key: cord-0715729-jej9kj0c
authors: Zhao, Jialin; Zhao, Fu; Li, Haolin; Xiong, Youlin; Cai, Shuangfei; Wang, Chen; Chen, Yunfa; Han, Ning; Yang, Rong
title: Magnet-assisted electrochemical immunosensor based on surface-clean Pd-Au nanosheets for sensitive detection of SARS-CoV-2 spike protein
date: 2021-12-23
journal: Electrochim Acta
DOI: 10.1016/j.electacta.2021.139766
sha: 6622829b9cea3ea373be05797b0c011feaf779f3
doc_id: 715729
cord_uid: jej9kj0c

Tracking and monitoring of low concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can effectively control asymptomatic transmission of current coronavirus disease 2019 (COVID-19) in the early stages of infection. Here, we highlight an electrochemical immunosensor for sensitive detection of SARS-CoV-2 antigen marker spike protein. The surface-clean Pd-Au nanosheets as a substrate for efficient sensing and signal output have been synthesized. The morphology, chemical states and excellent stable electrochemical properties of this surface-clean heterostructures have been studied. Functionalized superparamagnetic nanoparticles (MNPs) were introduced as sample separators and signal amplifiers. This biosensor was tested in phosphate buffered saline (PBS) and nasopharyngeal samples. The results showed that the sensor has a wide linear dynamic range (0.01 ng mL(−1) to 1000 ng mL(−1)) with a low detection limit (0.0072 ng mL(−1)), which achieved stable and sensitive detection of the spike protein. Therefore, this immunosensing method provides a promising electrochemical measurement tool, which can furnish ideas for early screening and the reasonable optimization of detection methods of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) outbreak has brought an unprecedented devastating impact on human life around the world [1, 2] . Until September 2021, the number of people infected with the virus has exceeded 220 million and continues to rise [3] . These days the SARS-CoV-2 virus, which can cause various degrees of upper respiratory disease, has been deeply analyzed [4] [5] [6] [7] [8] [9] . The β-coronavirus is a sequence of RNA genome with single-positive strand characteristics. It mainly encodes four structural proteins, including small envelope (E) protein, nucleocapsid (N) protein, spike (S) protein and matrix (M) protein [10, 11] . The infection route of SARS-CoV-2 can be achieved through a variety of contact or non-contact ways, and no obvious clinical symptoms appear in the early stage [12, 13] . Therefore, rapid virus screening can alleviate the fatal flaws of asymptomatic transmission that appeared in this pandemic.

At present, the detection of SARS-CoV-2 in the public health system is mainly through the PCR method targeting RNA and the ELISA method targeting antibody [14] [15] [16] [17] . However, the PCR method takes a long time, especially for such a large infection base; and the antibody detection technology of ELISA is not suitable for early screening of COVID-19, but is more conducive to the recovery period after treatment [1] .

Therefore, more and more attention is focused on the antigen coexisting in the virus as a potential detection target [18] . Among a variety of predicted antigenic biomarkers, the S protein that mediates adhesion to host cells has been reported to be one of the most valuable detection targets [9, 19] . S1 protein with receptor binding domain (RBD) located on the surface of the virus generally recognizes and binds to host cells, contributing to the development of clinical diagnostic kits suitable for sensitive detection in the field [20] . Therefore, the design and development of practical methods for tracking and detection S1 protein in respiratory samples are extremely important.

Electrochemical sensors that can convert changes in active biological substrates into capturable electrical signals have predictable potential [21] [22] [23] . The electrochemical detection method exhibits the advantages of high sensitivity and simple processing, and it is very promising to construct methods such as remote controllable measurement or on-site instant detection driven by a smartphone [24, 25] . The layered noble metal nanocomposite has high conductivity, large surface area and flexibly adjustable surface chemistry [26] . Among them, thin-layer metal nanosheets formed by embedding or converting other metal ions can provide many possibilities for the desired design and function. However, most thin-layer multi-metal nanosheets need to introduce a large amount of surfactants and stabilizers during the preparation process, and accompanied by harsh synthesis conditions, which greatly block their active sites and reduce their inherent activity. So far, simple preparation methods of layered bimetallic nanosheets with surface-clean heterostructures are quite rare. As a sensing substrate, the enhanced electrocatalytic performance by the synergistic effect and high chemical stability make it undoubtedly have important capabilities in the field of electrochemical measurement [27, 28] . In the constructed antigen-based sensor, the introduction of high charge concentration and appropriate electron mobility can promote the transfer of electrons from biomolecules to the sensor platform, enabling the detection of ultra-low levels of biological species [25] .

