Sandwich-type electrochemical immunosensor for ultrasensitive detection of prostate-specific antigen using palladium-doped cuprous oxide nanoparticles

Abstract

A new and facile sandwich-type electrochemical immunosensor is proposed for the ultrasensitive detection of prostate-specific antigen (PSA) based on Au nanoparticles (Au NPs) and palladium-doped cuprous oxide nanoparticles (Pd@Cu₂O NPs). In this study, Au NPs were used as a substrate material to immobilize primary antibodies (Ab₁) and to accelerate electron transfer. The Pd@Cu₂O NPs were obtained through the prepared cuprous oxide nanoparticles (Cu₂O NPs) by the in situ reduction of palladium chloride (PdCl₂). The immunosensor was employed as a label for secondary antibodies (Ab₂), presenting efficient electrocatalytic activity toward the reduction of hydrogen peroxide (H₂O₂). Under optimal conditions, the proposed immunosensor exhibited a wide linear response range from 10⁻⁵ to 100 ng mL⁻¹ with a low detection limit of 2 fg mL⁻¹. We thus established and demonstrated a simple, fast, and sensitive strategy for the ultrasensitive detection of PSA, which could find promising application in clinical diagnoses and other fields.

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1. Introduction

Because of the effects of the polluted ambient environment and the prevalence of food coloring in many foods, the incidence of cancer is rising year by year. For example, prostate cancer, which is representative of men's health, is at record high levels, especially in middle-aged men. Due to the high recurrence rate of prostate cancer after treatment, prostate-specific antigen (PSA) has become a vital analyte of prostate cancer in clinical diagnosis over the last decade and is also an efficient indicator to diagnose the recurrence of prostate cancer.^1,2 The concentration and activity of PSA in serum have been detected for decades in clinical measurements.³ In general, 4 ng mL⁻¹ of PSA is the cutoff value in serum, and when PSA levels in serum are above 4 ng mL⁻¹, it is generally thought to indicate a risk of prostate cancer.^4,5 It is therefore necessary and imperative to detect PSA with high accuracy and sensitivity in the field of modern clinical settings.

With an aim to avoid surgery and other invasive and expensive medical treatments for patients, researchers have been seeking methods to detect and treat PSA to foresee outbreaks of prostate cancers.⁶ A variety of immunoassays have also been fabricated to test PSA, such as the enzyme-linked immunosorbent assay (ELISA),⁷ electrophoretic immunoassay,⁸ micromechanical pillars,⁹ chemiluminescence immunoassay,¹⁰ and electrochemical immunoassay.¹¹ Among these methods, the electrochemical immunoassay has received a lot of interest despite it having some drawbacks, such as being time-consuming and needing expensive instruments as well as skillful operators. Electrochemical techniques are the most desired systems due to their excellent sensitivity, rapidity, low detection limits, and ease of handling.¹² Currently, electrochemical immunosensor technology has been spread worldwide in a fairly mature way. The underlying physicochemical principle in immunoassays is that captured antibodies are physically adsorbed or covalently immobilized on a solid surface in a random pattern,^13,14 and after antigen binding, signal antibodies can be used to produce a detectable signal, whereby a measurable electrical signal is represented by the label attached to a secondary antibody (Ab₂).¹⁵

Hunting strategies and new labels to fabricate electrochemical immunosensors are of great value. The choice of labels could address a critical need for the self-assembly of electrochemical immunosensors and help determine the sensor properties, including the programmable nature and high precision as well as ease of preparation. Pd NPs have a unique biocompatibility in biology and medicine. Ab₂ can be combined with Pd NPs by covalent binding to the surface to form a bioconjugate.^16–18 Among the limited transition metal oxides, Cu₂O NPs represent a credible catalyst, usually in the bulk form or particle form without fixed shapes, including as nanorods, cubes, octahedrons, and pyramids structures,^19–22 and is considered to be an efficient and reusable catalyst to generate primary aromatic amines and promote C–N and C–S cross-coupling reactions.^23,24 Our developed sandwich-type electrochemical immunosensor, with Pd@Cu₂O NPs as the label, presented excellent characteristics of both the palladium nanoparticles (Pd NPs) and cuprous oxide nanoparticles (Cu₂O NPs). The synthetic Cu₂O NPs depicted a rice-shaped morphology, unlike their previous appearance, and exhibited electrocatalytic ability toward H₂O₂. With the advantages of low cost, environment-friendliness, and their natural abundance, Pd@Cu₂O NPs are a promising material in the field of immunosensors. In assessing our sandwich-type electrochemical immunosensor, the fabrication of Pd@Cu₂O NPs by the doping method and their catalytic activity in H₂O₂ reduction were also studied.

