A sensitive electrochemical immunosensor for the detection of human chorionic gonadotropin based on a hierarchical nanoporous AuAg alloy

Abstract

A sensitive electrochemical immunosensor for the detection of human chorionic gonadotropin (hCG) is designed based on a novel signal amplification strategy. A hierarchical nanoporous (HNP) AuAg alloy with the advantages of a large surface area, excellent structure stability, and rich pore channels is used as an hCG antibody carrier for the preparation of a highly sensitive immunosensor. The HNP-AuAg alloy with bimodal ligament/pore size distribution is fabricated by means of simple dealloying and redealloying of the AuAgAl source alloy combined with an annealing operation. Graphene sheets (GS) and ionic liquid (IL) composites have been introduced as transducing materials to modify the conductivity as well promote the electron transfer in the immunosensor. Based on the dual-amplification effects of the HNP-AuAg alloy and the IL/GS composite film, the constructed immunosensor exhibits an enhanced performance for hCG detection compared with that based on single nanoporous (NP) Au, including a lower detection limit of 0.01 ng mL⁻¹ and wider linear range from 0.05 to 35.0 ng mL⁻¹. Moreover, the immunosensor exhibits respectable reproducibility and stability, indicating a potential application in clinical monitoring of hCG.

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1. Introduction

Human chorionic gonadotropin (hCG), a glycoprotein hormone produced by placental trophoblasts, is composed of 244 amino acids with a molecular mass of 36.7 kDa.^1–3 It exists in the blood and urine of pregnant women, so can be used as a tumor marker for the diagnosis of pregnancy and several cancers.^4–6 Therefore, sensitive detection of hCG plays an important role in clinical assay. At present, many methods have been developed to detect hCG, such as a resonance scattering spectral assay,⁷ fluorescence resonance energy transfer assay,⁸ and impedance immunosensor assay.⁹ Compared with the methods above, the electrochemical immunosensor assay that combined the specificity of an immunological reaction has attracted great attention due to its advantages of high sensitivity, inherent simplicity, and low cost. Hence, it is of significant importance to design sensitive electrochemical immunosensors for the detection of hCG. More importantly, the signal response of the electrochemical immunosensor is usually affected by the modification strategies of the electrode.^11,12 Some strategies based on sandwich-type immunosensors have been applied to the determination of hCG. However, sandwich-type immunosensors always involve tedious labeling operations and too many modifying procedures, which usually affect the signal reproducibility of the electrochemical immunosensor as well as the immobilization of the biological molecule.¹¹ In comparison to sandwich-type immunosensors, label-free immunosensors have the obvious advantages that its fabrication is simple, easy-handle, and low cost owing to its avoidance of tedious labeling operations. Therefore, it is necessary to develop a simple label-free electrochemical immunosensors to the determination of hCG.

In the design and fabrication of highly sensitive electrochemical immunosensors, one of the crucial steps is signal amplification. Many kinds of nanomaterials have been developed to amplify the electrochemical signal to improve the sensitivity of electrochemical immunosensor. Among various materials, graphene sheets (GS) have attracted great interesting to construct the electrochemical immunosensors due to its excellent electronic properties and large surface area.^13,14 However, the formation of irreversible agglomerates through strong π–π stacking and van der Waals interaction presents a key challenge for its application in electrochemical immunosensor. In recent years, ionic liquid (IL) made of molten organic cations and various anions have been used not only as the supporting electrolyte but also as the modifier in chemically modified electrode because they have many advantages in terms of wide electrochemical windows, thermal stability, high ionic conductivity, and low toxicity.^15–18 It has been found that a small amount of GS can dramatically enhance the conductivity of the IL.¹⁹ At present, the GS/IL composites are used to construct various electrochemical sensors not only in order to increase the conductivity of the electrode but also avoid the agglomerates of the GS.²⁰ The combination of GS and IL provides rich opportunities to construct highly sensitive electrochemical immunosensor by making good use of the advantages of these two materials.

