Electrochemical immunosensor with Cu(I)/Cu(II)-chitosan-graphene nanocomposite-based signal amplification for the detection of newcastle disease virus

An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification. Graphene (Gra) was used for both the conjugation of an anti-Newcastle disease virus monoclonal antibody (MAb/NDV) and the immobilization of anti-Newcastle disease virus polyclonal antibodies (PAb/NDV). Cu(I)/Cu(II) was selected as an electroactive probe, immobilized on a chitosan-graphene (Chi-Gra) hybrid material, and detected by differential pulse voltammetry (DPV) after a sandwich-type immune response. Because Gra had a large surface area, many antibodies were loaded onto the electrochemical immunosensor to effectively increase the electrical signal. Additionally, the introduction of Gra significantly increased the loading amount of electroactive probes (Cu(I)/Cu(II)), and the electrical signal was further amplified. Cu(I)/Cu(II) and Cu(I)/Cu(II)-Chi-Gra were compared in detail to characterize the signal amplification ability of this platform. The results showed that this immunosensor exhibited excellent analytical performance in the detection of NDV in the concentration range of 100.13 to 105.13 EID50/0.1 mL, and it had a detection limit of 100.68 EID50/0.1 mL, which was calculated based on a signal-to-noise (S/N) ratio of 3. The resulting immunosensor also exhibited high sensitivity, good reproducibility and acceptable stability.


Results and discussion
Morphological characterization of the nanocomposites. Figure 1 shows scanning electron microscopy (SEM) images and energy dispersive spectrometry (EDS) analyses of Gra, Chi-Gra and Cu(I)/Cu(II)-Chi-Gra. The image of Gra confirms that its structure had many folds (a). After Gra was modified with Chi, the folded structure was filled with Chi, and the surface of the Chi-Gra composite became smooth (b). The presence of Chi on Gra was confirmed by EDS analysis (e). N was observed in the sample because Chi is a natural, biocompatible polymer with many amino groups. Interestingly, the Cu(I)/Cu(II)-Chi-Gra nanocomposite exhibited many upturned folded edges and had a porous matrix (c). Due to this characteristic structure, the exposed surface of the Cu(I)/Cu(II)-Chi-Gra nanocomposite was larger than those of the Chi-Gra composite and Gra. The active surface area increased, resulting in a high surface/volume ratio for antibody immobilization. Furthermore, this porous structure facilitated electrochemical signal amplification. The successful incorporation of Cu(I)/Cu(II) into the Chi-Gra surface was also confirmed by EDS analysis (f).

Chemical characterization of the nanocomposites. Fourier transform infrared (FT-IR) spectra of
Chi, Gra, Chi-Gra, CuSO 4 and Cu(I)/Cu(II)-Chi-Gra are presented in Fig. 2. As shown in Fig. 2a (black line, Chi), the stretching vibrations of the -OH bonds in Chi were observed at 3,425 cm −1 , and this band overlapped with the -NH 2 stretching peaks 29 . The signals originating from the C-H stretching vibrations were observed at approximately 2,920 cm −1 and 2,878 cm −130 . The NH 2 group and γ-NH 2 bending vibrations appeared at 1653 cm −1 and 1596 cm −1 , respectively 31 . Furthermore, the peak at 1,424 cm −1 was attributed to the OH bending vibration. The stretching vibrations of the C-C-O bonds in the Chi backbone were observed at approximately 1,154 cm −1 , 1,081 cm −1 and 1,034 cm −1 . As shown in Fig. 2a (red line), the characteristic absorption bands of pure Gra appeared at 1555 cm −1 , 1,459 cm −1 , and 1,420 cm −1 (benzene ring backbone stretching vibrations); 1659 cm −1 (C=O stretching vibration); 2,916 cm −1 (C-H stretching vibration); and 3,406 cm −1 (O-H stretching vibration). Chi adsorption on Gra resulted in the appearance of the characteristic absorption bands of pure Gra in the FT-IR spectrum of Chi-Gra ( Fig. 2a; blue line), but compared with pure Gra, the characteristic absorption bands of Chi-Gra had lower intensities, which helped confirm that Chi was successfully adsorbed on Gra. Comparing the spectra of Chi-Gra and Cu(I)/Cu(II)-Chi-Gra ( Fig. 2b; red and blue lines) revealed some changes in the intensities and shifts in the peaks. Furthermore, the main absorption peaks of pure CuSO 4 ( Fig. 2b; black line) were also observed in the FT-IR spectrum of Cu(I)/Cu(II)-Chi-Gra ( Fig. 2b; blue line), providing evidence of the interaction between CuSO 4 and Chi-Gra. Chi-Gra binds Cu 2+ well because Chi-Gra contains many negatively charged groups (carboxylic (O=C-OH), hydroxyl (-C-OH) and carbonyl (-C=O)) that can strongly interact with the positively charged Cu 2+ ion in CuSO 4 .