Magnetic nanoparticles (MNPs) have advantages in virus detection, especially in this COVID-19 pandemic [29] [30] [31] . Magnetic labels can meet the requirements of rapid separation and enrichment, and low cost-effectiveness [32] . Although the magnetic properties of MNP have been extensively studied, the employment of MNP is obviously more direct and effective in rapid and sensitive screening such as SARS-CoV-2. MNPs can reduce the amount of sample processing while simplifying the method, and can also exert its signal amplification function in electrochemical biosensing.

Here, we designed a magnet-assisted electrochemical immunosensor based on Pd-Au nanosheets for sensitive detection of S1 protein (Fig. 1) . Pd-Au nanosheets are selected as the sensing and fixing substrate. The surface-clean Pd precursor is used as the sacrificial template. It is worth mentioned that the entire preparation system was carried out at room temperature without any surface agents or stabilizers. The excellent conductivity and large surface area greatly enhanced the electronic transport and signal output between the interface and the solution. Antibody-functionalized MNPs were introduced as sample separators and signal amplifiers. The results showed a wide linear range (0.01 ng mL -1 to 1000 ng mL -1 ) and an ultra-low detection limit (LOD, 0.0072 ng mL -1 ), which achieved stable and sensitive detection of the S protein. This is essential for the initial monitoring of the virus. Meanwhile, this electrochemical immunosensor showed good selectivity, repeatability and stability. Overall, the successful casting of electrochemical biosensors combining Pd-Au nanosheets and functionalized MNPs provides a valuable analysis and application approach for antigen detection. Organics (USA). SARS-CoV-2 S1 recombinant protein (S1 protein), nucleocapsid protein (N protein) and anti-SARS-CoV S1 antibody (Ab) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All aqueous solutions were made using ultrapure 18.2 MΩ cm water.

Scanning electron microscope (SEM, Hitachi-SU8220, Japan) and transmission electron microscope (TEM, Tecnai G2 F20 U-TWIN, USA) were used to characterize the morphology of Pd-Au nanosheets. The surface modification of MNPs was identified by fourier transform infrared spectroscopy (FT-IR, Spectrum One, USA). The potential measurement was carried out by the Zetasizer instrument (Zetasizer Nano ZS, England).

The crystal structures of nanomaterials were studied by X-ray powder diffraction (XRD， M18XHF, Japan). The chemical elements and structure of the nanosheets were analyzed by X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, England). All electrochemical measurements were recorded by Chenhua Electrochemical Instrument (CHI 660, China). The typical three-electrode system used includes counter electrode (platinum electrode), reference electrode (saturated calomel electrode) and working electrode.

The precursor Pd nanosheets were synthesized with modification according to the previously reported method [33] . First, 15.2 mg of Pd(acac) 2 was ultrasonic dissolved in 10 mL of glacial acetic acid. Then, CO gas was introduced into the mixed solution at a flow rate of 250 mL min -1 and maintained for 30 min; after that, the above reaction solution was sealed and allowed to stand for 24 h. During this process, the color of the solution changed from yellow to black, and Pd nanosheets gradually formed and settled slowly. Finally, the products were washed thoroughly with ethanol and re-dispersed in ultrapure water.

Pd-Au composite nanosheets were synthesized by a simple method of galvanic replacement without the use of additional reductants. First, 10 mL of ultrasonically dispersed Pd sheets was transferred to a flask and stirred vigorously, and then an aqueous solution containing 4 mL of 26.32 mM HAuCl 4 •3H 2 O was dropped into the flask at a flow rate of 33.3 mL min -1 using an auto-injector. Then the reaction system continued to stir for 1 h to fully carry out the displacement reaction. Finally, Pd-Au nanosheets were obtained by washing several times with ultrapure water.

The typical synthesis of MNPs was performed as previously reported [34] . In short,

MNPs with core-shell structure were prepared by a simple solvothermal method. First, 

The glassy carbon electrode (GCE) was pretreated according to our previous procedure [35, 36] . Next, 20 μL of 1 mg mL −1 Pd-Au nanosheets was cast on the surface of the GCE and dried at 40 °C. The modified electrode was immersed in 10 mM PBS solution and used for further experiments. Then, 15 μg mL -1 S1 protein antibody was irradiated with UV light of 300 mW/cm 2 for 30 s to generate sulfhydryl groups for immobilization. This process relies on simple and mature photochemical immobilization technology [37, 38] . After that, 10 μL S1 protein antibody was used to incubate the Finally, the formed MNPs-Ab-S1 were reacted and bound with the antibody on the interface for 40 min, then washed with PBS and used for measurement next.

Nasopharyngeal samples were collected using sterile swab sticks and invaded into 2.5 mL PBS. After standing for 1 h, the samples were centrifuged and diluted twice as a stock solution for the dilution of spike protein. Then, 1 mg mL -1 spike protein was successively diluted with stock solution and stored at 4 °C for later use.