The immobilization of antibody (Ab) is another important issue to achieve the much larger signal amplification and high sensitivity in electrochemical immunosensors. Gold nanoparticles as one of Au-based materials are the conventional bio-nanomaterials for the immobilization of antibody because of their large specific surface area, strong adsorption ability, well suitability, and good conductivity.^21–24 Compared with the conventional gold nanoparticles, nanoporous gold presents much more potential for the construction of electrochemical immunosensors because it can combine both the advantages of Au-based materials and nanoporous structure. The nanoporous structure with large surface area can promote the unblocked mass transport of medium molecules and improve the electron transport along the interconnected skeleton and channels.²⁵ In previous work, most researches have been focused on the fabrication of single-modal nanoporous Au.²⁶ It is known that the smaller pores are favorable for increasing the active surface area to achieve sensitive response, while larger pores are beneficial for the unlimited mass transfer of medium molecules to achieve rapid response.^27–29 Consequently, the combination of larger pores with smaller pores in nanoporous structure is desirable for not only providing rich channels for the transport of medium molecules but also increasing the surface active sites. Based on such understanding, it is considered to design the hierarchical nanoporous (HNP) Au-based structure with bimodal ligament/pore size distribution as hCG antibody carrier.

In current work, a simple dealloying strategy is employed to design and fabricate HNP-AuAg alloy with bimodal pore size distributions. Considering that the chemical property of Al is more reactive and distinctly different with Ag, AuAgAl ternary alloy was selected as the source alloy. The selective dissolution of Al from the AuAgAl alloy generates the first order ligaments and pores. Upon further being annealed and treated in HNO₃ solution, the second order ligaments/pores were sequentially generated on the nanoporous surface accompanied with part removal of Ag atoms. During the whole dealloying/annealing/redealloying process, a good control on the bimodal structure can be achieved in terms of simple operation, perfect yielding, and large-scale synthesis. With the abundant interconnected voids and nanoscaled skeleton, HNP-AuAg alloy was used as hCG antibody carrier in this electrochemical immunosensor for the hCG detection. Meanwhile, the immunosensor is constructed by assembling the hCG antibody on IL and GS composites film modified layer. Based on the dual-amplification effects of HNP-AuAg alloy and the IL/GS composites film, it exhibits the lower detection limit and wider liner range for hCG detection compared with that based on single nanoporous (NP) Au. This signal amplification strategy of the immunosensor is simple and convenient, which may provide promising applications for the sensitive detection of hCG.

2. Experimental section

2.1. Reagents

1-Butyl-3-methylimidazolium hexafluorophosphate as the IL was purchased from Sigma-Aldrich. Ascorbic acid (AA), uric acid (UA), dopamine (DA), and chitosan (CS) were also purchased from Sigma-Aldrich. hCG and Ab were purchased from Shanghai Lingcao Biotechnology Co., Ltd (China). H₂O₂ (30%) and glucose, bovine serum albumin (BSA), and N,N-dimethylformamide (DMF) were purchased from Sinopharm Chemical Reagent Co., Ltd (China). The phosphate buffer saline (PBS) solution was prepared using Na₂HPO₄ and KH₂PO₄. All the chemicals were of analytical reagents grade without further purification. Ultra-pure water (18.2 MΩ) was used throughout the whole experiments.

2.2. Apparatus

All electrochemical measurements were performed using CHI 760D electrochemical workstation (Shanghai CH Instruments Co., China). A conventional three-electrode cell was used with Pt foil as a counter electrode, mercury sulfate electrode as the reference electrode, and 4 mm-diameter glassy carbon electrode (GCE) as the working electrode. All potentials were provided with respect to the reversible hydrogen electrode (RHE). Electrochemical impedance measurements (EIS) were performed on a Zennium electrochemical workstation (Zahner, Germany). Powder X-ray diffraction (XRD) analysis was carried out on a Bruker D8 advanced X-ray diffractometer using Cu Kα radiation at a step rate of 0.04° s⁻¹. The morphology of the sample was characterized by a JEM-2100 transmission electron microscope (TEM) and a JSM-6700 field-emission scanning electron microscope (SEM) equipped with an Oxford INCA X-sight Energy Dispersive Spectrometer.

2.3. Preparation of HNP-AuAg alloy and NP-Au

The AuAgAl and AuAl alloy foils were made by melting high-purity (99.99%) Au, Ag, and Al in an arc-furnace, followed by melt-spinning in Ar atmosphere. AuAgAl alloy foils were firstly dealloyed in 1 M NaOH solution for 24 h at room temperature, annealed at 200 °C for 30 min in the presence of high purity N₂, and then immersed in concentrated HNO₃ for 1 h to obtain the HNP-AuAg alloy.³⁰ After dealloying, the foils were crushed to uniform grains using a pestle and mortar prior to characterization. NP-Au was prepared by etching AuAg alloy foil in 2 M HNO₃ solution at room temperature for 10 h.²⁶ HNP-AuAg alloy and NP-Au were dispersed in CS (1 wt%) under sonication to obtain a black suspension.

