Ternary Pt@Pd@Ru nanodendrite-decorated graphene oxide for sensitive electrochemical immunoassy of CEA

Abstract

Nobel metal nanoparticles have attracted intense attentions in biological immunoassay due to the inhereted good catalytic activity. In this study, a new kind of nanocomposite of ternary Pt@Pd@Ru nanodendrite-decorated graphene oxide (GO–Pt@Pd@Ru) was prepared as a signal label for ultrasensitive detection of carcinoembryonic antigen (CEA). With a sandwich type immunoassay, the established electrochemical immunosensor showed highly sensitive detection performance towards CEA in a linear range (0.1 pg mL⁻¹ to 9 ng mL⁻¹) with an ultralow detection limit of 0.05 pg mL⁻¹. The enhancing detection performance could be attributed to: ternary Pt@Pd@Ru nanodendrites have exhibited superior electrochemical reduction activity towards H₂O₂, then GO–Pt@Pd@Ru can combine all generated electrochemical reduction current towards H₂O₂ by the decorated Pt@Pd@Ru nanodendrites, which can significantly amplify the detection signal.

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1. Introduction

Sensitive and reliable detection of tumor biomarkers plays an important role in cancer diagnosis and therapy.¹ The carcinoembryonic antigen (CEA) has been taken as a broad-spectrum tumor biomarker for clinical detection of some kinds of cancers, such as colon cancer, breast cancer and lung cancer.² Therefore, there is an urgent desire to develop methods for sensitive detection of CEA. Currently, many immunoassay approaches, like magnetoimpedance immunosensor,³ Raman spectro-immunoassay,⁴ enzyme-linked immunosorbent assay,⁵ radioimmunoassay,⁶ chemiluminescence immunoassay⁷ and electrochemical immunoassay,⁸ have been used to detect CEA. Especially, electrochemical immunoassay has gained much more research interest owing to the advantages of portability, speedy analysis, cost effectiveness and high sensitivity.⁹ In order to enhance the sensitivity of electrochemical immunosensor, a variety of nanomaterials, such as graphene,¹⁰ carbon nanotubes,¹¹ metal nanoparticles,¹² were used achieving signal amplification for the detection of protein biomarkers. Among these nanomaterials, metal nanoparticles especially noble metal nano-alloy containing bimetallic and trimetallic nano-alloy attached considerable attentions.¹³ Compared with monometallic nanoparticles, nano-alloy presented higher catalytic performance.^14–16 Herein, we demonstrated an electrochemical immunosensor using ternary Pt@Pd@Ru nanodendrite-decorated graphene oxide (GO–Pt@Pd@Ru) for sensitive detection of CEA. Ternary Pt@Pd@Ru nanodendrites have exhibited superior electrochemical reduction activity towards H₂O₂ because Pt@Pd@Ru nanodendrites provide many catalytically active sites for catalytic reactions. Therefore, the new nanocomposite of GO–Pt@Pd@Ru can merge all generated electrochemical reduction current towards H₂O₂ by loaded Pt@Pd@Ru nanodendrites and play as signal labels for electrochemical immunoassay of CEA, which can significantly improve the sensitivity of the developed electrochemical immunosensor.

2. Experimental

2.1 Materials and apparatus

Graphene oxide (GO) was prepared by modified Hummer's method.¹⁷ Dopamine hydrochloride, potassium hexachloroplatinate (K₂PtCl₆) disodium tetrachloropalladate (Na₂PdCl₄) ruthenium chloride (RuCl₃) and nano-iron oxide (Fe₃O₄) were purchased from Aladdin-industrial-corporation. Ascorbic acid (AA) and bovine serum albumin (BSA) and Pluronic F127 were bought from Sigma Company. Carcinoembryonic antigen (CEA) and CEA antibody were purchased from Shanghai E. Star Biotechnology Co., Ltd. (Shanghai, China). Phosphate buffered solutions (PBS) were prepared by mixing 0.067 mol L⁻¹ Na₂HPO₄ and 0.067 mol L⁻¹ KH₂PO₄ stock solution. Ultrapure water was used throughout the experiments.

All electrochemical measurements were performed on a CHI760E electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd., China). Transmission electron microscopy (TEM) images were recorded with a Philips CM200 UT (Field Emission Instruments, USA). Conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GCE, 4 mm in diameter) as the working electrode, a platinum wire electrode as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode.

