key: cord-0843623-5ibd6g7a
authors: Hosseini, Morteza; Sobhanie, Ebtesam; Salehnia, Foad; Xu, Guobao; Rabbani, Hodjattallah; Naghavi Sheikholeslami, Mahsa; Firoozbakhtian, Ali; Sadeghi, Niloufar; Hossein Farajollah, Mohammad; Reza Ganjali, Mohammad; Vosough, Houman
title: Development of sandwich electrochemiluminescence immunosensor for COVID-19 diagnosis by SARS-CoV-2 Spike protein detection based on Au@BSA-Luminol nanocomposites
date: 2022-05-25
journal: Bioelectrochemistry
DOI: 10.1016/j.bioelechem.2022.108161
sha: 39cca376ec13c5b06d3b83942568ceb41bbc7a34
doc_id: 843623
cord_uid: 5ibd6g7a

Coronavirus disease (COVID-19) is a new and highly contagious disease posing a threat to global public health and wreaking havoc around the world. It's caused by the Coronavirus that causes severe acute respiratory syndrome (SARS-CoV-2). In the current pandemic situation, rapid and accurate SARS-CoV-2 diagnosis on a large scale is critical for early-stage diagnosis. Early detection and monitoring of viral infections can aid in controlling and preventing infection in large groups of people. Accordingly, we developed a sensitive and high-throughput sandwich electrochemiluminescence immunosensor based on antigen detection for COVID-19 diagnosis (the spike protein of SARS-CoV-2). For the spike protein of SARS-CoV-2, the ECL biosensor had a linear range of 10 ng mL(-1) to 10 µg mL(-1) with a limit of detection of 1.93 ng mL(-1). The sandwich ECL immunosensor could be used in early clinical diagnosis due to its excellent recovery in detecting SARS-CoV-2, rapid analysis (90 min), and ease of use.

The new acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak put people's physical and mental health all over the world in danger. The virus's rapid transmission speed and high infectious capacity have resulted in a considerable increase in the number of affected people every day. As a result, early detection of SARS-CoV-2 is critical for human survival, health, and security.

Early clinical diagnosis of SARS-CoV-2 is quite challenging. The gold standard method of RT-PCR requires complex types of equipment as well as skilled personnel, resulting in a costly and sensitive assay. The procedure requires sample pretreatment as it operates on the genome of the virus [1, 2] . Elimination of the many steps involved not only lessens the duration of the process but also reduces the chance of human error in it. This would also eliminate the need for trained personnel and cut expenses. Biosensors as highly sensitive, selective, and accurate monitoring tools are the alternatives to the traditional methods of detection. These sensors are capable of ultralow determination of analytes in biological samples. Among them, electrochemical biosensors have the advantages of ultra-sensitive detection and a wide range of detection [3, 4] .

Electrochemiluminescence (ECL)-based sensors are classified as one of electrochemical sensors featuring good sensitivity as well as stability. ECL technology has become more popular in recent years as a strong analytical technique for the detection of biomolecules in clinical, environmental, and industrial settings [5] [6] [7] . The ECL method has also been used in the analysis of disease-related biomarkers. It offers high sensitivity, low cost, simple operation, and easy miniaturization and intelligence to answer clinical treatment development needs [8, 9] .

The antigen-antibody specific interactions are considered as one of the most selective and sensitive interactions. The employment of antibodies as bioreceptors in immunosensors is responsible for the high sensitivity and selectivity of these types of sensors enabling ultra-low detection of analytes in biological samples. Immunosensors detect antibody binding to a specific target molecule and can be used to detect the presence and quantity of specific antibodies in order to find whether a patient has been previously infected and to assess the disease's prevalence [10] [11] [12] . The most common detection strategy in immunosensors is the sandwich immunoassay type. The detected antigen is sandwiched between two antibodies, one of which, the capture antibody, is attached to the transducer surface. On the other hand, the detection antibody is usually labeled through which the amount of antigen is measured [13, 14] .

Smartphone-based sensing systems have recently received much attention since they provide a semi-automated user interface that even non-technical individuals can utilize [15] . Sensing systems can be designed within a smartphone with customized hardware and software incorporated, allowing the system to be miniaturized and carried out in any location. They can be operated by anyone who is semi-trained [16, 17] . The RGB 1 color space is regarded as the most expanded and accepted color system. Virtually every visible spectrum can be represented as RGB three colors in a mixture of different proportions and intensities [18] [19] [20] .

Noble metal nanocomposites have been used in ECL-based detections of biomarkers due to their low toxicity and enhancing effects on the ECL signal. In research done by Jia's team, Ag@BSAluminol-Ab 2 nanocomposites were employed for the detection of carbohydrate antigen 19-9 [21] .