2.4. Preparation of GS/IL composites

GS were prepared from graphite oxide (GO) through a thermal exfoliation method.³¹ GO powders were synthesized from graphite by a modified Hummer's method.³² In brief, 23 mL concentrated H₂SO₄ was added into a mixture of 0.5 g graphite flakes and 0.5 g NaNO₃ in the temperature of 0 °C. Then 3 g KMnO₄ was slowly added into the solution and stirred for 2 h. The reaction was improved to 95 °C and poured onto 3 mL H₂O₂ (30%). Subsequently, the filtrate was centrifuged, and the remaining solid material was washed in succession with 200 mL water, 200 mL 30% HCl, and 200 mL ethanol. The solid obtained on the filter was vacuum-dried overnight at room temperature. As-purified GO suspension was then dispersed in water by ultrasonication for over 0.5 h and then the GO suspension was reduced with hydrazine at 100 °C for over 24 h. Finally, the black precipitates were filtrated and washed with water. The resulting solid was dried to obtain GS. 2 mg GS was dispersed in 1 mL CS. IL was dispersed in DMF. GS/IL composites were prepared by mixing the above mixture under sonication to obtain a homogeneous dispersion.

2.5. Construction of the immunosensor

Fig. 1 displays the stepwise procedure for the construction of the immunosensor. The GCE was polished to a mirror finish and then thoroughly washed ultrasonically in ultrapure water. GS/IL composites were dropped on the surface of the GCE. Next, HNP-AuAg alloy was dropped onto the electrode surface to immobilize the Ab. Subsequently, 6 μL Ab (10.0 μg mL⁻¹) was added onto the modified working electrode and incubated for 12 h. The modified working electrode was then washed with PBS to remove unbounded biomolecules and immersed in 1% BSA solution for 1 h at 4 °C to block nonspecific binding sites. Finally, the electrode was incubated in varying the concentrations of hCG solution for 2 h. The electrode was stored in the refrigerator prior to use.

Fig. 1 Illustration of the fabrication process of the electrochemical immunosensor.

3. Results and discussions

3.1. Characterization of HNP-AuAg alloy and GS

Previous works have demonstrated that selective etching of the more reactive element from a well-designed alloy can generate the porous structure with nanosized ligaments and pores.^33,34 For dealloying the AuAgAl source alloy in NaOH solution, selective dissolution of the more reactive Al atoms can produce the open bicontinuous nanoporous structure. The following annealing operation is applied to release the surface strain as well make the Au and Ag atoms reassemble again to become more uniform. Upon further redealloying the annealed sample in concentrated HNO₃ solution to dissolve away the part of Ag atoms from the first order ligaments, the second order pores can be generated on the nanosized ligaments. The component of the dealloyed sample was examined by X-ray energy-dispersive spectroscopy (EDS). As shown in Fig. S1a,† after dealloying AuAgAl alloy in NaOH solution, most of Al atoms are leached away, while the bimetallic ratio of Ag and Au are around 3.3 : 1. After annealing and further dealloying the sample in concentrated HNO₃ solution, Al was etched to an undetectable level as well as most of the Ag atoms were leached away with 11.12 at% remained (Fig. S1b†). Hence, after dealloying the sample in the concentrated HNO₃ solution, the weight percentage of the HNP-AuAg alloy is 37.56%.

The structure formation and evolution of the HNP-AuAg alloy during the dealloying process was monitored by the SEM images. As shown in Fig. 2a, the open bicontinuous nanoporous structure with the typical ligament size around 90 nm was shown in the first dealloyed sample prepared by dealloying the AuAgAl source alloy in 1 M NaOH solution for 24 h. After annealed the first dealloyed sample at 200 °C for 30 min, a well maintained and uniform nanoporous structure is formed without evident structure coarsening (Fig. 2b). Upon second free corrosion step in the concentrated HNO₃ solution for 1 h, the dissolution of the part of Ag atoms produces new porous structure on the first order ligaments as well with the first order nanoporous structure well maintained (Fig. 2c). The TEM image in Fig. 2d further demonstrates that the resulted sample has the hierarchical nanoporous structure. The clear contrast between the dark skeletons and bright pores demonstrates the formation of interconnected hollow channels embedded in the bicontinuous nanoscaled skeleton. It is obvious that further dealloying the sample in HNO₃ solution generates the hierarchical nanoporous structure with bimodal pore size distributions.³⁰ In addition, the TEM image of the NP-Au is added for comparison (Fig. S2a†). Similar with the HNP-AuAg alloy, the NP-Au is characterized by the nanoporous structure with ligament sizes about 15 nm. The morphology of GS was investigated by the TEM. As shown in Fig. S2b,† the structure of the GS was wrinkled and transparent with irregular size.