Additionally, Au NPs have good biocompatibility, good adhesive ability, relatively good dispersibility, and good electroconductibility.²⁵ It could enhance electrical signal about catalytic reduction toward H₂O₂. Thus, in a controlled assembly, we utilized Au NPs as the substrate material, Pd@Cu₂O NPs as the label, and PSA as the model analyte to fabricate a novel electrochemical immunosensor. The immunosensor exhibited a wide response range and good selectivity and sensitivity, which indicated that the immunosensor has certain superiority over the detection of cancer biomarkers.

2. Experimental

2.1. Apparatus and reagents

Prostate-specific antigen (PSA), primary antibodies (Ab₁), secondary antibodies (Ab₂), carcino-embryonic antigen (CEA), squamous cell carcinoma antigen (SCCA), and alpha fetal protein (AFP) were acquired from Beijing Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China). Human serum was obtained from the University of Jinan's campus Hospital. Bovine serum albumin (BSA, 96–99%) HAuCl₄·4H₂O, PdCl₂, and Cu(CH₃COO)₂·H₂O were purchased from Sigma-Aldrich (Beijing, China) and used as received. All the other chemicals were of analytical reagent grade and were used without further purification. Phosphate buffered saline (PBS) was prepared using 0.1 mol L⁻¹ Na₂HPO₄ and 0.1 mol L⁻¹ KH₂PO₄ stock solution as the electrolyte for all the electrochemistry measurements. All the solutions were prepared with ultrapure water (18.2 MΩ cm⁻¹).

All the electrochemical measurements were carried out on a CHI 760D electrochemical workstation (Shanghai CH Instruments Co., China). A traditional three-electrode system was used for the experiments, which consisted of a glassy carbon electrode (GCE, 4 mm diameter) as the working electrode, a platinum wire electrode as the auxiliary electrode, and a calomel electrode as the reference electrode. A transmission electron microscopy (TEM) image was obtained using an H-800 microscope (Hitachi, Japan). A scanning electron microscopy (SEM) image was obtained using a field emission SEM system (ZEISS, Germany). X-ray diffraction (XRD) patterns were gathered using a D8 Advance X-ray diffractometer (Bruker AXS, Germany) using Cu Kα radiation (40 kV, 30 mA) of 0.154 nm wavelength.

2.2. Synthesis of the Au NPs

Au NPs were prepared in accordance with the previous literature.²⁶ 100 mL of a solution comprising 4.12 mL HAuCl₄ (1 wt%) and 95.88 mL ultrapure water was refluxed at 100 °C. Then, 10 mL sodium citrate (38.8 mmol L⁻¹) was added into the solution with magnetic stirring and maintained reflux for 15 min.

2.3. Synthesis of Cu₂O NPs

Cu₂O NPs were prepared by reducing Cu(CH₃COO)₂·H₂O with N₂H₄·H₂O according to the procedures reported in the literature.²⁷ Briefly, 0.159 g of Cu(CH₃COO)₂·H₂O was dissolved in 40 mL of ultrapure water in a flask and stirred with a magnetic stirrer to obtain a clear solution. 8.0 mL of N₂H₄·H₂O (0.1 mol L⁻¹) solution was speedily added into the mixture under vigorous stirring, which aroused an immediate color change from blue to bright yellow. The mixture was further stirred for 30 min to complete the reaction. Then, the acquired yellowish-brown precipitates were separated by centrifugation, washed sufficiently with ultrapure water and absolute anhydrous ethanol several times each, and dried in an oven at 60 °C for 4 h.