In addition, X-ray photoelectron spectroscopy (XPS) was used to identify the valence state of Cu. The XPS spectrum of Cu(I)/Cu(II)-Chi-Gra is shown in Fig. 3a. The formation of Cu 2 O was confirmed by the presence of the Cu 2p 3/2 peak at 931.73 eV and the Cu 2p 1/2 peak at 951.39 eV 32 . Furthermore, the presence of Cu 2p 3/2 and Cu 2p 1/2 peaks with binding energies of 933.26 eV and 953.14 eV, respectively, proved the formation of CuO 32 Fig. 4b, which was obtained with the BSA-MAb/NDV-AuNP-Chi-Gra film-modified GCE, the background current was low, and no CV redox waves were observed because of the absence of electrochemically active substances in the working solution. After the immunosensor was incubated with 10 5.13 EID 50 /0.1 mL NDV and sandwiched for the immunoreaction with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra, stable redox peaks were observed at 0.13 and − 0.08 V vs. saturated calomel electrode (SCE) (curve b-2 in Fig. 4b), due to the redox reaction of Cu(I)/Cu(II). The peak at 0.13 V was caused by the oxidation of Cu(I) to Cu(II), and the reduction of Cu(II) to Cu(I) produced the peak at − 0.08 V. These results indicated the efficient redox activity of Cu(I)/Cu(II)-functionalized Gra.
Comparison of different signal amplification strategies. Signal amplification strategies are very important for immunosensors. Two signal label materials (PAb/NDV-Cu(I)/Cu(II)-Chi-Gra and PAb/NDV-Cu(I)/Cu(II)-Chi) were prepared, and differential pulse voltammetry (DPV) was performed from − 0.3 to 0.4 V at a 50 mV/s scan rate using a 10 2.13 EID 50 /0.1 mL sample to evaluate the effects of the signal amplification materials. The results are shown in Fig. 4c. As indicated by curve c-1, in the absence of a signal labelling material, a low background current was obtained, and no anodic peak was observed for the immunosensor. In contrast, the immunosensor conjugated with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra (curve c-3) exhibited a greater current shift than the immunosensor conjugated with PAb/NDV-Cu(I)/Cu(II)-Chi (curve c-2). The increase in the cur- www.nature.com/scientificreports/ rent shift was due to the use of Gra, which has with a high surface/volume ratio, as the carrier, leading to the immobilization of Cu(I)/Cu(II) on the GCE and facilitating electrochemical signal amplification. These results confirmed that the immunosensor with Gra could load more of the electroactive signal labelling material and PAb/NDV than the immunosensor without Gra. Accordingly, the signal of the immunosensor was greatly amplified by using Gra.