Fig. 2 SEM images of (a) the sample upon dealloying AuAgAl alloy in 1 M NaOH for 24 h, (b) the dealloyed sample followed by annealed at 200 °C for 30 min. (c) SEM and (d) TEM images of the resulted HNP-AuAg sample obtained by redealloying the annealed sample in concentrated HNO₃ for 1 h.

The crystal structure of the resulted sample upon two-step dealloying was examined by XRD. As shown in Fig. 3, a set of five diffraction peaks emerged around 38.3, 44.5, 64.8, 77.7, and 81.9 (2θ), which can be assigned to the face centered cubic (fcc) AuAg alloy structure. Based on the experimental observations, it is concluded that hierarchical nanoporous AuAg alloy with bimodal ligament/pore size distributions can be successfully prepared by a simple dealloying/annealing/redealloying process.

Fig. 3 XRD pattern of the HNP-AuAg sample. The standard patterns of pure Au (JCPDS 65-2870) and Ag (JCPDS 65-2871) are attached for comparison.

3.2. Electrochemical characteristics of the modified electrode

It is noted that the construction of the hCG immunosensor always involves many steps concerning multiple modified materials in the previous reports.^13,35–37 However, too many procedures in the process of modifying the electrodes usually affect the signal reproducibility of the electrochemical immunosensor as well as the immobilization of the biological molecule.¹¹ So it is necessary to develop a simple signal amplification strategy to improve the performance of electrochemical immunosensors. In current work, HNP-AuAg alloy with high porosity is used as hCG antibody carrier for the preparation of a highly sensitive immunosensor. GS and IL composites have been introduced as the electrode modified layer to increase the conductivity of the immunosensor. The immunosensor is constructed by assembling the hCG antibody on IL and GS composites film modified layer. The influences of the GS/IL/HNP-AuAg on the sensitivity of the fabricated immunosensor are first investigated by the cyclic voltammetry (CV) in PBS solution containing 5 mM K₃[Fe(CN)₆] and 0.2 M KCl (Fig. 4a). A couple of reversible redox peaks could be observed on the bare GCE, indicating a reversible electrochemical process by taking potassium ferricyanide as the electrochemical probe. It is found that the peak current of GS/IL or HNP-AuAg electrode was larger than that of the bare GCE, suggesting that the GS/IL and HNP-AuAg played an important role to enhance the conductivity as well increase the active area of the electrode.¹⁰ It is clear that the peak current based on the HNP-AuAg alloy was larger compared with that on the NP-Au. The enhanced performance of the HNP-AuAg alloy may be attributed to the hierarchical nanoporous structure as well as the synergistic catalytic effect between Au and Ag in the alloy. The result suggests the promising application of the HNP-AuAg alloy for constructing the sensitive immunosensor. In addition, the electrochemical performance of the GS/IL/HNP-AuAg was also superior to that of the HNP-AuAg and GS/IL electrodes, which could be attributed to the synergistic amplification effect of the two kinds of nanomaterials. Based on this observation, the GS/IL composites film was suitable for the effective load matrix for HNP-AuAg.

Fig. 4 (a) CVs of (A) GCE/GS/IL/HNP-AuAg, (B) HNP-AuAg/GCE, (C) GS/IL/GCE, (D) NP-Au/GCE, and (E) bare GCE in PBS (pH 7.2) containing 0.2 M KCl and 5 mM K₃[Fe(CN)₆]. (b) EIS of (A) GCE, (B) GCE/GS/IL/Ab, (C) GCE/GS/IL/HNP-AuAg/Ab/BSA, (D) GCE/HNP-AuAg/Ab, (E) GCE/GS/IL/HNP-AuAg, and (F) GCE/GS/IL/HNP-AuAg/Ab in PBS (pH 7.2) containing 0.2 M KCl and 5 mM K₃[Fe(CN)₆]. Effects of (c) the pH value, (d) the incubation time of the Ab, (e) the concentration of IL, and (f) the concentration of HNP-AuAg on the electrochemical signal of the immunosensor.