2.4. Synthesis of the Pd@Cu₂O NPs

The procedure for the synthesis of the Pd@Cu₂O NPs involved only one further step than in the preparation of Cu₂O NPs. The amounts of 0.5, 1.0, 1.5, 2.0, and 3.0 mL of PdCl₂ (0.05 mol L⁻¹) solution were added dropwise into separate Cu₂O-containing mother solutions. The suspension was stirred for another 30 min before centrifugation. The samples prepared were referred to as Pd@Cu₂O NPs.

2.5. Preparation of the Ab₂–Pd@Cu₂O NPs

Ab₂–Pd@Cu₂O NPs were synthesized as follows: the as-prepared Pd@Cu₂O NPs were dispersed in ultrapure water (0.5 mL) by ultrasonication, followed by the addition of Ab₂ (200 μL, 10 μg mL⁻¹) and PBS (200 μL, pH 7.4). Next, the mixture was continuously shaken for 12 h at 4 °C. Then, BSA (100 μL, 1 wt%) was added to the modified Pd@Cu₂O NPs suspension to block the possible remaining active sites and the mixture was shaken overnight at 4 °C. The Ab₂-conjugated Pd@Cu₂O NPs were stored at 4 °C until used.

2.6. Preparation of the electrochemical biosensor

A schematic diagram of the stepwise self-assemble procedure of the proposed immunosensor is given in Scheme 1.

Scheme 1 Schematic of the fabrication process of the immunosensor.

First, GCE was polished carefully with alumina slurry to a mirror-like surface and then dried in air. Then, 10 μL of Au NPs solution was dropped onto and immobilized on the electrode. This was allowed to disperse to capture sufficient Ab₁ of PSA through physical and chemical binding. Then, 6.0 μL of 1 μg mL⁻¹ Ab₁ was tightly immobilized onto the electrode with Au NPs through a Au–NH₂ bond. The resulting electrode was washed away with PBS (0.1 mol L⁻¹, pH 7.4) and ultrapure water, respectively, to eliminate the physically adsorbed materials. Subsequently, 6.0 μL of 1 wt% bovine serum albumin (BSA) solution was used to seal the nonspecific sites on the particle surface. Then, the electrode was rinsed with ultrapure water to wash away the excessive BSA. Thereafter, 6.0 μL of a specific concentration of PSA was dropped onto and maintained on the surface for 30 min at 4 °C, which was then washed again with PBS (0.1 mol L⁻¹, pH 7.4). Finally, 6.0 μL of prepared Ab₂–Pd@Cu₂O NPs solution was dropped onto the surface, which was then washed once more with PBS (0.1 mol L⁻¹, pH 7.4). Finally, the sandwich-type construction of Ab₂–Pd@Cu₂O NPs/PSA/BSA/Ab₁/Au NPs was prepared and stored at 4 °C until used.

2.7. Electrochemical measurement of the immunoassay

As shown in Scheme 1, the sandwich-type immunoassay was used for PSA detection. The electrochemical detection was performed in a 25 mL electrochemical cell containing 10.0 mL of PBS (pH 7.4) at room temperature. The amperometric responses of the immunosensor were recorded by an amperometric i–t curve with E = −0.4 V. After the background current was stabilized, 5.0 mmol L⁻¹ H₂O₂ was added into the buffer solution and the current change was directly recorded.

3. Results and discussion

3.1. Characteristics of the nanomaterials

Fig. 1A and B are the SEM images of the Au NPs and Cu₂O NPs. Many evenly distributed Au NPs can be observed in Fig. 1A, while Fig. 1B shows the synthetic Cu₂O NPs with a rice-shaped morphology and a uniform size diameter of about 120 nm. An energy dispersive spectroscopy (EDS) image of the Pd@Cu₂O NPs is presented in Fig. 1C, where a visible and sharp peak from the Pd NPs can be seen, which indicated indirectly that the Pd NPs are well-adsorbed on the Cu₂O NPs. Fig. 1D shows an image of the surface of Cu₂O NPs covered with amorphous Pd NPs, where a rough and angular surface was obtained following the adsorption and interaction of each of the NPs. An irregular shape of Pd@Cu₂O was observed, and the average diameter of the Pd@Cu₂O NPs was approximately 150 nm. In order to see the NPs more clearly, TEM images of the Pd@Cu₂O NPs at higher resolution were obtained and are presented in Fig. 1E. Additionally, Fig. 1F gives the XRD patterns of the Cu₂O NPs and Pd@Cu₂O NPs. Compared to Cu₂O NPs, a Pd NPs peak has emerged and the peak intensity of the Pd@Cu₂O NPs is decreased significantly because of the surface of the Cu₂O NPs encapsulated by Pd NPs, which indicates that the Pd@Cu₂O NPs were successfully synthesized. These results indicate the method of fabricating Pd@Cu₂O NPs by reducing PdCl₂ in a mother solution of Cu₂O NPs was feasible.