Optimization of the experimental conditions. During NDV capture and the specific reaction with the signal labelling material (PAb/NDV-Cu(I)/Cu(II)-Chi-Gra), the incubation time is an important factor. Thus, the incubation times of NDV and PAb/NDV-Cu(I)/Cu(II)-Chi-Gra were optimized separately. To optimize the NDV incubation time, different incubation times (5,10,15,20,30,40,50, and 60 min) were used, and after incubation with NDV, the immunosensors were incubated with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra for 60 min. Finally, the immunosensors were used for DPV detection. Each test was repeated five times. The results are shown in Fig. 4d, curve d-1. As the NDV incubation time was increased up to 30 min, the electrochemical response increased; after 30 min, a constant value was reached, indicating that the immunoreaction was complete, and all the NDV in the sample was captured by the immunosensor. Thus, the optimal incubation time for NDV was 30 min.
Analytical performance of the immunosensor. The response of the prepared immunosensor was measured at different concentrations of NDV (F48E9) under the optimal experimental conditions. The results are shown in Fig. 5a. The electrochemical response current increased as the concentration of NDV increased, and the peak of the electrochemical response current was proportional to the concentration in the range of 10 0.13 to 10 5.13 EID 50 /0.1 mL. The linear regression equation, which is shown in Fig. 5b, was I (μA) = 0.75 log EID 50 /0.1 mL + 1.05, with a correlation coefficient of 0.97075, and the limit of determination for NDV was 10 0.68 EID 50 /0.1 mL, which was calculated based on a signal-to-noise ratio of 3 (S/N = 3). These results demonstrated that the immunosensor was sensitive enough to quantitatively monitor NDV.
The results for the immunosensor with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra as the signal label were compared with those for the immunosensor with PAb/NDV-Cu(I)/Cu(II)-Chi as the signal label, and the results obtained with PAb/NDV-Cu(I)/Cu(II)-Chi are shown in Fig. 5c. The electrochemical response current increased linearly with increasing NDV concentration, and the calibration curve in the range of 10 0.13 to 10 5.13 EID 50 /0.1 mL (Fig. 5d) was: I (μA) = 0.15 log EID 50 /0.1 mL + 1.10. The limit of determination for NDV was 10 2.09 EID 50 /0.1 mL (S/N = 3). This result indicated that Gra can improve the immunosensor sensitivity. In addition, as shown in Fig. 5c (curve c-2), the background signal was high when PAb/NDV-Cu(I)/Cu(II)-Chi was used as the signal label because without Gra, the excess Chi could not be removed from PAb/NDV-Cu(I)/Cu(II)-Chi by centrifugation, and the excess Chi chelated with Cu(I)/Cu(II) was attached to the GCE by non-specific binding.
Under the optimal experimental conditions, equivalently prepared immunosensors were used to detect 10 3.13 EID 50 NDV 20 times to evaluate the repeatability of the developed immunosensor, and the results are shown in Fig. 6b. The relative standard deviation was 2.58%, demonstrating the good repeatability of the immunosensor. The reproducibility of the immunosensor was evaluated by preparing six different batches of the immunosensor Scientific RepoRtS | (2020) 10:13869 | https://doi.org/10.1038/s41598-020-70877-3 www.nature.com/scientificreports/ independently. A series of six different batches of the immunosensor were prepared for the detection of 10 3.13 EID 50 NDV, and the results are shown in Fig. 6c. The relative standard deviation was found to be 2.84%, showing the excellent reproducibility. Long-term storage stability tests show the robustness of an immunosensor. The current responses of the developed immunosensor were periodically checked to evaluate its stability. The immunosensor was stored in PBS (pH = 7.4) at 4 °C when it was not in use. Every week, electrochemical measurements were performed with the developed immunosensor, and the average value was calculated based on five assays. The results shown in Fig. 6d indicated that the immunosensor response current decreased by only 4.1% after 2 weeks. After four weeks, the immunosensor current response decreased by 9.5% relative to its initial current, which indicated that the immunosensor had acceptable storage stability.

Application of the proposed immunosensor for the detection of NDV.
Oral and cloacal swab samples, which were gently collected from fowls at different live bird markets in Guangxi Province, were used as clinical samples. A viral transport medium composed of 0.05 mmol/L PBS containing 10 mg/mL gentamycin, 10 mg/mL kanamycin, 10 mg/mL streptomycin, 5% (v/v) foetal bovine serum and 10,000 units/mL penicillin was used to prepare the clinical samples, and the clinical samples were placed in an ice box.
