Yibiao Liu*,
Guangli He,
Huili Liu,
Hang Yin,
Fengli Gao,
Jian Chen*,
Shouren Zhang and
Baocheng Yang
Department of Henan Key Laboratory of Nanocomposites and Applications, Institute of Nanostructured Functional Materials, Huanghe Science and Technology College, Zhengzhou 450006, China. E-mail: liuyibiao12345@126.com; jianchen@infm.hhstu.edu.cn
First published on 24th February 2020
An ultrasensitive sandwich-type electrochemical immunosensor based on AuBP@Pt nanostructures and AuPd-PDA nanozyme was developed for the detection of apolipoprotein E4 (APOE4) which was an important risk factor for Alzheimer's disease (AD). In this work, gold nanobipyramid coated Pt (AuBP@Pt) nanostructures were prepared and applied to electrochemical immunosensors as a substrate material. AuBP@Pt nanostructures have advantages of electrical conductivity and large electroactive area, which could greatly increase electron transfer rate. In previous work, we designed AuPd alloy modified polydopamine (AuPd-PDA) nanozyme which catalyzed the decomposition of hydrogen peroxide (H2O2). AuPd-PDA nanozyme was used to label detection antibody due to excellent catalytic capability and stability in this new paper. And the concentration of APOE4 could be detected quantitatively by variation for transient current. As a result, the electrochemical immunosensor based on AuBP@Pt and AuPd-PDA exhibited a wide linear range from 0.05 to 2000 ng mL−1 and low detection limit of 15.4 pg mL−1 (S/N = 3). Furthermore, the designed biosensor displayed good selectivity in phosphate buffer saline (PBS) buffer solution or commercial goat serum, which provided a promising tool for early diagnosis of AD.
In the past few years, some traditional techniques such as mass spectrometry (MS),6,7 enzyme-linked immunosorbent assays (ELISA) and western-blot analysis8,9 were used to detect APOE. However, these methods are flawed due to being low sensitivity, time-consuming, with high cost and requirement of specific equipment. In recent years, some new technologies including microarray technology,10 surface plasmon resonance (SPR),11 and electrochemistry immunosensors12 were applied to detect human APOE. Among these methods, electrochemical immunosensors have caused widespread attention due to many superiorities including rapid detection, high sensitivity and low cost.13,14 With the rapid development of nanotechnology, nanomaterials have been widely applied to electrochemical biosensing by reason of their large electroactive area, excellent biocompatibility and unique physical/chemical properties, which makes ultrasensitive detection possible. So far, a lot of studies proved that many nanomaterials with interesting morphologies, such as 3D metal–organic frameworks,15 nanoparticles,16 nanowires,17 quantum dots,18 fractal structures12 and large amounts of nanocomposites,19–21 significantly enhanced the sensitivity of the electrochemical biosensor and decreased the detection limit.
Recently, bimetallic nanostructures have caught more attention due to their integration of physicochemical properties including plasmonic functionality, optical property, magnetism, catalytic performance and electrical conductivity.22,23 For instance, Au–Pd alloy could catalyze organic reactions.24 Yan Liu et al. designed a sandwich-like electrochemical immunosensor for the detection of carbohydrate antigen based on hierarchical AuPd nanochain networks.25 Recently, our group reported a H2O2 electrochemical sensor based on AuPd alloy-modified polydopamine nanotubes (AuPd-PDA).26 AuPd-PDA nano-enzyme showed excellent catalytic activities and good stability, which could be applied to biosensor in replace of HRP in some ways. Not only that, PDA nanotubes also had many advantages including good biocompatibility, large specific surface area and unique optoelectronic properties,27,28 which could be applied for signal acquisition and signal amplification as a carrier and thus improved the sensitivity of electrochemical immunosensors.