Fig. 1 SEM images of the prepared Au NPs (A) and Cu₂O NPs (B), EDS image of the Pd@Cu₂O NPs (C), TEM images of the Pd@Cu₂O NPs (D and E), XRD pattern of the prepared Cu₂O NPs and Pd@Cu₂O NPs (F).

3.2. Testing of the different Pd@Cu₂O NPs toward H₂O₂ reduction

For a sandwich-type immunosensor, the strength and sensitivity of the signal depends largely on the label used. Herein, the acquired electrochemical signals from our immunosensor were based on the excellent catalytic ability of Pd@Cu₂O NPs for the reduction of H₂O₂. To obtain superior catalytic activity, the catalytic performances of different Pd@Cu₂O NPs prepared by Cu₂O with the addition of 0.5, 1.0, 1.5, 2.0, and 3.0 mL PdCl₂ (0.05 mol L⁻¹) were investigated toward H₂O₂ reduction. The electrochemical responses for H₂O₂ reduction are shown in Fig. 2. Cu₂O NPs had a current response (curve a) for the catalytic reduction of H₂O₂, while, the current response of Cu₂O NPs prepared with the addition of 0.5 mL PdCl₂ (0.05 mol L⁻¹) was slightly enhanced (curve b), which indicated that the Pd NPs contribute to the Cu₂O NPs catalyzing the H₂O₂. In addition, when PdCl₂ was added continually to the Cu₂O NPs synthesis process, the generated amount of Pd@Cu₂O NPs increased, and this effect is obvious clearly from curves c–f in the figure. These results show that Cu₂O doping with different levels of Pd NPs exhibited good catalytic performance toward H₂O₂ reduction due to the synergistic effect between Pd and the Cu₂O NPs. A large current response (curve f) close to saturation occurred, indicating that the Pd NPs and Cu₂O NPs could simultaneously catalyze H₂O₂ well in this condition. The resultant Cu₂O with the addition of 3.0 mL PdCl₂ (0.05 mol L⁻¹) was selected as the chosen label for the immunosensor. According to the literature,^28,29 the mechanism for H₂O₂ electro-reduction could be expressed by the following:

H₂O₂ + e⁻ → OH_ad + OH⁻; (1)

OH_ad + e⁻ → OH⁻; (2)

2OH⁻ + 2H⁺ → 2H₂O; (3)

Fig. 2 Amperometric responses of Pd@Cu₂O NPs formed by Cu₂O with different added amounts of PdCl₂ (0.05 mol L⁻¹) for H₂O₂ reduction in pH 7.4 PBS: (a) Cu₂O, (b) Cu₂O with the addition of 0.5 mL PdCl₂, (c) Cu₂O with the addition of 1.0 mL PdCl₂, (d) Cu₂O with the addition of 1.5 mL PdCl₂, (e) Cu₂O with the addition of 2.0 mL PdCl₂, and (f) Cu₂O with the addition of 3.0 mL PdCl₂.