With the permission of the owners of the live bird markets, a total of 120 clinical samples were collected from chickens, the samples were assayed using the proposed immunosensor, and seven NDV-positive samples were detected. Virus isolation 3 was employed to confirm the test results. The positive results detected by the developed immunosensor were in agreement with the results of virus isolation, and the results are summarized in Table 1b,c. To test the recovery by the proposed immunosensor, NDV standards were added to the clinical samples that had been confirmed as positive. The results (Table 1d) showed that the fabricated immunosensor www.nature.com/scientificreports/ had acceptable recovery (96.28 ~ 104.49). Considering the acceptable recovery in real samples, the immunosensor was found to be practical for sample detection.

Materials and methods
Reagents and materials. MAb/NDV and PAb/NDV were purchased from Abcam (Cambridge, UK).
Instruments. SEM was performed on a HITACHI UHR FE-SEM SU8000 Series (SU8020) instrument.
FT-IR spectra were collected on a Nicolet IS10 instrument. XPS analysis was performed on an X-ray photoelectron spectrometer (ESCALAB 250Xi, Thermo Scientific). A CHI660D electrochemical workstation (Beijing CH Instruments, Beijing, China) with a standard three-electrode cell (a working electrode, an SCE as the reference electrode and a platinum wire as the auxiliary electrode) was employed to study the electrochemical characteristics. Electrochemical detection was performed at room temperature (25 ± 0.5 °C).

Gra synthesis.
A modified Hummers method was used to prepare Gra oxide 34 . In short, NaNO 3 (2.5 g) and graphite powder (1.0 g) were added to concentrated H 2 SO 4 (100 mL) and stirred for 2 h. KMnO 4 (5 g) was slowly added to the mixture under continuous stirring, and the mixture was then cooled with ice. Next, the mixture was stirred at 35 °C for 24 h. Double-distilled deionized water (100 mL) was slowly added to the reacted slurry, which was then stirred at 80 °C for another 3 h. Next, more double-distilled deionized water (300 mL) was added to  www.nature.com/scientificreports/ the reacted slurry. Then, 6 mL of H 2 O 2 (30%) was added (bubbles appeared, and the slurry immediately turned bright yellow). The resulting solution was continuously stirred for 3 h and then precipitated for 24 h at room temperature. The supernatant was subsequently decanted. The resulting yellow slurry was washed with 0.5 mol/L HCl (500 mL) and centrifuged. The solution was washed with double-distilled deionized water and centrifuged until the pH of the solution was neutral (pH = 7.0). Gra oxide was obtained after the solution was ultrasonicated for 2 h. To obtain Gra, Gra oxide was reduced at 95 °C for 3 h using NaBH 4 as a reducing agent.
Preparation of the Chi-Gra nanocomposite. Chi-Gra was prepared according to a previously reported method 23 . Briefly, Chi powder was dissolved in a 1.0% (v/v) acetic acid solution under stirring for 0.5 h at room temperature until it was completely dispersed. The Chi solution (0.5 wt.%) was thus prepared. Then, Gra (10 mg) was added to the Chi solution (10 mL), ultrasonicated for 1 h, and stirred for 24 h at 25 °C. Finally, the Chi-Gra nanocomposite was obtained.