In order to gain insight into the fabrication process of the immunosensor, Fig. 4b represents the Nyquist plots based on the electrochemical impedance spectroscopy (EIS) of the modified electrodes. The semicircle portion at high frequencies is associated with the electrochemical process related to the electron transfer, where the larger diameter corresponds to the higher resistance.¹ Therefore, the resistance change could be judged by observing the diameter change of semicircle portion. EIS of the modified electrode was performed in PBS containing 5 mM K₃[Fe(CN)₆] and 0.2 M KCl. As shown in Fig. 4b, the bare GCE showed a relatively large resistance. After the electrode was modified by the HNP-AuAg, remarkable resistance decay was observed, implying that the HNP-AuAg could promote the electron transfer. However, Ab with weak conductivity could inhibit the electron transfer kinetics of the redox probe at the interface of the electrode. As a result, the insulating layer is generated, which will hinder the diffusion of the redox marker with the increase of resistance. Similarly, the capture of BSA resulted in the increase of the electrode impedance. After BSA was employed to block the non-specific binding sites, a successive decrease in the current was observed due to the hindering effect of the protein on electron transfer. Based on the above results, it was confirmed that the fabrication program was feasible and the proposed immunosensor was successfully fabricated.

In order to obtain the best analytical performance of the immunosensor for hCG detection, differential pulse voltammetry (DPV) was recorded before and after antigen–antibody reaction in PBS containing 5 mM K₃[Fe(CN)₆] and 0.2 M KCl in various experimental conditions. The ferricyanide was used as the redox indicator.^38,39 The pH of the working buffer has great effects on both the bioactivity of immobilized proteins and the electrochemical performance of the electrode. In order to optimize the pH, a series of PBS buffers with the pH values from 5.8 to 8.0 were prepared, and the immunosensor was incubated in 10.0 ng mL⁻¹ hCG. The amperometric signal decreased in strong acidic and alkaline solutions. It might be on account of the irreversible behavior of the denaturation of proteins involved in the process which was caused by the pH.¹⁰ The optimal response was obtained at pH 7.2 (Fig. 4c), which indicated that the weak alkaline environment was more conducive for the Ab to be in operation. Thus, the 7.2 was chosen as the optimal pH value for the determination of hCG.

It is known to us that the reaction between the antibodies and antigens depends on the incubation time. As shown in Fig. 4d, the results demonstrated that with increasing the incubation time, the amperometric signal increased during the first 80 min, and then tended to level off after 2 h due to the formation of the equilibration state of antigen–antibody complexes. Therefore, an incubation time of 2 h was chosen for the determination of hCG. In order to achieve an optimal electrochemical signal, it was necessary to investigate the response changes of different concentrations of IL. Different concentrations of IL were used to fabricate the immunosensors. As shown in Fig. 4e, the response gradually increased, and reached a maximum value at the concentration of 5.0 mg mL⁻¹. Therefore, 5.0 mg mL⁻¹ was used as the optimal concentration of IL. In addition, the optimal concentration of GS was found to be 6.0 mg mL⁻¹ (Fig. 4f) upon exploring its effect. Under the optimal conditions, the designed immunosensor could exhibit an optimal electrochemical signal for the quantitative detection of hCG.

3.3. Calibration curve, specificity, reproducibility, and stability of the constructed immunosensor

Under optimal conditions, the immunosensor was used to detect different concentrations of hCG. DPVs of the immunosensor incubated with different concentrations of hCG in PBS containing 2 mM K₃[Fe(CN)₆] and 0.2 M KCl. The relationship between the current changes and the concentrations of hCG was shown in Fig. 5a. The current changes increased linearly with the increase of hCG concentration over the range of 0.5–25.0 ng mL⁻¹ by using GS/IL/NP-Au as the platform. The corresponding linear equation is y = 0.98x + 0.51 with a linear correlation coefficient of 0.9965 and a detection limit of 0.2 ng mL⁻¹ (S/N = 3). Compared with the immunosensor based on NP-Au, it exhibits the wider linear range of 0.05–35.0 ng mL⁻¹ and a lower detection limit of 0.01 ng mL⁻¹ by using GS/IL/HNP-AuAg as the platform. The corresponding linear equation for the currents based on HNP-AuAg alloy is y = 1.67x + 0.28 with a linear correlation coefficient of 0.9980. The immunosensor based on HNP-AuAg alloy shows enhanced performances for hCG detection compared with the immunosensor based on NP-Au, indicating the evident advantage of the HNP-AuAg alloy for hCG detection. In addition, the proposed method for the determination of hCG is compared with the previously reported methods in Table 1.^1,4,8,9 The proposed immunosensor based on HNP-AuAg alloy shows a relatively wide linear range and low detection limit, suggesting a promising sensitive method to quantify the hCG. The excellent performance is considered to originate from the excellent charge-transfer bridge to facilitate the electrode transfer rate of GS/IL composites. In addition, the interconnected nanosized skeleton in HNP-AuAg alloy provides the substantial immobilization for Ab, which is beneficial for improving the amperometric signal.