3.3. Characterization of the immunosensor

A common analysis technique, namely electrochemical impedance spectroscopy (EIS), was used to monitor the fabrication process of the immunosensor on the working electrode surface. An electrochemical impedance plot was made up, which consisted of a semicircle and a linear portion. The linear portion was associated with the diffusion process, and the semicircle diameter corresponded to electron-transfer resistance. The impedance spectra were recorded in a solution of 5.0 mmol L⁻¹ [Fe(CN)₆]^3−/4− and 0.1 mol L⁻¹ KCl in the frequency range from 0.1 to 10 000 Hz at 0.187 V. The amplitude of the alternating voltage was 5 mV. Electrochemical impedance Nyquist plots of the diverse modified electrodes are shown in Fig. 3A. For bare platinum carbon (as a glassy carbon electrode (GCE)), a small semicircle domain (curve a) emerged. Au NPs (curve b) also displayed a very small resistance, which had no obvious difference with curve a. Compared with curve b, the semicircle diameter significantly increased after the electrode was further modified with Ab₁ (curve c), which indicated that Ab₁ was successfully anchored on the surface of the electrode functionalized by Au NPs and inhibited electron transfer of the system. An obviously increased electron-transfer resistance (curve d) emerged when the electrode was blocked with BSA. After that, the resistance increased again (curve e), indicating the successful capture of PSA. When the Ab₂–Pd@Cu₂O NPs were incubated further on the electrode, the resistance reached the maximum (curve f), which indicated that the assembly process of the electrode was successful.

Fig. 3 EIS in the presence of 5.0 mmol L⁻¹ [Fe(CN)₆]^3−/4− solution containing 0.1 mol L⁻¹ KCl (A) and CVs scanned from −0.2 to 0.6 V in 5.0 mmol L⁻¹ K₃[Fe(CN)₆] containing 0.1 mol L⁻¹ KCl at a scan rate of 100 mV s⁻¹ (B) obtained for each immobilized step: (a) GCE, (b) Au NPs/GCE, (c) Ab₁/Au NPs/GCE, (d) BSA/Ab₁/Au NPs/GCE, (e) PSA/BSA/Ab₁/Au NPs/GCE, and (f) Ab₂–Pd@CuO₂ NPs/PSA/BSA/Ab₁/Au NPs/GCE.

Furthermore, cyclic voltammetry (CV) measurements were also recorded to monitor the performance of the immunosensor throughout construction (Fig. 3B). The CVs of the different modified electrodes were scanned for two cycles from −0.2 to 0.6 V at a 100 mV s⁻¹ scan rate in 5.0 mmol L⁻¹ K₃[Fe(CN)₆] solution. The results are shown in Fig. 3B. As can be seen, a pair of reduction/oxidation peaks of the GCE were observed (curve a). Au NPs may facilitate the electron transfer (ET) process between the probe and the electrode. When Au NPs were decorated on the GCE, a larger reduction/oxidation peak current emerged (curve b). Subsequently, Ab₁ was bound on the surface of the electrode functionalized by Au NPs through the formation of Au–NH₂ bonds. Here, the peak current of Ab₁/Au NPs decreased clearly (curve c). In order to block the possible nonspecific bonding sites at the Ab₁/Au NPs, BSA was applied to the surface of the electrode, and thus the peak current decreased again (curve d). Afterwards, PSA (1 ng mL⁻¹) was introduced into the electrode, causing a further decrease of the peak current (curve e). Finally, the Ab₂–Pd@Cu₂O NPs were immobilized on the electrode, and the redox peaks further decreased (curve f). These results agree with the EIS, thus confirming the successful assembly process of the immunosensor.

3.4. Optimization of the experimental conditions and performance of the immunosensors

To obtain the best analytical performance for PSA, the experimental detection conditions were optimized. Because the immobilized protein would be damaged by overly acidic or alkaline surroundings, pH values of PBS from 4.5 to 8.5 were investigated by an amperometric i–t curve. As shown in Fig. 4A, pH 7.4 PBS was optimal and was consequently used throughout this whole study.

Fig. 4 Effects of (A) pH and (B) chronoamperometry potential on the response of the sandwich-type immunosensor, (C) the i–t curves of the modified electrodes toward different concentrations of PSA, from a to i: 0, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², 10⁻¹, 1, 10, 100 ng mL⁻¹, (D) calibration curve of the immunosensor toward a series of concentrations of PSA. Error bar = RSD (n = 5).

