NPs/PB@PANI nanocomposites. Subsequently, benefiting from the advantages of core-shell structure nanoprobes and gold-platinum bimetallic nanoflower (AuPt NF), a sandwich-type electrochemical immunosensor for CEA detection was constructed, which provided a wide linear detection range from 1.0 pg·mL−1 to 100.0 ng·mL−1 and a low detection limit of 0.35 pg·mL−1 via DPV (at 3σ). Moreover, it displayed a satisfactory result when the core-shell structure nanoprobe-based immunosensor was applied to determine CEA in real human serum samples.


Introduction
As we all know, carcinoembryonic antigen (CEA) is a tumor marker for colon cancer, breast cancer, ovarian cancer, and other cancers, which can provide reliable information for the early diagnosis and treatment of tumor patients [1][2][3]. Consequently, the highly sensitive determination of CEA is a pressing need by virtue of accurate and efcient analytical techniques. Immunosensors based on antibody-antigen interaction are one of the most widely used analytical techniques in the quantitative detection of biomarkers [4]. Among them, electrochemical immunosensors have attracted much attention due to their characteristics of high specifcity, good sensitivity, short time consumption, low energy consumption and trace detection, and more suitable for the detection of biomarkers in low concentration [5,6]. More recently, lots of electrochemical immunosensors have been constructed for quantitation of CEA. For example, Wang et al. Jozghorbani et al. and Wang et al. designed three kinds of label-free CEA immunosensors with detection limits of 0.005, 0.05, and 0.0429 ng·mL −1 , respectively [7][8][9]. Especially, a large number of reports have focused on the sandwich-type electrochemical immunosensor for CEA detection [10][11][12][13], thanks to their distinct advantages such as lower background noise, higher sensitivity, and importance, and they have higher selectivity for analytes through two specifc reactions, which are superior to label-free counterparts [14][15][16].
Prussian blue (PB) with a face-centered cubic lattice structure is popular as a kind of electrochemical redoxactive species in electrochemical biosensors because of its high electrochemical/electrocatalytic properties and low redox potential [17,18]. Unfortunately, the poor stability and low conductivity of PB limit its further applications in biosensors [19]. To minimize these problems, some conducting materials can be introduced to meet the requirements mentioned above. Polyaniline (PANI) as one of the most desired materials with good chemical stability, low cost, and good electrical conductivity can not only provide a conductive substrate but also can form an efective coating layer on "the core" such as carbon nanotubes (CNTs) or PB [20,21]. Te combination of PB and PANI (PB@PANI) can play a signifcant synergistic efect and improve the electrocatalytic performance, conductivity, and stability [21]. Furthermore, to further enhance the conductivity and biocompatibility of PB@ PANI, gold nanoparticles (Au NPs) were introduced over the surface of PB@PANI by Au-N bonds between Au NPs and -NH 2 from PANI to form Au NPs/PB@PANI nanocomposites [15]. To the best of our knowledge, the preparation and application of Au NPs/PB@PANI nanocomposites in electrochemical immunosensors have not been reported. Hence, the PB@PANI loaded with Au NPs (Au NPs/PB@PANI) would be a promising nanoprobe.
For sandwich-type electrochemical immunosensors, to achieve signal amplifcation and high sensitivity, it is a more important key to efectively immobilize the primary antibody (Ab 1 ) [22,23]. Gold and platinum nanoparticles are the most promising bimetallic materials for various applications due to their superior biocompatibility, higher specifc surface area, and superior electrocatalytic properties towards the reduction of H 2 O 2 [24][25][26]. Tey can be decorated onto the surface of the L-cysteine (L-Cys)-modifed electrode to provide an available microenvironment for loading amounts of Ab 1 , thus greatly improving the sensitivity of the immunosensor.
Tis work was developed by electrodeposition of AuPt bimetallic nanofower (AuPt NFs) on the GCE modifed with L-Cys as a sensing platform and Au NPs/PB@PANI as a novel label prepared by a facile low-temperature method. Te existence of AuPt NFs not only fxed the primary antibody but also accelerated the electron transfer. Furthermore, the as-developed Au NPs/PB@PANI nanoprobes were used as a tracer for the generation and amplifcation of electrochemical signals and easily captured second antibodies (Ab 2 ) via Au-N bond. As expected, a sandwich-type electrochemical immunosensor capable of achieving large signal amplifcation was developed by connecting Ab 1 -AuPt NFs/L-Cys with Ab 2 -Au NPs/PB@PANI.