2.2 Synthesis of amino functionalization of Fe₃O₄ (NH₂-Fe₃O₄) and conjugated Ab₁

10 mg mL⁻¹ of Fe₃O₄ was dispersed in 100 mL of dopamine hydrochloride solution (0.5 M). After that, this mixture was sonicated for 1 h and the product (NH₂-Fe₃O₄) was washed with ultrapure water three times collected by magnet. 2 mg of NH₂-Fe₃O₄ was dispersed in 1 mL PBS (pH 7.4) after adding 1 mL 0.25% glutaraldehyde and reacting for 12 h. Then, the mixture was separated by magnet. Afterwards, the product was shocked for 12 h after adding 1 mL of 200 ng mL⁻¹ Ab₁. Then, the solution was centrifuged to remove the unbonded antibody and then the product was redispersed in 1 mL PBS (pH 7.4) for electrochemical detection.

2.3 Synthesis of GO–Pt@Pd@Ru nanocomposites

Pt@Pd@Ru nanodendrites were prepared following the previous work.¹⁸ Thereafter, 2.0 mg of Pd@Pt@Ru nanodendrites were added into 5 mL of GO (2 mg mL⁻¹) solution and shocked for 12 h. Then, the resulted GO–Pt@Pd@Ru nanocomposites were collected after centrifugation and washed with ultrapure water for three times. Finally, the nanocomposites were dried under vacuum. Pt@Pd and Pt@Ru nanoparticles were prepared by the similar procedure of Pt@Pd@Ru, but there was no precursor solution of RuCl₃ and Na₂PdCl₄, respectively.

2.4 Synthesis of GO–Pt@Pd@Ru conjugated Ab₂ as labels

2.0 mg of GO–Pt@Pd@Ru were dispersed in 2 mL of Ab₂ (100 μg mL⁻¹). The mixture was shocked at 4 °C for 12 h and washed with PBS (pH 7.4) in consecutive centrifugation. The final product (GO–Pt@Pd@Ru-Ab₂) were redispersed in 1 mL of PBS (pH 7.4) and kept at 4 °C until used.

2.5 Fabrication of immunosensor

The fabrication process of electrochemical immunosensor can be illustrated as Scheme 1. A GCE was polished with alumina slurry to obtain a mirror-like surface and dried at temperature. 6.0 μL of Fe₃O₄-Ab₁ (1.5 mg mL⁻¹) was dropped on the surface of GCE and left to dry naturally at room temperature. Then 3.0 μL of 1%wt BSA was put onto the electrode surface to block nonspecific active sites. Afterwards, the electrode was washing with PBS (pH 7.4) to remove the excess BSA and then 6.0 μL of CEA with different concentrations was added onto the electrode surface for one hour. Thereafter 6.0 μL of GO–Pt@Pd@Ru-Ab₂ was added onto the electrode surface for one more hour, and the electrode was washed thoroughly with PBS (pH 7.4) for electrochemical detection.

Scheme 1 Illustration the fabrication process of the immunosensor.

3. Results and discussion

3.1 Characterization of Fe₃O₄, GO–Pt@Pd@Ru and Pt@Pd@Ru

As shown in Fig. 1A, Fe₃O₄ nanoparticles exhibited irregular lamellar structure and its average diameter was 200 nm approximately. It was clearly observed that Pt@Pd@Ru had dendritic structure which could provide many catalytic active sites and present higher catalytic performance (Fig. 1B). It was obviously seen that Pt@Pd@Ru nanoparticles were uniformly decorated on the surface of GO, shown in Fig. 1C, indicating that GO–Pt@Pd@Ru were synthesized successfully.

Fig. 1 TEM images of Fe₃O₄ nanoparticles (A), Pt@Pd@Ru nanodendrites (B) and GO–Pt@Pd@Ru (C).

As revealed in Fig. 2, it was cyclic voltammetry (CV) curves of bare GCE (black curve) and the electrode modified with 1.5 mg mL⁻¹ of NH₂-Fe₃O₄ (red curve) in 0.1 mol L⁻¹ KCl solution containing 5.0 mM Fe(CN)₆³⁻. It was evident that the CV of bare GCE and NH₂-Fe₃O₄ modified electrode were both shown a well-defined reversible redox behavior of [Fe(CN)₆]^3−/4−. And the peak current of NH₂-Fe₃O₄ modified electrode was higher than that of bare electrode, indicating that NH₂-Fe₃O₄ modified electrode had better conductivity, which could improve the sensitivity of electrochemical immunosensor.