The potential of chronoamperometry is another key parameter that determines current signals and electrochemical catalytic behaviors. H₂O₂ was used as the redox probe to record the electron transfer generated from H₂O₂ to the electrode when the immunological reaction occurred. The optimum potential to catalyze H₂O₂ was from −0.4 to −0.2 V. The sensitivity of the Pd@Cu₂O-modified electrode to H₂O₂ increased the more negative, such as −0.6 V, the background of the electrode became and as O₂ reduction interfered with H₂O₂ detection.³⁰ Thus, in this study, cyclic voltammograms were recorded from −0.5 to 0.1 V to show the electrochemical response (shown in Fig. 4B). Finally, −0.4 V was employed as a suitable potential.

Under optimized conditions, the fabricated sandwich-type immunosensor was applied to detect a series of concentrations of PSA, and the current responses toward the PSA concentrations are shown in Fig. 4C. The testing by amperometric i–t curve can be further seen from the equation of the calibration plot (Fig. 4D). As shown in Fig. 4D, the calibration plot exhibited a good linear range from 10⁻⁵ to 100 ng mL⁻¹ between the current intensity and logarithm of the PSA concentration. The equation of calibration curve was ΔI = 145.89 + 25.42 lg[c], R² = 0.9985. The limit of detection was estimated to be 2 fg mL⁻¹, which was lower than that with previously reported methods (Table 1). The low detection limit may be attributed to the following factors: (1) the high biocompatibility and electron transfer of Au NPs means Ab₁ was tightly anchored and the signal was enhanced, (2) the catalytic activity of the Pd@Cu₂O NPs arising from the synergistic effect between Cu₂O and Pd NPs toward H₂O₂ reduction.

Table 1 Comparisons of the proposed method with other reported electrochemical immunosensors for PSA

Methods	Linear range/ng mL^−1	Limit of detection/pg mL^−1	References
This immunosensor	10^−5 to 100	0.002	—
rGO–MWCNT–Pd	5 × 10^−4 to 15	0.17	31
Au–PAMA–aptamer	10^−4 to 90	0.010	32
GOx/GNR	10^−2 to 8	8	33
PtAg@CNCs	10^−3 to 50	0.6	34
RBITC–AuNPs	10^−4 to 1.05 × 10^−1	0.032	35

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3.5. Stability, repeatability, and selectivity

The stability of the fabricated immunosensor was evaluated and is presented in Fig. 5A. The immunosensor for the detection of 1.0 ng mL⁻¹ PSA was prepared and was stored at 4 °C, and then the immunosensor current was detected and recoded after every week. The signal strength decreased only 6.8% after one month. The small noticeable changes in the of recorded current response indicated the excellent stability of the proposed immunosensor.

Fig. 5 (A) Current responses of the immunosensor at different times, (B) the responses of the interference of (1) 1.0 ng mL⁻¹ PSA + H₂O, (2) 1.0 ng mL⁻¹ PSA + 100 ng mL⁻¹ carcino-embryonic antigen (CEA), (3) 1.0 ng mL⁻¹ PSA + 100 ng mL⁻¹ BSA, (4) 1.0 ng mL⁻¹ PSA + 100 ng mL⁻¹ squamous cell carcinoma antigen (SCCA), (5) 1.0 ng mL⁻¹ PSA + 100 ng mL⁻¹ alpha fetal protein (AFP). Error bar = RSD (n = 5).

To test the repeatability of the biosensor, five electrodes were equally prepared to detect 1.0 ng mL⁻¹ PSA. All the tests were operated under the same conditions. As a result, a relative standard deviation (RSD) of 3.7% was obtained. The results indicated that the precision and reproducibility of the immunosensor were acceptable.

BSA, carcino-embryonic antigen (CEA), squamous cell carcinoma antigen (SCCA), and alpha fetal protein (AFP) were used in the selectivity tests for the immunosensor under the optimal conditions. Here, a mixture of 1.0 ng mL⁻¹ of PSA solution with 100 ng mL⁻¹ of the interfering substances was prepared and the relevant measurements were made. No obvious change was obtained in comparison with the result without interfering substances, and the RSD of the measurement was 3.4% (Fig. 5B). The results suggested that the selectivity of the immunosensor was acceptable.