Another ECL-based biosensor for the detection of human chorionic gonadotropin was reported [22] . The sensor benefits from a sandwich assay signaling format employing Au star@BSAluminol-Ab 2 as the signal reporter. The great water solubility and low toxicity of the nanocomposites combined with their enhancing effect on the ECL intensity of the luminol have made them a good candidate for this work.

Here, for the first time we report the ECL-based detection of SARS-CoV-2 in a signal-on sensing format in untreated samples through its surface spike proteins. The immunosensor was fabricated by the addition of gold nanoparticles (AuNPs), 11-mercapto undecanoic acid (MUA), and 3mercapto propionic acid to a glassy carbon electrode surface. The Ab 1 was then immobilized on the surface of the electrode through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) functionalization of the electroe. The detecting antibodies (Ab 2 ) and luminous substance luminol were immobilized using flower-like Au@BSA nanoparticles with excellent biocompatibility. The determination steps were evaluated using the development of an immunocomplex between the antigen and the ECL labeled antibody.

Moreover, visual detection of targets based on Smartphone Imaging is being investigated for future application of this system. The polyclonal antibody anti-SARS-CoV (made in rabbit, 1.3 mg mL -1 ) (Ab 1 ), as well as the polyclonal antibody SARS-CoV Spike (manufactured in goat, 1.8 mg mL -1 ) (Ab 2 ), which identifies the S protein, were bought from Padzacompany (Iran). Inactivated virus (vero cell) vaccine (Coronavac; Sinovac, 1mg mL -1 ). Antigens and antibodies were prepared and diluted in PBS 1X buffer, pH 7.4.

A three-electrode setup was used for all electrochemical and ECL studies, with the modified glassy carbon electrode (GCE, 2 mm) serving as the working electrode, a saturated KCl Ag/AgCl electrode serving as the reference electrode, and a Pt wire serving as an auxiliary electrode. A DropSens Stat400 Bipotentiostat/Galvanostat was used to perform cyclic voltammetry (CV) analyses (Asturias, Spain). A photomultiplier LS 50 (Perkin-Elmer) was coupled to operate as an ECL detector with a bias potential of -800 V. The working electrode was vertically placed in a 4 mL quartz cell for the ECL measurements. The AUTOLAB PGSTAT 30 was used to perform electrochemical impedance spectroscopy (EIS) experiments (The Netherlands). A Metrohm device was used to take the pH readings. Microscopy images and morphology studies were obtained using a MIRA3 TESCAN-XMU Scanning Electron Microscope.

The Au nanoparticles employed in the synthesis process were obtained according to previously published work with minimal modifications [23, 24] . Briefly, 5 mL trisodium citrate solution (38 mM) was added to 25 mL boiling HAuCl 4 (1 mM) solution under constant stirring for 8-10 minutes until the color of the solution changed to red wine, indicating the practical synthesis of gold seeds.

Following that, BSA was used to create various functional groups via the interaction of thiol groups with Au nanomaterials. Freshly generated BSA solution (500 mg) was first denatured by reacting with the chemical reagent sodium borohydride (40 mg) with vigorous stirring for one night to transform the disulfide bonds into thiol groups and generate additional thiol groups onto the surface of BSA [21, 22] . After that, the obtained products were centrifuged and washed multiple times with double-distilled water and ethanol, respectively. Before usage, the compounds were freeze-dried and stored at 4 °C in the refrigerator.

The luminol and Ab 2 modified Au@BSA were produced using the chemical bonding approach in this study. Ultrasonication was used to dissolve 2 mg of Au@BSA into 1 mL double-distilled water. After that, 400 µL of 12.5% GA was promptly added to the above solution and shaken for 3 hours to ensure GA was attached to the Au@BSA surface. Subsequently, the GA-linked Finally, 40 L detection antibodies (1.8 mg mL -1 ) were added to the mixture and stirred slowly for 8 hours in an ice bath to effectively conjugate the Ab 2 to the surface of the Luminol-Au@BSA.

The produced luminol-Au@BSA-Ab 2 nanocomposites were separated by centrifugation (6000 rpm) and washed with double-distilled water to eliminate any remaining reagents. The produced luminol-Au@BSA-Ab 2 nanomaterials were dispersed in 1 mL PBS (0.1 M, pH 7.4) for further application.