All the electrochemical measurements are carried out using a standard three-electrode system. 

The microstructure and surface morphology of the Pd-Au nanosheets were characterized by TEM and SEM. The TEM images revealed the thin-layer structure of Pd-Au hybrid nanosheets ( Fig. 2A, 2B and Fig. S1 ). In the high-resolution TEM image (Fig. 2C) ; the lattice spacing of Pd was 0.221 nm, while the gold nanostructure showed a larger lattice spacing of 0.235 nm, which was consistent with the face-centered cubic structure (111) of Au [34, 39] . The SEM images (Fig. 2D, 2E and Fig. S2 ) described the overall morphology of Pd-Au nanosheets, which were flower-like thin-layer structures with large surface areas. The EDS mapping images exhibited that Pd and Au coexist in Pd-Au nanosheets ( Fig. 2F and Fig. S3 ), and the Pd element was significantly higher than Au; while the Pd nanosheet only showed Pd element and strong Si element, which was attributed to the SiO 2 film (Fig. S4) . XPS survey spectrum was used to further analyze the chemical elements and existing states of Pd-Au nanocomposite. Fig. 2G and Fig. S5 revealed the overall survey and individual XPS element scan results. In the Pd 3d spectrum, Pd 3d 3/2 and Pd 3d 5/2 pertained to the peaks at 340.3 and 335.0 eV respectively, while the peaks at 335.8 and 341.2 eV were attributed to Pd 2+ (Fig. S5A) . The 87.3 and 83.6 eV peaks of Au were located in Au 4f 5/2 and Au 4f 7/2 (Fig. S5B ) [40] . The peaks of C 1s and O 1s were revealed at 284.8, 285.9 and 532.0 eV (Fig. S5C, S5D) , respectively, which originated from CO and glacial acetic acid in simple synthesis [41] . The crystal structure of the synthesized material was further explored by XRD. As shown in Figure   1H , 

First, TEM was employed to study the morphology of the MNP. Fig. 3A and 3B showed a relatively uniform MNP of 170 nm, consisting of a dark Ag core and a dense Finally, the purified S1 protein and Ab were analyzed by Western Blotting (Fig. S6) . The results showed that S1 antigen had strong binding to the Ab. 

Here we evaluated the application ability of Pd-Au nanosheets as substrates in electrochemical sensing. First, the CV response was measured at different scanning rates (10 mV s -1 -100 mV s -1 ) in 5 mM [Fe(CN) 6 ] 3-/4solution (Fig. 4A) , the oxidation and reduction peak currents increased with the increase of scanning rate. There was a good linear relationship between the peak current and the square root of the scanning rate (R 2 (Ox) = 0.9988, R 2 (red) = 0.9986), as shown in Fig. 4B , indicating that the electron transfer in [Fe(CN) 6 ] 3-/4was reversible and the reaction was a diffusion controlled process [42, 43] . The diffusion coefficient calculated by the Randles-Sevcik equation was 6.77 × 10 -6 cm 2 /s, which was comparable to the previous reports [44] [45] [46] . After that, the catalase activity of Pd-Au nanosheets was identified by a simple visual method (Fig. S7 ).

In 

Then, the catalytic ability of Pd-Au nanosheets to substrate H 2 O 2 was tested by i-t method (Fig. 4C ). The modified electrode showed a step-up current response in different concentrations of H 2 O 2 (0 -20 mM), proving the highly efficient catalytic ability for

In addition, the stability of the nanomaterials was studied (Fig. 4D) . The volt-ampere curve was stable after 30 times of CV scanning, demonstrating that the substrate Pd-Au nanosheets have good stability. 

The electrochemical properties of the modified interface were studied by EIS and CV ( Fig. 4E and 4F ). According to the modification procedure, compared with bare GCE, the load of Pd-Au nanosheets caused a decrease (∆R ct = 198 Ω) in the diameter of the Nyquist plots (R ct value), the increase of the CV peak current and a lower peak potential separation value (ΔEp), showing that this process greatly enhanced the electron transport between the interface and the solution. This is due to the large surface area of the Pd-Au nanosheets, which increased the contact and penetration sensitivity of [Fe(CN) 6 ] 3-/4-. The modification of antibody and BSA as a blocking agent resulted in an increase (∆R ct = 107 Ω) in Rct and a decrease in CV peak current, indicating that this process hindered the reversible delivery of the redox probe [35, 41] . However, the process exhibited lower spikes. This result can be attributed to the electronic coupling of the reduced probe [Fe(CN) 6 ] 3with negatively charged proteins (spike protein, BSA and antibody) in cathodic scanning and the greater polarization effect [47, 48] . The binding of MNP-Ab-S1

nanocomposite on the surface of GCE further strengthened the above process. These results proved the effectiveness of the construction method. In addition, CV and DPV procedures were performed in PBS solution containing 10 mM H 2 O 2 to verify the feasibility of this strategy ( Fig. 4G and 4H) . The results revealed that the modified Ab showed a reduced current response relative to the Pd-Au nanosheets, and the DPV curve collected the reduction current of H 2 O 2 at -0.074 mV. After the MNP-Ab was bound to the target S1 protein, the modified electrode measured the reduced CV and DPV signals.