Fig. 5 (a) Calibration curve of the immunosensor for the detection of different concentrations of hCG. (b) Amperometric response of the immunosensor based on HNP-AuAg alloy containing (A) 10.0 ng mL⁻¹ hCG, (B) 10.0 ng mL⁻¹ hCG + 50.0 ng mL⁻¹ glucose, (C) 10.0 ng mL⁻¹ hCG + 50.0 ng mL⁻¹ AA, (D) 10.0 ng mL⁻¹ hCG + 50.0 ng mL⁻¹ UA, (E) 10.0 ng mL⁻¹ hCG + 50.0 ng mL⁻¹ DA, and (F) 10.0 ng mL⁻¹ hCG + 50.0 ng mL⁻¹ H₂O₂.

Table 1 Comparison of analytical parameters for the detection of hCG

hCG-Detection methods	Additional material	Linear range	Detection limit	Ref.
Epitaxial graphene immunosensor	APTES/graphene/SiC	0.62–5.62 ng mL^−1	0.62 ng mL^−1	1
Surface plasmon resonance assay	Liquid crystals	—	2 nM	4
Chemiluminescence resonance energy transfer	Graphene	0.1–10 mIU mL^−1	0.06 mIU mL^−1	8
Impedance immunosensor	Polypyrrole-pyrolecarboxylic acid copolymer	0.1–1 ng mL^−1	2.3 pg mL^−1	9
Electrochemical immunosensor	GS/IL/HNP-AuAg	0.05–35 ng mL^−1	0.01 ng mL^−1	This work

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The response of the immunosensor on different interferents, such as glucose, AA, UA, DA, and H₂O₂, were investigated to examine the selective determination of target analytes in analyzing biological samples. The immunosensors were separately exposed to 10.0 ng mL⁻¹ hCG solutions with and without 50.0 ng mL⁻¹ interfering substance. As shown in Fig. 5b, the current variation upon adding the interfering substances was less than 4% of that without interferences. The results indicated that the specificity of the present immunoassay protocol was satisfactory.

Reproducibility is a key factor for developing a practical immunosensor. The reproducibility of the immunosensor was evaluated by detecting the responses of five electrodes prepared by the same conditions independently in 10.0 ng mL⁻¹ hCG. The relative standard deviation (RSD) was 5.6%, suggesting that the reproducibility of the designed immunosensor were acceptable.

The stability of the immunosensor was also investigated by periodically recording the current responses of the immunosensor. The response of the immunosensor maintained 88% of the initial response after 2 weeks, suggesting the long-term stability of the as-made immunosensor.

3.4. Application of hCG immunosensor in human serum samples

The feasibility of the immunoassay for practical applications was investigated the recoveries of different concentrations (1.0, 5.0, 10.0, and 20.0 ng mL⁻¹) of hCG by standard addition methods in human serum. The analytical results are shown in Table S1.† The recovery was in the range of 96–102%, and the RSD was in the range of 1.8–3.2%, indicating the satisfactory application of the proposed immunosensor to the clinical determination of hCG in real samples.

4. Conclusions

In this work, a simple and highly sensitive electrochemical immunosensor for hCG detection has been successfully developed based on a novel signal amplification strategy by using GS/IL/HNP-AuAg as a sensor platform. HNP-AuAg with good biocompatibility and high active surface area could enhance the loading area of Ab. The GS/IL composites with high electron conductivity and high surface area could modify the electron transfer between solution and electrode. Experimental observations demonstrated that the HNP-AuAg alloy represents an interesting class of promising candidate for the construction of highly sensitive and selective biosensors with the advantages of simple preparation, unique structure feature, and high sensing performance. The proposed strategy provides a potential application for hCG detection.

Acknowledgements

This work was supported by the National Science Foundation of China (21271085).

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Footnote

† Electronic supplementary information (ESI) available: The TEM images of GS and nanoporous Au, the EDS data of the sample upon first dealloying AuAgAl alloy and the resulted HNP-AuAg alloy, and the hCG detection in serum by the constructed immunosensor. See DOI: 10.1039/c5ra24300a

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