In this work, another bimetallic nanostructure, gold nanobipyramid coated Pt (AuBP@Pt) was prepared. AuBP@Pt nanostructures have good conductivity and could increase the electroactive area due to porous surface though its catalytic activity was bad (Fig. 3a and S1†). Therefore, AuBP@Pt was used to modify the electrode as a substrate material, which could greatly increase the sensitivity of sensor. Furthermore, detection antibody labelled AuPd-PDA (Ab2 label) was used to amplify signal again as a whole because of the excellent catalytic capabilities for H2O2 (ref. 26) and large surface areas. The fabrication procedure for Ab2 label was shown in Fig. 1a. In the sensing system, AuPd-PDA replaced the traditional bio-enzyme which had drawback of easier deactivation. According to previous report,29 the combination of AuPd-PDA and Ab2 was characterized by FTIR as seen in Fig. S2.† The detailed description was shown in ESI.†
Fig. 1 The constructed process of Ab2 label (a) and the schematic illustration of the APOE4 electrochemical immunosensor (b). |
Based on AuBP@Pt nanomaterials and AuPd-PDA nanozyme, we constructed an APOE4 electrochemical immunosensor, which exhibited high sensitivity and low detection limit in phosphate buffered saline (PBS) or commercial goat serum. The schematic illustration of the fabricated APOE4 electrochemical immunosensor was shown in Fig. 1b. The electrochemical immunosensor included working electrode and electrochemical detection system. Firstly, Au nanoparticles that had advantages of good biocompatible and excellent conductivity were electrodeposited on the surface of GCE, which increased the binding site of primary antibody (Ab1) and accelerated the electron transfer.30,31 Secondly, AuBP@Pt nanoparticles were dropped on the surface of electrode, and then the working electrode was obtained. In the electrochemical detection system, AuPd-PDA, as a nanozyme, could catalyze hydrogen peroxide (H2O2) decomposition, during which the change of current was monitored.26 The detection limit of our designed electrochemical immunosensor is 15.4 pg mL−1 and the available linear range is from 0.05 ng mL−1 to 2000 ng mL−1. This result made a tremendous progress compared to our previous work, which provided potential detection method for early diagnosis of AD.
Monoclonal human APOE4 antibody (Ab1), polyclonal human apolipoprotein E4 antibody (Ab2), human APOE4 protein and bovine serum albumin (BSA) were purchased from Novus Biologicals Ltd. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O), palladium chloride (PdCl2), chloroplatinic acid (H2PtCl6·6H2O) and dopamine were obtained from Sigma-Aldrich. The phosphate-buffered saline (PBS, 0.01 M, pH = 7.4) was used as incubating and washing buffer solution. All chemicals were of analytical grade and used without further purification. All solvents were ultrapure water (Milli-Q, 18.2 MΩ cm).
The selectivity of fabricated electrochemical biosensor was investigated by the same procedure in PBS buffer containing other proteins, such as BSA, APOE2 and APOE3. Moreover, commercial goat serum containing various proteins was used to prove the specificity and potential application value. The detailed procedures were as follows. 10 μL Ab1 (100 μg mL−1) was dropped onto the surface of GCE/Au/AuBP@Pt electrode and incubated at 37 °C for 1.0 h. Then, 6 μL BSA (1 mg mL−1) was applied to block the non-specific adsorption sites. Thirdly, a 6 μL diluted goat serum containing different concentration of APOE4 (1 ng mL−1, 10 ng mL−1, 100 ng mL−1) was dropped onto the surface of electrode at 37 °C for 1 h. Finally, 10 μL Ab2 label (50 μg mL−1) were added and incubated at 37 °C for 1 h. Beyond the incubation, other experiments were all performed at room temperature.
Fig. 2 The EDX mapping analysis (a–d) and TEM image (f) of AuBP@Pt nanostructures. (e) The SEM image of AuBP. (g) The schematic illustrating the variation from AuBP to AuBP@Pt. |
And then, the AuBP@Pt nanostructures were prepared by adding Au BPs into a mixed solution including CTAB, ascorbic acid, and H2PtCl4 at 65 °C for 6 h. The detailed concentration of each component was seen in the Experimental section. The characterization of the AuBP@Pt nanostructures was shown in Fig. 2. Overall, the AuBP@Pt exhibited a nano bipyramid morphology with about 100 nm length and 50 nm width (Fig. 2f). The surface of AuBP@Pt nanostructures was composed of many Pt nanoparticles with a diameter of 2 nm according to the typical TEM images Fig. 2f, which was porous. The porous nanostructures further increased the binding sites of primary antibody. Not only that, the porous nanostructures also enhanced the electron transfer rate, which contributed to enhance the sensitivity of sensor. Moreover, the EDX mapping analysis was carried out and the distribution of Au and Pt elements on the surface was shown in Fig. 2a–d. The result of EDX spectrum showed that the atomic ratio of Au/Pt on the surface of AuBP@Pt was about 48:52 (Fig. 2d). No other metallic element was observed, which proved the purity of the AuBP@Pt nanostructures.