Fig. 2 Cyclic voltammetry curves of bare GCE (black) and the electrode modified with 1.5 mg mL⁻¹ of NH₂-Fe₃O₄ (red) in 0.1 mol L⁻¹ KCl solution containing 5.0 mM Fe(CN)₆³⁻.

Compared with the CV of bare GCE (curve a) in pH 7.4 of PBS, it could be seen that the CV of GO–Pt@Pd@Ru modified electrode (curve b) was shown a wider range, indicating GO–Pt@Pd@Ru presents good conductivity (Fig. 3A). Additionally, it could be seen that a reduction peak of H₂O₂ was at −0.3 V (curve c) with relative high reduction current. Fig. 3B presented the i–t curves of the electrodes modified by the same concentration of Pt@Ru (a), Pt@Pd (b), Pt@Pd@Ru (c) and GO–Pt@Pd@Ru (d) in pH 7.4 PBS with 5 mM H₂O₂ at −0.3 V. Obviously, GO–Pt@Pd@Ru showed the best electrochemical reduction performance towards H₂O₂. As a result, we established the sensitive electrochemical immunosensor for CEA detection using high catalytic GO–Pt@Pd@Ru as signal labels.

Fig. 3 (A) Cyclic voltammetry curves of bare GCE (a) and GO–Pt@Pd@Ru modified electrode (b) in pH 7.4 PBS without H₂O₂, GO–Pt@Pd@Ru modified electrode with 5 mM H₂O₂ in pH 7.4 PBS (c). (B) i–t curves of the electrodes modified by the same concentration 2 mg mL⁻¹ Pt@Ru (a) Pt@Pd (b), Pt@Pd@Ru (c) GO–Pt@Pd@Ru (d) in pH 7.4 PBS with 5 mM H₂O₂ at −0.3 V.

3.2 Characterization of the assembly procedure of immunosensor

The assembly procedure of electrode was characterized by electrochemical impedance spectroscopy (EIS). The impedance spectra consist of a semicircle portion corresponding to the electron-transfer limited process and a linear portion representing the diffusion-limited process and the semicircle diameter was presented electron-transfer resistance.¹³ As shown in Fig. 4, it could be seen that the resistance of GCE (curve a) was the smallest one. After modifying Ab₁-NH₂-Fe₃O₄ (curve b), the resistance of electrode was increased obviously. The reason was that antibody as a kind of protein could hinder electro transfer. Similarly, because protein molecules could hinder electron transfer, the corresponding resistances (curve c, d and e) would increase sequentially when the electrode was following modified by BSA, CEA and GO–Pt@Pd@Ru-Ab₂, which also indicated that the electrode was well-modified. In conclusion, the electrochemical immunosensor was established successfully.

Fig. 4 EIS recorded for the modified electrodes in 0.1 mol L⁻¹ KCl solution containing 5.0 mmol L⁻¹ Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ (1 : 1). (a) GCE, (b) Fe₃O₄-Ab₁/GCE, (c) BSA/Fe₃O₄-Ab₁/GCE, (d) CEA/BSA/Fe₃O₄-Ab₁/GCE and (e) GO–Pt@Pd@Ru-Ab₂/CEA/BSA/Fe₃O₄-Ab₁/GCE.

3.3 Optimizing conditions

In order to achieve good performance of the electrochemical immunosensor, experimental conditions of concentration of Fe₃O₄, concentration of GO–Pt@Pd@Ru and pH were optimized. It is clear to see that the current value reaches the maximum at the 1.5 mg mL⁻¹ of Fe₃O₄ as shown in Fig. 5A (black line). Similarly, the maximum current response of the immunosensor occurred at 2.0 mg mL⁻¹ of GO–Pt@Pd@Ru, pH 7.4 of PBS and 60 minutes of incubation time, seen from Fig. 5A (red line), Fig. 5B and C, respectively. Thus, 1.5 mg mL⁻¹ of Fe₃O₄, 2.0 mg mL⁻¹ GO–Pt@Pd@Ru and pH 7.4 of PBS were selected as the optimized conditions for the fabricated electrochemical immunosensor.

Fig. 5 Current responses of the fabricated immunosensor in pH 7.4 PBS solution with 5 mM H₂O₂ towards: (A) different concentrations of Fe₃O₄ (black line) and GO–Pt@Pd@Ru (red line), respectively; (B) different pH PBS solution; (C) different incubation time.

