Recently, the hepatitis B virus (HBV) has become a leading cause of death worldwide [1
]. Obviously, the earlier the diagnosis, the more treatment options are available and the greater the possibility of healing. Therefore, the highly sensitive detection of viruses in human serums is considered to be a key point in the processes of treatment of HBV [2
]. Furthermore, once you have been infected with chronic hepatitis B and this is not been arrested in a timely manner and effectively, it will further evolve into acute hepatitis, chronic hepatitis and cirrhosis, and will even lead to serious complications such as liver cancer, eventually leading to death [4
]. Therefore, the prevention of hepatitis has gradually become an important public health problem all over the world [5
HBsAg is one of the main markers of HBV infection, and it usually occurs after being infected for 1–2 weeks [6
]. Many methods have been exploited to detect HBsAg in the past few decades [7
], including time-resolved fluoroimmunossay (TRFIA), electro-chemiluminescence immunoassay (ECLIA), gold immunochromatography assay (GICA), ELISA, etc. [8
]. In particular, the ELISA test has since been seen as the “gold standard” for comparison against all newly developed immunoassays and immunosensors [11
]. However, the methods above usually need expensive instruments, skilled operation and strict culture conditions, but have low sensitivity and a narrow range [12
]. In contrast with this, as an innovation for conventional determination methods, electrochemical immunosensors are a kind of analytical method that combines electrochemical sensors with immunoassays [13
]. Immunoassays make specific and sensitive measurements of target analytes by harnessing the high specificity of the antigen–antibody interaction [14
]. Therefore, electrochemical immunosensors have attracted increasing attention in recent years due to their stable operation, convenient use, high precision and satisfactory effectiveness in practical application, etc. [15
]. Accordingly, the electrochemical immunoassay is expected to become an ideal strategy among various measurement techniques for HBsAg in the future.
Moreover, electrochemical immunosensors can be classified into two types: label-free (direct assay) and labeled (brief assay) [16
]. The labeled immunosensor is used to label the analyte target before detection, and monitor the immunoassay response by quantifying the labeled product. However this type of sensor’s non-specific response is small, and the detection process is complex [17
]. The label-free electrochemical immunosensor vastly simplifies the preparation and operation procedures by directly measuring the physical and chemical changes during the formation of antigen–antibody complexes [18
]. Based on those, label-free electrochemical immunosensors have been developed rapidly in recent years, and the further exploration of electrochemical immunosensors still has huge space for development and tremendous development potential [19
Signal amplification and antibody immobilization are the crucial steps in the design and fabrication of highly sensitive electrochemical immunosensors [20
]. Thereby, searching for ideal materials for immobilizing identifiable redox probes as trace labels is another important issue in developing a successful immunosensor [21
]. Meanwhile, profound advances in nanotechnology and nanomaterials have offered powerful tools for the design of electrochemical immunosensors [22
]. Graphene is a new, two-dimensional carbon nanomaterial, and it has promising application prospects in energy, materials science and biomedicine [24
]. Graphene oxide (GO) is the oxidation product of graphene, and its oxygen-containing functional groups are chemically active [26
]. Therefore, the special structural characteristics of GO have proved to be a promising material in designing and preparing electroactive nanocomposites, due to advantages such as an impressive surface area, high conductivity, anti-toxicity and good electron mobility [27
]. Due to the unique crystalline forms and the morphologies of Fe3
nanoparticles (NPs), it exhibits unique physical and chemical properties. Under the influence of the nano-effect, Fe3
nanomaterials with different morphologies have different properties, which makes it possible to prepare some new functional materials with special performance requirements [30
]. They are widely used in electromagnetics, the chemical industry, catalysis, sensors, acoustics, medicine, environmental protection and other related fields. In particular, Fe3
NPs are widely used in the field of electrochemical immunosensors due to the excellent catalytic performance, impeccable magnetism, good biocompatibility and large specific surface area [31
]. Prussian blue has great potential in many fields and has been widely used in electrocatalysis, biosensors, chemical sensors, rechargeable batteries and electroanalytical chemistry [34
]. Therefore, PB is a well-studied material that has been extensively studied in the field of electrochemical sensors and biosensors [35
]. In addition, there are some research works which have shown that metal NPs can also can generatse a synergistic effect with GO, such as AuNPs [37
] and PtNPs [38
]. Normally, AuNPs could improve electrical conductivity and provide more active sites for the binding of antibodies due to their excellent physicochemical properties [39
]. In addition, AuNPs have been widely used to improve signal intensity [41
In this work, a novel label-free electrochemical immunosensor based on redox-active conductive PB/Fe3O4/GO nanocomposites and AuNPs was constructed for the detection of HBsAg. A stable PB/Fe3O4/GO composite with electrocatalytic activity was first coated on the carbon working electrode, while PB was used as the redox probe. Subsequently, AuNPs were attached onto the modified electrode by electrodeposition, which offered an interface for HBsAb immobilization. The principle of the proposed immunosensor was based on the fact that the decrease in the peak currents of PB is proportional to the quantity of HBsAg captured on the modified immunosensor. Furthermore, the proposed method can be employed to detect HBsAg in real human serum samples with satisfactory results, which provides promising potential applications in clinical immunoassays.