3.6. Real sample analysis

To study the analytical feasibility and application potential of the proposed immunosensor for PSA, standard solutions of different concentrations (1.00, 5.00, and 10.00 ng mL⁻¹ PSA) were added into the human serum samples to test the recoveries. As shown in Table 2, the recovery rate for the PSA recovery test was between 99.2% and 101.2% and RSD was in the range of 1.0–2.1%. These results suggest that our proposed method is suitable enough to accomplish PSA detection in real samples and indicated our approach was a promising approach for clinical research and diagnostic applications.

Table 2 Real sample analysis and recovery tests

Content of PSA in serum sample (ng mL^−1)	The addition content (ng mL^−1)	The detection content (ng mL^−1)	Average value (ng mL^−1)	RSD (%)	Recovery (%)
2.37	1.00	3.26, 3.38, 3.42, 3.40, 3.30	3.35	2.1	99.4
	5.00	7.56, 7.54, 7.32, 7.48, 7.41	7.46	1.3	101.2
	10.00	12.42, 12.16, 12.18, 12.40, 12.20	12.27	1.0	99.2

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4. Conclusion

A facile and novel sandwich electrochemical immunosensor was proposed and applied to the sensitive detection of PSA. In this system, Pd@Cu₂O NPs were acquired by an in situ reduction of PdCl₂ in a mother solution of Cu₂O NPs. This method could be easily implemented with simple handling and without a complicated process. Pd@Cu₂O NPs as a label presented the excellent characteristics of both palladium nanoparticles (Pd NPs) and cuprous oxide nanoparticles (Cu₂O NPs). Hence, the obtained immunosensor had strong selectivity, good repeatability, acceptable stability, and exhibited a wide linear response range (10⁻⁵ to 100.0 ng mL⁻¹) with a low detection limit (2 fg mL⁻¹) for PSA. This new and simple sandwich-type immunosensor has the advantages of utilizing simple equipment and a convenient operation, and represents a promising prospect for clinical research and diagnostic applications.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 21375047, 21377046, 21575050 and 21505051), the Science and Technology Plan Project of Jinan (No. 201307010), the Science and Technology Development Plan of Shandong Province (No. 2014GSF120004), the Special Project for Independent Innovation and Achievements Transformation of Shandong Province (No. 2014GSF120004), the Special Project for Independent Innovation, Achievements Transformation of Shandong Province (No. 2014ZZCX05101), and Q. Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).