A 0.05 M alumina slurry was used to polish the GCE (2 mm). After that, the mirror-like surface was cleaned with distilled water and allowed to dry at room temperature. By immersing the electrode in an electrochemical cell containing a solution of 10 mg mL -1 HAuCl 4 in 0.1 M KNO 3 , followed by a 60-second potential step of -0.2 V, AuNPs were deposited on the electrode surface.

Then, the electrode was washed with distilled water and dried at room temperature. The and -SH) [21, 26] . Additionally, EDS analysis and FT-IR were also used to characterize the produced Au@BSA. The EDS examination revealed the presence of gold, carbon, nitrogen, and oxygen components ( Figure 1B) , confirming that the Au@BSA nanoparticles were successfully constructed. As shown in Figure 1C the typical stretching vibrations of -OH and -NH caused the wide peak at 3450 cm -1 , and the peak at 1520 cm -1 was also caused by -NH vibration.

Furthermore, the characteristic peaks at 1646 and 1172 cm -1 were due to the -COO group vibration, implying that the synthesized Au@BSA could provide a variety of functional groups, including -COOH and -NH 2 groups.

Here Figure 1 The ferri/ferrocyanide electron transfer kinetics alter from one step to the next, as seen by cyclic voltammograms (Figure 2A) . The ferri/ferrocyanide electron transfer kinetics of the bare GCE has a well-defined reversible peak. The peak current was enhanced when Au NPs were deposited on the electrode due to Au NPs' excellent electron transfer ability (curve b). After modification with MUA/MPA, the peak currents decreased confirming the modification of the electrode surface. modification with EDC/NHS and further on Ab 1 also led to a decrease in the current intensities confirming the successful immobilization of the capture antibody. The addition of SARS-CoV-2 also dropped the peak current of the CV curves. The addition of the produced nanocomposite also had a decreasing effect on the current intensity of the CV curves. All these results indicate that the proposed immunosensor has been successfully built.

Moreover, Figure 2B clearly shows that the EIS of the bare GCE has a low electron transfer resistance. After depositing Au-Nps, a substantially smaller resistance (curve b) was detected, which was attributed to Au-Nps good electron transfer capacity and excellent conductivity. The diameter of the semicircle and electron transfer resistance increased after modification of the surface with MUA-MPA (curve c), antibody1 (curve d), and SARS-CoV-2 antigen curve e) due to the resistance of non-conductive bioactive compounds. When Luminol-Au@BSA-Ab 2 (curve f) was dropped on the electrode, the diameter of the semicircular in the EIS increased even more.

This result confirms the CV data, indicating that the sandwich immunosensor was successfully assembled.

Here figure 2 

A series of optimization experiments were done based on sandwiched immunoassay to improve COVID-19 detection capability. The intensity of the ECL signal was studied against the incubation time of Ab 1 for 75 minutes in time intervals of 15 minutes. As can be seen from Figure S1a the intensity of the signals increased with the increasing time of incubation to 60 minutes and reduced afterwards suggesting that the optimum time for its incubation could be 60 minutes. Further more the ECL intensity was investigated against the reaction time between the analyte and Ab 1 where results suggested that a good-enough signal can be generated after 45 minutes ( Figure S1b ). The incubation time of the reporter nanocomposite was also investigated for a better signal intensity.

As depicted in Figure S1c the optimum duration for this reaction was found to be 60 minutes. The relationship between the ECL intensity and pH value was investigated over a pH range of 7.0 to 9.5. Figure S2b shows that the ECL signal increased considerably with the increase in pH from 7.0 to 8.5 and then decreased. The maximum ECL intensity was observed at pH 8.5. Thus, phosphate buffer solution of pH 8.5 was chosen for further ECL immunosensor determination.

The performance of the ECL immunosensor was further assessed by evaluating the ECL response at various concentrations of the SARS-CoV-2 antigen. Under the optimized conditions, the sandwich ECL biosensor was utilized to detect SARS-CoV-2 antigen. Figure 3A depicted that the ECL signal intensity increased as the concentrations of SARS-CoV-2 antigen increased from 10 ng mL -1 to10 µg mL -1 . Furthermore, the change in ECL signal intensity was linearly related to antigen concentration, with a linear relationship of y = -415.54x+3015.9, R 2 = 0.9823 ((inserted picture of Figure 3A) ). The LOD was calculated to be 1.93 ng mL -1 using the limit of detection

Moreover, visual detection of target was investigated for further application of this system. The Here Figure 3 3.6. Specificity, stability, and reproducibility Stability, selectivity, reproducibility, and long-term stability of the biosensing device are important for use in clinical diagnosis. So, the ECL immunosensor's selectivity for the SARS-CoV-2 antigen should be examined. We compared the sensor's specificity to that of other related viruses, including SARS-CoV, MERS-CoV, and influenza A. The biosensor was immersed in 100 times SARS-CoV-2, 100 X SARS-CoV, 100 X MERS-CoV, 100 X influenza, and blank before being modified with Au@BSA-Luminol-Ab 2 . The biosensor processed by SARS-CoV-2 antigen showed a significant difference compared to the biosensor processed by other viruses, as shown in Figure 4A . As a result, the biosensor has excellent selectivity.