Devices.
Electrochemical measurements were made at the CHI 660E electrochemical workstation (Shanghai Co., Ltd., China). Te morphology of nanomaterials was obtained by a scanning electron microscopy (SEM, JSM-7100F, Japan) and a transmission electron microscopy (TEM, JEM-6700F, Japan).

Synthesis of PB Nanocubes.
PB nanocubes were synthesized according to a previous work despite a minor modifcation [27]. 8.0 mmoL·L −1 Na 4 Fe(CN) 6 .8H 2 O, 4.0 mL hydrochloric acid (37%), and 2.0 g polyvinyl pyrrolidone (M w ∼ 40000) were added into deionized water (400 mL) and stirred for 30 min, and then were refuxed at 60°C for 6 h. After that, the blue products were washed with deionized water and dried in vacuum at 100°C for 24 h.

Preparation of PB@PANI Core-Shell Nanocubes.
PB@PANI core-shell nanocubes were synthesized with reference to previous work with slight variations [21]. Briefy, 0.6 g·PB, 0.5 g polyvinyl pyrrolidone (M w ∼ 40000) and 100 μL aniline monomer were dissolved in 100 mL hydrochloric acid (1 moL·L −1 ) with sonication for 1 h. Ten, ammonium persulfate solution (1.6 mmoL·L −1 ) was added dropwise into the above solution and stirred for 18 h in an ice bath. Finally, the PANI-coated PB (core-shell structure PB@PANI) was collected by centrifugation and washed with deionized water and ethanol for several times.

Preparation of Ab 2 -Au NPs/PB@PANI Bioconjugates.
15 mg PB@PANI nanocubes were added into the concentrated Au NPs colloidal solution (5.0 mL, 1.0 mg·mL −1 ) and reacted 30 min under shaking. Au NPs were assembled on the surface of PB@PANI to form the Au NPs/PB@PANI nanocomposites by the chemical bond of Au-NH 2 [15]. Followed by centrifugation and washing twice with deionized water, the obtained precipitate was redispersed into PBS solution (pH 7.0). Subsequently, 500 μL CEA-Ab 2 (2.0 mg·mL −1 ) was added into the Au NPs/PB@PANI suspension (2 mL) and stirred for 12 h at 4°C. To get rid of nonspecifc adsorption, 100 μL of 1% BSA was mixed with the obtained conjugates for 6 h at 4°C. Finally, the bioconjugates were centrifuged and redispersed in PBS (pH 7.0, 10 mmoL·L −1 ), and then stored at 4°C.

Fabrication of the Immunosensor.
For polymer nanocomposite flm deposition, polished glass carbon electrode (GCE) was frst immersed in 0.5 mmoL·L −1 cysteine containing PBS (pH 7.0) and was scanned in the potential range of −0.0 to 1.7 V at 50 mV/s for 3 cycles. Te obtained electrode was dried in air, then dipped into 0.2 moL·L −1 H 2 SO 4 and the solution containing 0.5 mmoL·L −1 HAuCl 4 and 0.5 mmoL·L −1 H 2 PtCl 6 , and electrodeposited with chronoamperometric method at −0.2 V vs. Ag/AgCl for 600 s at room temperature. After rinsing, the AuPt NFs/L-Cys/GCE-modifed electrode was immersed in primary antibody (CEA-Ab 1 ) solution at 4°C overnight, and Ab 1 was connected to the modifed electrode. Next, to remove excess binding sites, the modifed electrode was immersed in 1 wt% BSA solution at room temperature for 40 min. Finally, the obtained electrode was washed three times with PBS and stored at 4°C. Scheme 1(a) displays the schematic of the designed immunosensor, and Scheme 1(b) shows the preparation procedure of Ab 2 -Au NPs/PB@PANI bioconjugates.

Electrochemical Detection.
Te prepared immunosensors were incubated in the solutions of CEA antigen with diferent concentrations for 40 min and then continued incubated in Ab 2 -Au NPs/PB@PANI solution at 37°C for the same time. Subsequently, diferential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were performed in PBS (pH 6.5) and 5 mmoL·L −1 [Fe (CN) 6 ] 3-/4-, respectively.