This was because of the MNP, which was a poor conductor and hindered the contact and reaction of Pd-Au nanosheets with H 2 O 2 in the solution. Furthermore, the surface morphology of the interface of the modified MNPs-Ab-S1 was characterized by SEM, as shown in Fig. S8 . Compared with that one before modification (Fig. S8A-C) , the interface after modification of MNPs-Ab-S1 showed that the nanocomposite was bound to the surface of the 3D Pd-Au nanosheet substrate (Fig. S8D-F 

In order to obtain the best electrochemical detection performance, the experimental conditions were optimized, including the incubation concentration of Ab on the interface, 

First, the comparative experiments between Pd-Au+Ab+S-MNP with the blanks (bare GCE, Pd-Au, Pd-Au+Ab) were carried out to detect S1 protein (Fig. S10) , the results indicated that compared with the blanks, Pd-Au+Ab+S-MNP showed an observably suppressed DPV current, which proved the importance of this construction strategy. Then, the performance of the magnet-assisted electrochemical immunosensor was analyzed by DPV program under optimal conditions. Different concentrations of S1 protein in PBS were tested, and each test was performed 3 times. As shown in Fig. 5A , as the target concentration increased the DPV response at -0.074 mV gradually decreased. Fig. 5B showed the relationship between S1 protein concentration and DPV peak current.

The inset showed that the current signal gradually decreased with the logarithmic value of the target concentration from 0.01 ng mL -1 to 1000 ng mL -1 , and exhibited an excellent linear relationship over a wide range (R 2 = 0.9916). The calibration curve was y = 46.066 -11.439 lg x, where y was the peak DPV current and x was the target antigen concentration. The LOD of 0.0072 ng mL -1 was calculated by the signal-to-noise ratio of 3 (S/N = 3), according to the means of 3's blank criterion. In addition, various methods for detecting S protein and its related proteins were summarized and compared (Table S1) , and the results demonstrated that this electrochemical sensor was superior to most of them. This is also due to the high catalytic performance of Pd-Au nanosheets and the signal amplification effect of MNPs. These results confirmed that this detection strategy has an excellent ability to detect S protein and can achieve ultra-sensitive detection of the target. (B) The relationship between S1 protein concentration and DPV peak current. Inset: Plot of current vs logarithm of S1 protein concentration.

To evaluate the repeatability and stability of this method, multiple prepared immunosensors were stored in a dry environment at 4°C for different periods of time, and then DPV responses were tested under the same conditions (Fig. 6) . The results showed that the current signals between different GCEs did not show significant differences (Fig.  6A ). After 10 days of storage, the electrochemical response of the modified electrode was close to its original current signal (Fig. 6B) . We noticed that there is some variation in the mean valves within 10 days. Yet compare with the exiting literature, this magnet-assisted electrochemical immunosensor based on Pd-Au nanosheets has relative ultrasensitivity, simplicity, and a wide detection range [49] [50] [51] . 

We evaluated the selectivity of this detection method using 10 different proteins or small molecules (Fig. 7A) . The results revealed that except the S1 protein, other proteins or molecules exhibited almost constant DPV signal; and after incubating the target protein, the current was greatly reduced (Fig. 7B ). It should be pointed out that the concentration of non-target substances set was 5 times of the concentration of target substances. The above results proved that this strategy has good selectivity. In order to test the possible applications of the proposed immunoassay method, SARS-CoV-2 S1

protein was detected in the nasopharyngeal samples collected from healthy people by standard addition method. Fig. 7C showed the DPV responses caused by incubation with

signal strongly dependent on the concentration. The determination results in the complex samples were visually compared with those in PBS, and similar results were obtained (Fig. 7D) . The calculated recovery rate was 84.545% -103.520%, and the RSD was between 1.833% and 8.406% (Table S2 ), indicating that the feasibility of the proposed sensor for practical applications. It is important to further validate the accuracy of the proposed sensor for future clinical samples. 

In general, an electrochemical immunosensor was fabricated to detect SARS-CoV-2 S1

protein, achieving a LOD as low as 0.0072 ng mL -1 and wide detection range, which was 

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