Preparation of the AuNP-Chi-Gra nanocomposite. The AuNP-Chi-Gra nanocomposite was prepared as previously described 23,35 . Furthermore, 0.5 mL of HAuCl 4 (1 mM) was added to Chi-Gra (   of the Cu(I)/Cu(II)-Chi-Gra nanocomposite obtained from the above preparation method was centrifuged (12,000 rpm, 10 min), the supernatant was discarded, and the residue was washed with double-distilled deionized water three times to remove the excess Chi, Cu 2+ and SO 4 2− that did not combine with Gra. Then, 5.0 mL of a PBS buffer (pH = 7.4) was added to the residue to disperse the Cu(I)/Cu(II)-Chi-Gra nanocomposite, and the mixture was sonicated for 10 min to obtain a homogeneous suspension. Next, 1 mL of PAb/NDV (10 µg/mL) was added to the homogeneous suspension, and the mixture was vigorously stirred for 5 min at 4 °C. Then, 1 mL of 1% glutaraldehyde was slowly added to the solution under continuous stirring. The solution was subsequently incubated at 4 °C for 8 h. The reaction mixture was washed with PBS (pH = 7.4) and centrifuged (12,000 rpm, 10 min) three times. The supernatant was discarded, the resulting mixture was dispersed in PBS (5.0 mL, pH = 7.4), and 1 mL of a 2.0% (w/v) BSA solution was added to the suspension, which was then incubated at 4 °C for 8 h. The obtained PAb/NDV-Cu(I)/Cu(II)-Chi-Gra nanocomposite was stored at 4 °C for further use.
Fabrication of the electrochemical immunosensor. First, 0.05 mm alumina was used to polish a GCE (Ø = 3 mm) until it had a mirror-like surface. Then, the GCE was rinsed with double-distilled deionized water and ultrasonicated in baths of double-distilled deionized water, ethyl alcohol, and double-distilled deionized water to remove any physically adsorbed substances. Next, the GCE was placed in H 2 SO 4 (0.05 M) and chemically cleaned until the background signal stabilized. Finally, the GCE was thoroughly rinsed with double-distilled deionized water and dried with nitrogen gas to obtain a clean GCE. Figure 7 shows the procedures used to construct the immunosensor. The process was as follows: the AuNP-Chi-Gra (8 μL) nanocomposite was dropped onto the clean GCE surface, dried at 4 °C overnight to obtain the modified electrode (AuNP-Chi-Gra-GCE), washed with double-distilled deionized water, immersed in a 1 µg/ mL (200 µL) MAb/NDV PBS solution (pH = 7.4) and incubated at 4 °C for 8 h. The resulting electrode (MAb/ NDV-AuNP-Chi-Gra-GCE) was immersed in a 1.0% (w/w) BSA solution for 1 h at 37 °C to block the remaining active sites. The final modified electrode was stored at 4 °C when not in use.

Electrochemical immunosensor detection.
A well-known sandwich immunoassay was used to detect NDV. First, the MAb/NDV-AuNP-Chi-Gra-GCE immunosensor was incubated with 15 μL of the sample for 30 min and then washed with a PBS buffer (pH = 7.4) to remove non-specifically adsorbed conjugates. Next, the modified electrode was incubated with 200 μL of the PAb/NDV-Cu(I)/Cu(II)-Chi-Gra nanocomposite for Ethics statement. The authors confirm that relevant guidelines were followed for the care and use of animals. This work was approved and conducted by the Animal Ethics Committee of the Guangxi Veterinary Research Institute, which supervises all live bird markets in Guangxi Province. Oral and cloacal swab samples, which were gently collected from fowls at different live bird markets in Guangxi Province, were used as clinical samples. Before sampling, the fowls were not anaesthetized, and after sampling, they were returned to their cages and observed for 30 min.

conclusions
In summary, AuNP-Chi-Gra was used as a platform, and PAb/NDV-Cu(I)/Cu(II)-Chi-Gra was used as a label for signal amplification in this work. Based on the well-known sandwich immunoreaction, a novel electrochemical immunosensor was developed for the quantitative detection of NDV. It exhibited a linear response over a wide range (10 0.13 to 10 5.13 EID 50 /mL), had a low detection limit (10 0.68 EID 50 /0.1 mL), and was more sensitive than an immunosensor with PAb/NDV-Cu(I)/Cu(II)-Chi as the signal label (the limit of detection for NDV was 10 2.09 EID 50 /0.1 mL). This newly designed immunosensor might have widespread application potential because it had acceptable reproducibility, selectivity and stability; could be obtained by a facile fabrication procedure; and was ultrasensitive for the detection of NDV.