Results and Discussion
3.1. Characterization of the Nanomaterials. SEM images of the PB, the PB@PANI, and the Au NPs/PB@PANI are displayed in Figures 1(a)-1(c). Te as-prepared PB nanocomposites were made up of cubes with side lengths in the range of 300-320 nm (Figure 1(a)), which had a rough surface to provide abundant nucleation sites for the uniform growth of PANI shell [28]. It was clearly seen that the nanocubes of PB were completely covered by the PANI shell, and the thickness of the PANI was about 10 nm after the reaction time of 18 h (Figure 1(b)). When Au NPs with diameters of about 15 nm were attached to the surface of PB@PANI, we clearly observed that Au NPs with uniform globular morphology were absorbed on the surface of PB@PANI core-shell nanocubes (Figure 1(c)), which can also immobilize CEA-Ab 2 through Au-N bonds. Figures 1(d) and 1(e) show the morphology of the electrode. As exhibited in Figure 1(d), the surface of the bare GCE was not smooth. By electrodeposition, the L-Cys flm was deposited on the surface of GCE (Figure 1(e)), which maybe infuence on the size and crystalline structure of AuPt bimetallic nanofowers (AuPt NFs) according to previous reports [29]. At the same time, the presence of L-cysteine helped to bind the AuPt NFs on the surface of GCE. Quasi-spherical fowerlike AuPt NFs with diameters of about 50 nm were observed after the electrodeposition on L-Cys/GCE (Figure 1(f )).
Meanwhile, Au, Pt, C, N, and S elements were found in the energy dispersive spectroscopy (EDS) of AuPt NFs/L-Cys/GCE ( Figure S1A). EDS can further prove that Au NPs/ PB@PANI nanocomposites were composed of Au, Pt, K, N, C, O, and Fe elements (as shown in Figure S1B).

Characterization of the Constructed Immunosensor.
CV and EIS measurements were utilized to monitor the stepwise assembly process of the immunosensors [30]. As shown in Figure 2(a), the peak current of L-Cys flm modifed GCE electrode by one-step electropolymerization was decreased clearly (curve b) compared with the bare GCE electrode (curve a), indicating L-Cys flm hindered electron transfer. In addition, modifcation of AuPt NFs onto the flm of L-Cys caused an increase in peak current benefting from excellent conductivity of the AuPt NFs (curve c). After Ab 1 being adsorbed on the modifed electrode (curve d), blocking with BSA (curve e), and modifying with Ag (curve f ), a further decrease in the peak currents was seen, due to the efect of biomacromolecules hindering electron transport. However, the peak currents increased signifcantly after the modifed electrodes were combined with Ab 2 -Au NPs/PB@PANI bioconjugates (curve g), indicating that Ab 2 -Au NPs/PB@PANI bioconjugates can greatly enhance electron transfer. Moreover, EIS results of each step of electrode modifcation were in accordance with those of CV.

Optimization of Experimental Conditions.
To improve the sensitivity and analysis efciency of the proposed immunosensor, the experimental conditions were optimized. As shown in Figure 3(a), when incubation time was 20 min-80 min, the DPV response increased gradually and then reached a plateau after 40 min because the combination of the antigen with Ab 1 reached equilibrium in approximately 40 min.
Te pH value of bufer solution has a signifcant infuence on the stability and activity of electrochemical biosensors. As can be seen from Figure 3(b), with the change of pH, the signal of DPV response also changed. In the range of pH 5.0-8.0, DPV value frst increased and then slightly decreased. Au NPs/PB@PANI nanoprobes possessed optimal activity and stability in approximately neutral or acid environment due to an efective coating layer of conducting PANI. Terefore, we chose PBS solution with a pH of 6.5 as the appropriate electrolyte.
In the meantime, the deposition time of AuPt NFs had a signifcant efect on DPV response as well. As displayed in Figure 3 from 200 to 1000 s, the current signal increased gradually and then reached a plateau at 600 s. Terefore, the deposition time of AuPt NFs was selected as 600 s for the preparation of the immunosensor. Similar to reported biosensor, the performance of immunosensor was highly afected by the concentration of Au NPs/PB@PANI. As shown in Figure 3(d), with the increasing concentration from 0.5 mg·mL −1 to 2.0 mg·mL −1 , the current response increased rapidly, but when the concentration was higher than 2.0 mg·mL −1 , the current response reached a plateau. Terefore, 2.0 mg·mL −1 became the optimum concentration for this study.