3.4 Analytical performance

Under the optimal conditions, the immunosensor was fabricated for the detection of CEA. The current responses towards electrochemical reduction of H₂O₂ at −0.3 V were increased proportionally with the increasing concentrations of CEA, shown in Fig. 6A. Accordingly, a calibration curve towards the current responses for different concentrations of CEA was built: ΔI = 195.17 + 31.131 log C, R² = 0.9836 (Fig. 6B). It is satisfied to see that the electrochemical immunosensor presented a broad linear range of 0.1 pg mL⁻¹ to 9 ng mL⁻¹ with an ultralow detection limit of 0.05 pg mL⁻¹ (S/N = 3). As shown in Table 1, this method presented more sensitive performance towards CEA detection comparing with other reported methods.

Fig. 6 (A) Current responses of the immunosensor in proportion to different concentrations of CEA in pH 7.4 PBS solution with 5 mM H₂O₂ at −0.3 V. (B) Calibration curve of the immunosensor in the linear concentration range of CEA (0. 1 pg mL⁻¹ to 9 ng mL⁻¹).

Table 1 Comparison the detection performance of CEA by different methods

Method	Linear range (ng mL^−1)	Detection limit (pg mL^−1)	Reference
Pb-alginate	0.01–100	8.6	19
AuNps	0.01–80	2.36	20
AgNps	0.001–50	0.27	21
Au–Ag/BMNPs	0.001–50	0.3	22
This method	0.0001–9	0.05

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3.5 Selectivity, stability and reproducibility

To investigate the selectivity of the developed electrochemical immunosensor, interference study was conducted using BSA, alpha-fetal protein (AFP), glucose and vitamin C (Vc) as interfering substances. 0.1 ng mL⁻¹ of CEA adding 100 ng mL⁻¹ of individual interfering substance were tested by the immunosensor, respectively. According to the results shown in Fig. 7A, the relative standard deviation (RSD) of current response with or without interfering substances was within 3%, indicating that the selectivity of the immunosensor was fairly good.

Fig. 7 Amperometric response of the immunosensor for the detection of 0.1 ng mL⁻¹ CEA in pH 7.4 PBS solution with 5 mM H₂O₂: (A) coexisting different interfering substances: (1) blank, (2) 100 ng mL⁻¹ BSA, (3) 100 ng mL⁻¹ AFP, (4) 100 ng mL⁻¹ glucose and (5) 100 ng mL⁻¹ Vc; (B) during the storage at 4 °C and (C) to five different electrodes, error bar = RSD (n = 5).

To evaluate the stability of the immunosensor, the immunosensor was periodically measured the current response during the storage at 4 °C (Fig. 7B). Even after four weeks, the immunosensor could generate around 90% of the initial current. This result proved that the immunosensor had good stability.

To check the reproducibility of the immunosensor, 0.1 ng mL⁻¹ of CEA was detected by a series of five electrodes (Fig. 7C). The measured RSD was within 4%, which means good reproducibility for the developed immunosensor.

In order to demonstrate the feasibility of the electrochemical immunosensor for clinical diagnosis, we measured the recovery of different concentrations of CEA by standard addition methods. As shown in Table 2, the recovery was within the range of 98.0–102%, revealed that the immunosensor could be applied into clinical analysis of CEA.

Table 2 Recovery of the fabricated immunosensor in human serum

Sample	Added (ng mL^−1)	Found (ng mL^−1)	Recovery (%)
1	0.5	0.51 ± 0.02	102.0
2	1.0	0.98 ± 0.05	98.0
3	1.5	1.52 ± 0.1	101.3
4	2.0	2.04 ± 0.3	102.0
5	2.5	2.49 ± 0.4	99.6

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4. Conclusions

In summary, Pt@Pd@Ru nanodendrites-decorated graphene oxide as a new kind of nanocomposite has been taken as signal label for enhancing sensitive detection of CEA. The excellent performance of the fabricated immunosensor could be contributed to: ternary Pt@Pd@Ru nanodendrites exhibited superior electrochemical reduction activity towards H₂O₂, and then Pt@Pd@Ru nanodendrite-decorated graphene oxide integrated all generated electrochemical reduction current towards H₂O₂ from each decorated Pt@Pd@Ru nanodendrite, which can significantly enhance the detection signal.

Acknowledgements

The authors would like to thank the National Natural Science Foundation of China (No. 21245007 and 81000976) and Postdoctoral Science Foundation of China (No. 2012M521295) for the financial support.

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