The ECL stability of the proposed sandwich immunosensor was examined at various SARS-CoV antigen concentrations (10 -6 , 2 10 -7 , 1.5 10 -7 , 10 -7 g mL -1 ) under successive three potential × × scans ( Figure 4B ). It was discovered that the ECL signal enhanced when the quantity of SARS-CoV-2 spike protein increased. Furthermore, ECL curves were relatively stable at all concentrations. The relative standard deviation (RSD) for SARS-CoV-2 spike protein detection was 2.51 %, 3.05 %, 5.13 %, and 1.52 %, respectively, indicating that the as-prepared ECL immunosensor has excellent stability. The ability of the created immunosensor for long-term cyclic use was also investigated since this factor was an important parameter for the immunosensors application. The ECL responses of the developed immunosensor could keep their ECL signal after four weeks (81.45%) when stored at 4 0 C, as shown in Figure 4C . Also, the biosensor's (GCE/AuNps/MUA-MPA/Ab 1 /BSA) and produced electrodes for measuring SARS-CoV-2 antigen (1µg mL -1 ). The RSD was calculated to be 5.75 %, indicating that the biosensor is repeatable ( Figure 4D ).

Here figure 4 

The direct identification of SARS-CoV-2 infected patients would be ideal for this sensing technology. So, saliva and nasopharyngeal swabs from ten positive and negative human subjects were examined with GCE/AuNps/MUA-MPA/Ab 1 /BSA/target/Luminol-Au@BSA-Ab 2 our proposed immunosensor and the gold-standard RT-PCR, respectively, to assess the efficiency of the created sandwich Immunosensor using clinical samples. As shown in Table 1 , our sensor can produce results that are comparable to gold-standard RT-PCR methods, but much faster. Figure 5 shows the ECL response of the GCE/AuNps/MUA-MPA/Ab1/BSA immunosensor incubated in pharyngeal swabs of patients (without any treatment) and then modified with Luminol-Au@BSA-Ab 2. Photomultiplier tubes (PMT) and smartphones as detectors were used to measure ECL signals derived from sandwich immunosensor.

Here Table 1 Here figure 5 For further investigation, the signal response of various concentrations of SARS-CoV-2 antigen added in pharyngeal swabs according to the standard addition method was detected in recovery studies. The experimental recoveries ranged from 93% to 99.1%, with RSD values ranging from 1.41% to 4.94%, as shown in Table 2 .

Here table 2

In this study, a sandwich ECL immunosensor for sensitive diagnosis of SARS-CoV-2 antigens was proposed based on flower-like Au@BSA nanoparticles. Because of their notable electroconductivity, good biocompatibility, and large surface area, flower-like Au@BSA nanoparticles not only have sufficient binding sites for loading plenty of Ab 2 but also adsorb large amounts of luminol molecules, resulting in enhancement of luminol ECL intensity, as well as good stability, excellent reproducibility, favorable selectivity, and a wide dynamic range of this developed immunosensor. The fast (90 min) and sensitive detection of SARS-CoV-2 antigens was recorded with a detection limit of 1.93 ng mL -1 . In addition, this sandwich ECL sensor is capable of detecting spike protein antigen of SARS-CoV-2 in real nasal swabs from patients and has an acceptable sensitivity and specificity. Significantly, the ECL images can be quickly recorded using a smartphone, and the results for SARS-CoV-2 detection were outstanding. The smartphone-based sensor platform is a field-deployable device that is portable, cost-effective, and user-friendly. In the future, it may be a useful tool for the early identification of SARS-CoV-2. 

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Mohammad Hossein Farajollah, Mohammad Reza Ganjali, Houman Vosough, declare no conflict of interest. Highlights:  Fabrication of a COVID-19 diagnostic ECL-based immunosensor

 Highly sensitive and inexpensive detection of the virus

 Excellent selectivity for the surface spike protein of the virus

 Applicable to real clinical sera with satisfactory results

The authors of this paper would like to express their gratitude to the Iran National Science Foundation and Chinese Academy of Sciences (INSF 99008701, CAS-VPST Silk Road Science, GJHZ202125) and for the financial support. We also gratefully acknowledge the support from the University of Tehran.