Quantitative Detection of CEA.
Under optimal experimental conditions, CEA was quantifed by using the developed immunosensor. As shown in Figure 4, the DPV signal increased gradually as the CEA concentration increased. Tere was a good linear relationship between the current intensity and the logarithm of CEA concentration in the range of 0.001-100 ng·mL −1 . Te regression equation was I P (μA) � 4.6652 + 0.7570x (R 2 � 0.9981) with 0.35 pg·mL −1 detection limit. In comparison with other nanoprobe-based immunosensors [31][32][33][34][35][36][37], the detection limit of the prepared immunosensor was lower (Table 1). Te reasons for the excellent analytical performance of the designed immunosensor  may be summarized as follows: frst, PANI, as the conductive shell of PB@PANI core-shell structure, maintained the structural stability of PB and efectively improved the electron transfer from PANI to PB due to the intimate adhesion. Second, a large amount of Au NPs distributed on the PB@ PANI surface provided a large specifc surface area for the binding of antibodies, which enhanced the immobilization of CEA-Ab 2 . In addition, the Au NPs/PB@PANI nanoprobes with the unique structure and synergetic contributions exhibited good conductivity and high stability, increased the conduction efciency of signal molecules, and can be used as a signal amplifcation system to improve the sensitivity of detection. Finally, AuPt NFs with multifunctionality were used as an ideal sensing platform, not only can capture more Ab 1 and improve conductivity but also constituted a dual signal amplifcation system together with Au NPs/PB@PANI nanoprobes.

Specifcity, Stability, and Reproducibility of the
Immunosensor. In order to investigate the specifcity of the constructed immunosensor targeting CEA, 100.0 ng·mL −1 of  four interferences including alpha fetoprotein (AFP), ascorbic acid (AA), glucose (Glu), and BSA was added to CEA (1.0 ng·mL −1 ) solution, respectively. As displayed in Figure 5, these interferences made a neglectful infuence to the target, suggesting the proposed biosensor presented high specifcity for the specifc recognition of CEA.
Te stability experiments of the immunosensor were carried out by storing several immunosensors at 4°C for 30 days, which were tested for CEA every 5 days (Figure 6(a)). After 30 days, the DPV value retained 88.6% of its initial response. Te efective fxation of Ab 2 on the Au NPs/PB@PANI nanoprobe and the efcient protection of PB in the PANI coating made the developed immunosensor have high stability.
To further verify the reproducibility of the immunosensor, six immunosensors were incubated in 10.0 ng·mL −1 of CEA for DPV determination. As shown in Figure 6(b), the RSD was 3.1% after measurement, demonstrating the reproducibility of the immunosensor was acceptable.

Determination of Real Serum Sample.
To certify analytical reliability and application potential of the developed immunosensor, ten clinical serum specimens containing CEA were analyzed. As displayed in Table 2, the relative   Journal of Analytical Methods in Chemistry error between the two methods was in the range from −5.26% to 6.94%, and these data revealed that the developed immunosensor had good practicability for clinical serum analysis.

Conclusions
In conclusion, an electrochemical immunosensor based on gold nanoparticles functionalized PB@PANI core-shell nanocubes as a signal amplifcation strategy was fabricated for CEA detection. Te high sensitivity of the immunosensor was attributed to the reasons as follows. First, the abundant N-species in PANI can be coupled with PB to efectively protect the structural degradation of PB in basic media. Second, Au NPs/PB@PANI nanocubes containing abundant electron mediators provided a large signal of DPV. Finally, AuPt NFs were dotted on the L-Cys/GCE electrode to increase the specifc surface area and electrical conductivity, which not only immobilize more antibodies but also accelerate the electron transfer. Moreover, the designed immunosensor showed the especial merit that avoided the addition of mediators to the bufer or separated the immobilization of mediators onto the electrode. Promisingly, the strategy can be easily extended to the application scope of Au NPs/PB@PANI nanocomposites.

Data Availability
Te data supporting this study are from previously reported studies and datasets, which have been cited.

Disclosure
A preprint of the manuscript has previously been published [38].

Conflicts of Interest
Te authors declare that they have no conficts of interest. Journal of Analytical Methods in Chemistry 7