References

1. C. S. Thaxton, R. Elghanian, A. D. Thomas, S. I. Stoeva, J.-S. Lee, N. D. Smith, A. J. Schaeffer, H. Klocker, W. Horninger and G. Bartsch, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 18437–18442.
2. B. Kavosi, A. Salimi, R. Hallaj and F. Moradi, Biosens. Bioelectron., 2015, 74, 915–923.
3. M. Emami, M. Shamsipur, R. Saber and R. Irajirad, Analyst, 2014, 139, 2858–2866.
4. R. De La Rica, D. Aili and M. M. Stevens, Adv. Drug Delivery Rev., 2012, 64, 967–978.
5. L. Tang, S. Li, L. Xu, W. Ma, H. Kuang, L. Wang and C. Xu, ACS Appl. Mater. Interfaces, 2015, 7, 12708–12712.
6. I. A. Asangani, V. L. Dommeti, X. Wang, R. Malik, M. Cieslik, R. Yang, J. Escara-Wilke, K. Wilder-Romans, S. Dhanireddy, C. Engelke, M. K. Iyer, X. Jing, Y.-M. Wu, X. Cao, Z. S. Qin, S. Wang, F. Y. Feng and A. M. Chinnaiyan, Nature, 2014, 510, 278–282.
7. J. L. Powers, K. D. Rippe, K. Imarhia, A. Swift, M. Scholten and N. Islam, J. Chem. Educ., 2012, 89, 1587–1590.
8. Z. He, N. Gao and W. Jin, Anal. Chim. Acta, 2003, 497, 75–81.
9. M. Tardivo, V. Toffoli, G. Fracasso, D. Borin, S. Dal Zilio, A. Colusso, S. Carrato, G. Scoles, M. Meneghetti and M. Colombatti, Biosens. Bioelectron., 2015, 72, 393–399.
10. L. Deng, H.-Y. Chen and J.-J. Xu, Electrochem. Commun., 2015, 59, 56–59.
11. K. Chuah, L. M. Lai, I. Y. Goon, S. G. Parker, R. Amal and J. Justin Gooding, Chem. Commun., 2012, 48, 3503–3505.
12. J. Tang, D. Tang, R. Niessner, G. Chen and D. Knopp, Anal. Chem., 2011, 83, 5407–5414.
13. T. M. Squires, R. J. Messinger and S. R. Manalis, Nat. Biotechnol., 2008, 26, 417–426.
14. T. Lakshmipriya, M. Fujimaki, S. C. Gopinath, K. Awazu, Y. Horiguchi and Y. Nagasaki, Analyst, 2013, 138, 2863–2870.
15. L. Wang, X. Jia, Y. Zhou, Q. Xie and S. Yao, Microchim. Acta, 2010, 168, 245–251.
16. Y. Wei, Y. Li, N. Li, Y. Zhang, T. Yan, H. Ma and Q. Wei, Biosens. Bioelectron., 2016, 79, 482–487.
17. Q. Zhou, Y. Lin, Y. Lin, Q. Wei, G. Chen and D. Tang, Biosens. Bioelectron., 2016, 78, 236–243.
18. W. Xu, P. Jing, H. Yi, S. Xue and R. Yuan, Sens. Actuators, B, 2016, 230, 345–352.
19. J. Zhang, J. Liu, Q. Peng, X. Wang and Y. Li, Chem. Mater., 2006, 18, 867–871.
20. Q. Hua, D. Shang, W. Zhang, K. Chen, S. Chang, Y. Ma, Z. Jiang, J. Yang and W. Huang, Langmuir, 2010, 27, 665–671.
21. Y. Zhao, J. Zhao, D. Ma, Y. Li, X. Hao, L. Li, C. Yu, L. Zhang, Y. Lu and Z. Wang, Colloids Surf., A, 2012, 409, 105–111.
22. H. Ma, Y. Li, Y. Wang, L. Hu, Y. Zhang, D. Fan, T. Yan and Q. Wei, Biosens. Bioelectron., 2016, 78, 167–173.
23. X. Guo, H. Rao, H. Fu, Y. Jiang and Y. Zhao, Adv. Synth. Catal., 2006, 348, 2197–2202.
24. H.-J. Xu, X.-Y. Zhao, J. Deng, Y. Fu and Y.-S. Feng, Tetrahedron Lett., 2009, 50, 434–437.
25. M.-C. Daniel and D. Astruc, Chem. Rev., 2004, 104, 293–346.
26. J. Han, L. Jiang, F. Li, P. Wang, Q. Liu, Y. Dong, Y. Li and Q. Wei, Biosens. Bioelectron., 2016, 77, 1104–1111.
27. W. Zhang, X. Yang, Q. Zhu, K. Wang, J. Lu, M. Chen and Z. Yang, Ind. Eng. Chem. Res., 2014, 53, 16316–16323.
28. Y. Zhou, G. Yu, F. Chang, B. Hu and C.-J. Zhong, Anal. Chim. Acta, 2012, 757, 56–62.
29. F. Meng, X. Yan, J. Liu, J. Gu and Z. Zou, Electrochim. Acta, 2011, 56, 4657–4662.
30. Q. Wei, Z. Xiang, J. He, G. Wang, H. Li, Z. Qian and M. Yang, Biosens. Bioelectron., 2010, 26, 627–631.
31. L. Tian, L. Liu, Y. Li, Q. Wei and W. Cao, New J. Chem., 2015, 39, 5522–5528.
32. B. Kavosi, A. Salimi, R. Hallaj and F. Moradi, Biosens. Bioelectron., 2015, 74, 915–923.
33. S. Xu, Y. Liu, T. Wang and J. Li, Anal. Chem., 2011, 83, 3817–3823.
34. M. Zhang, W. Dai, M. Yan, S. Ge, J. Yu, X. Song and W. Xu, Analyst, 2012, 137, 2112–2118.
35. D. Liu, X. Huang, Z. Wang, A. Jin, X. Sun, L. Zhu, F. Wang, Y. Ma, G. Niu and A. R. Hight Walker, Journal, 2013, 7, 5568–5576.

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