2. Materials and Methods
2.1. Materials and Reagents
Graphene oxide (GO) was purchased form Xianfeng Nano Materials Tech Co. Ltd. (Nanjing, China). Ethylene glycol (CH2OH2)2, ethanol (C2H6O), ferric chloride (FeCl3.6H2O), sodium acetate trihydrate (CH3COONa) and hydrochloric acid (HCl) were purchased from Xilong Scientific Company (Guangdong, China). Potassium ferricyanide (K3Fe (CN)6), L-cysteine and cholesterol were purchased from Aladdin Company (Shanghai, China). Chloroauricacid (HAuCl4∙4H2O), bovine serum albumin (BSA), carcinoembryonic (CEA), A-fetoprotein (AFP), and human serum albumin (HSA) were purchased from Sangon Biotech (Shanghai, China). Hepatitis B surface antibody and hepatitis B surface antigen were purchased from Huayang Zhenglong Biochem. Lab. (Chengdu, China). SPEs were purchased from Nanjing Yunyou Biotechnology Co, Ltd. (Nanjing, China), as well as the bare SPE (working electrode: carbon; counter electrode: carbon; reference electrode: silver/silver chloride). All other chemicals employed were of analytical grade. Double-distilled water was used in all work.
All electrochemical measurements were carried out on a CHI660D electrochemical Workstation (Chenhua Instrument Co, Shanghai, China). Constant temperature Biochemical Culture was carried out on a BSD-100 (Shanghaibo xunshiye Company, Shanghai, China). A conventional three-electrode system was used for all electrochemical measurements and a bare SPE or modified SPE was served as the measurement electrode. The pH measurements were carried out on a PHS-3E (Shanghai INESA Scientific Instrument CO. Ltd., Shanghai, China). Centrifugal processes were all accomplished by using the high-speed freezing centrifuge TGL-20M (Hunan Xiangyi Development Co. Ltd., Yiyang, China).
2.3. Preparation of Fe3O4/GO Nanocomposites
/GO nanocomposites were synthesized based on the previous reported method with a little modification [43
]. Briefly, 15 mg GO was ultrasonic dispersed in 75 mL (CH2
for 2.5 h. Afterwards, 0.81 g FeCl3
was added to the above GO aqueous solution and dissolved thoroughly. Then, 1.23 g CH3
COONa was added to the mixture and stirred vigorously for 30 min, until the color changed from black to yellow brown. The obtained mixture was moved into a Teflon-lined stainless-steel autoclave and heated to 180 °C for 11 h. After it was cooled to room temperature, the obtained Fe3
/GO nanocomposites were isolated in the magnetic field and washed several times with ethanol and ultrapure water. Finally, the products were dried in a vacuum at 60 °C for 12 h.
2.4. Preparation of PB/Fe3O4/GO Nanocomposites
A total of 2 mg Fe3O4/GO nanocomposites were dispersed homogeneously in 2 mL ultrapure water by sonication. Then, 2 mL of the above aqueous solution was added into a 2 mL aqueous solution (pH 1.5, adjusted with HCl) containing 15 mmol L−1 K3Fe (CN)6 and 15 mmol L−1 FeCl3∙6H2O. After vigorously stirring for 5 h, the color changed from yellow brown to dark cyan, which indicated that GO/Fe3O4 nanocomposites were completely synthesized. The final mixture was separated by a magnet and washed several times and then dispersed in 2 mL ultrapure water.
2.5. Fabrication of the Immunosensor
In total, 1.5 μL GO/Fe3O4/PB of the nanocomposites (1 mg/mL) were dropped onto the surface of the SPE and dried at a constant temperature in a biochemical incubator (25 °C). The modified SPE was immersed in 50 μL (0.5 mM) HAuCl4, which was performed by using the electrochemical workstation to complete the electrodeposition system at a potential of −0.5 V for 180 s. A total of 5.0 μL HBsAb (0.25 mg/mL) was dropped onto the modified electrode and stored at 4 °C for 12 h. For the further fabrication of the immunosensor, it was blocked through incubation in 10 μL 1% BSA for 30 min to avoid possible nonspecific adsorption. After each step, the fabricated SPE was thoroughly cleaned with PBS and dried at room temperature prior to use.
2.6. Electrochemical Measurements
In total, 5 μL standard HBsAg solution at different concentrations was dipped on the proposed immunosensor and incubated at 25 °C for 40 min, followed by washing with PBS buffer. After that, the electrochemical measurements were performed in 50 μL PBS (0.01 M, PH = 7.4) by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The change in the peak currents was proportional to the concentrations of HBsAg captured on the modified electrode immunosensor.