As Fig. 3a showed, when Au nanoparticles were electrodeposited on the surface of GCE, the electroactive area significantly increased. After the AuBP@Pt was dropped onto the Au nanoparticles surface, the electroactive area further got larger. Therefore, the introduction of Au/AuBP@Pt nanostructures enhanced the sensitivity of the sensor to some extent. After the construction of the GCE/Au/AuBP@Pt electrode, the electrochemical immunosensor was constructed through successively self-assembly procedures and the process was studied by CV measurements and electrochemical impedance spectroscopy (EIS). As shown in Fig. 3b, the peak current gradually reduced from curve a to curve f. The GCE/Au electrode showed a peak current of 75 μA (Fig. 3b, curve a). After the GCE/Au/AuBP@Pt electrode was modified with Ab1, the peak current decreased significantly (Fig. 3b, curve b), which demonstrated that Ab1 hindered the electron transfer of electrode surface in some way. And then BSA was used to block the non-specific adsorption sites on the surface. At the moment, the peak current was down to 50% of initial value (Fig. 3b, curve c), which indicated that non-specific binding sites on the surface had been blocked. As shown blue and cyan curve (curve d and e) in Fig. 3b, the peak current decreased again with the modification of APOE4 protein and Ab2 to the GCE/Au/AuBP@Pt surface.
To further proved the process of assembly, electrochemical impedance spectroscopy (EIS) was carried out as shown in Fig. S7.† The bare GCE/Au/AuBP@Pt exhibits a small resistance (Fig. S7,† curve a). When Ab1 (Fig. S7,† curve b), BSA (Fig. S7,† curve c), APOE4 (Fig. S7,† curve d) and Ab2 label (Fig. S7,† curve e) were immobilized layer by layer on the surface of electrode, the resistance increased gradually due to the modified protein. This result was consistent with the conclusion of the CVs. According to above analysis, it was inferred that the sensing interface was constructed successfully. At last, the quantification of APOE4 was monitored by chronoamperometry in the presence of H2O2.
Under the optimal conditions, the developed electrochemical immunosensor based on AuBP@Pt and AuPd-PDA was applied to detect quantitatively APOE4. According to our previous report,26 the AuPd-PDA nanotubes could catalyze hydrogen peroxide (H2O2) decomposition and the optimal potential was −0.25 V, during which the changes of electrochemical signal (reductive current) could be monitored. Based on this principle, the APOE4 could be detected quantitatively by the change value of current.
The change value of current response (ΔI) were monitored by chronoamperometry when different concentration of human APOE4 were added. According to our previous study, the optimized concentrations of Ab1 were 100 μg mL−1 and the concentration of Ab2 was about 50 μg mL−1. As shown in Fig. 4a, the transient current reached a steady state rapidly with add of H2O2 and the change value enhanced with the increase of APOE4 concentration. Fig. 4b showed the relationship between change value of current and logarithm of human APOE4 concentration. The ΔI and logarithm of APOE4 concentration from 0.05 ng mL−1 to 2000 ng mL−1 existed linear relationship. And the detection limit of our developed electrochemical immunosensor was about 15.4 pg mL−1 (S/N = 3). According to previous report, the concentration of human APOE4 in serum was about μg mL−1 level for the individuals who had APOE4 allele.34 And thus, the detection limit of our designed electrochemical immunosensor based on AuBP@Pt and AuPd-PDA could satisfy the requirements of clinical application in this respect.
Furthermore, compared to the previous methods for the detection of APOE or APOE4, the designed electrochemical immunosensor had a wider linear range from 0.05 ng mL−1 to 2000 ng mL−1 and lower detection limit of 15.4 pg mL−1. And the detailed comparison was shown in Table S1.† In this work, porous Au/AuBP@Pt substrate materials could increase the binding sites of antibody and electron transfer rate, which enhanced the sensitivity of sensor. What's more, AuPd-PDA, as a nanoenzyme, could catalyze hydrogen peroxide (H2O2) decomposition replacing the traditional bio-enzyme, which was more stable than traditional bio-enzyme. Therefore, our designed APOE4 electrochemical immunosensor based on AuBP@Pt nanomaterials and AuPd-PDA nanoenzyme exhibited excellent sensing performance.
To evaluate reproducibility and clinical potential of the electrochemical immunosensor, the commercial goat serum diluted with PBS buffer was used as electrolyte to detect the APOE4. Three groups of GCE/Au/AuBP@Pt electrodes (each group include 5 electrodes) were used to detect different concentration of human APOE4 protein (1.0, 10 and 100 ng mL−1) in goat serum. As shown in Fig. 5b and c, a good response for human APOE4 protein in goat serum was observed, which further proved that the APOE4 electrochemical immunosensor had a good selectivity. Moreover, the recovery and RSD for five electrodes were calculated and the result was shown in Table S2.† The RSD of the three groups was all less than 6%, which manifested that the sensor had a good reproducibility and potential application value in the real biological sample.
Footnote |
† Electronic supplementary information (ESI) available: The Reproducibility, selectivity, stability of the electrodes and some additional supporting information. See DOI: 10.1039/d0ra00298d |
This journal is © The Royal Society of Chemistry 2020 |