key: cord-0901579-hb5zl3ky
authors: Ghazizadeh, E.; Neshastehriz, Ali; Firoozabadi, Ali Dehghani; Yazdi, Mohammad Kaji; Saievar-Iranizad, Esmail; Einali, Samira
title: Dual electrochemical sensing of spiked virus and SARS-CoV-2 using natural bed-receptor (MV-gal1)
date: 2021-11-26
journal: Sci Rep
DOI: 10.1038/s41598-021-02029-0
sha: b268e39c263328a42f833492fe83f60d98faf54c
doc_id: 901579
cord_uid: hb5zl3ky

It has been necessary to use methods that can detect the specificity of a virus during virus screening. In this study, we use a dual platform to identify any spiked virus and specific SARS-CoV-2 antigen, sequentially. We introduce a natural bed-receptor surface as Microparticle Vesicle-Galactins1 (MV-gal1) with the ability of glycan binding to screen every spiked virus. MV are the native vesicles which may have the gal-1 receptor. Gal-1 is the one of lectin receptor which can bind to glycan. After dropping the MV-gal1 on the SCPE/GNP, the sensor is turned on due to the increased electrochemical exchange with [Fe(CN)(6)](−3/−4) probe. Dropping the viral particles of SARS-CoV-2 cause to turn off the sensor with covering the sugar bond (early screening). Then, with the addition of Au/Antibody-SARS-CoV-2 on the MV-gal1@SARS-CoV-2 Antigen, the sensor is turned on again due to the electrochemical amplifier of AuNP (specific detection).For the first time, our sensor has the capacity of screening of any spike virus, and the specific detection of COVID-19 (LOD: 4.57 × 10(2) copies/mL) by using the natural bed-receptor and a specific antibody in the point of care test.

| (2021) 11:22969 | https://doi.org/10.1038/s41598-021-02029-0 www.nature.com/scientificreports/ the impedance decreased by covering of the glycosylation bond. Dropping the Au@Anti-SARS-CoV-2 spike caused to turn on the sensor by electrochemical amplifier of AuNP, again. As a result, we reported a natural bed/receptor (MV-gal1) with Au@Anti-SARS-CoV-2 spike to double sensing of SARS-CoV-2 Antigen with high sensitivity in ~ 5 min.

Materials. All of the materials as analytical grade potassium ferrocyanide, potassium ferricyanide, sulfuric acid, hydrogen peroxide, sodium chloride, and potassium chloride were prepared from Novin Tech Company, IRAN. We used as Virus Transport Medium (VTM) from Nedashimi Co, IRAN. This is a liquid media for the transport of specimens to the laboratory for transport of viruses (including COVID19). We also used as SCPE/ GNP which is functionalized with gold nanoparticles on the ceramic substrate were purchased from DropSens Inc. Gold Nano Particle-Carbon (GNP-carbon) working electrode; a carbon counter electrode and a silver reference electrode are components of the electrode. Electrochemical impedance spectroscopy (EIS) and Differential pulse voltammetry (DPV), were evaluated by SP-300 Instruments (SP-300) Texas, USA. DPV was done in the presence of 1 mM [Fe(CN) 6 ] −3/−4 in phosphate buffer saline in the potential window − 0.4 V to + 0.4 V at a scan rate 50 mV s −1 , and impedance measurement was done between 100 kHz to 1 Hz of [Fe(CN) 6 ] −3/−4 in phosphate buffer saline pH = 7.4. ZSimpWin 3.22 Software (Princeton Applied Research) was used for measuring the EIS spectra with the help of equivalent circuit using, and the data were presented in Nyquist plots. AFM was done for the analysis of the surface roughness on a Dimension 3000 instrument (Digital Instruments/Aveco Science). TEM images were done by TecnaiG220 instruments from FEI Company, Hillsboro, USA. We determinate the particle sizes and zeta potentials by Horiba nanoparticle size analyzer, Malvern Nano SZ-100 at wavelength 532 nm. 

Immobilization of MV-gal1 was done by dropping 5.2 µL of MV-gal1 solution in 50 mM phosphate buffered saline (phosphate buffer saline, pH 7.4) onto the SCPE/GNP and incubated overnight at 4 °C. After incubation, excess MV-gal1 was removed by the phosphate buffer saline. Following rinsing, 50 µL of blocking solution (1% BSA in phosphate buffer saline for 1 h) was added onto the electrode surface to prevent the nonspecific binding and incubated at 4 °C. Then we use as SARS-CoV-2 Antigen as SARS-CoV-2 Antigen Protein stock (ProSci Incorporated, Co) which was diluted to 100 fold a 5 µL of this diluted solution was dropped on the MV-gal1/SCPE and incubated overnight at 4 °C. 3% BSA was added to the antibody solutions for blocking and minimize the non-specific absorption (NSA). Then, electrochemical tests were done at every stage.

A mixture of 100 µL of Anti-SARS-CoV-2 spike (50 µg/mL in 5 mM KH 2 PO 4 , pH 7.5) (MyBiotech Co) and 700 µL of 0.1% Au nanoparticle solution was prepared a kept for 50 min at room temperature. We add 50 µL of 1% PEG in 5 mM KH 2 PO 4 solution (pH = 7.5) and 100 µL of 10% BSA in 50 mM KH 2 PO 4 solution (pH 9.0) to block any uncovered surface on the AuNPs. The AuNP conjugated Anti-Cov-2 (Au/Anti-SARS-CoV-2 spike) was then collected via centrifugation (8000g for 15 min at 4 °C). Au/Anti-SARS-CoV-2 spike were suspended in 1 mL of preservation solution (1% BSA, 0.05% PEG 20000, 0.1% NaN 3 and 150 mM NaCl in 20 mM Tris HCl buffer, (pH = 8.2), and centrifuged again to collect the Au/Anti-SARS-CoV-2 spike. and stored as stock solution. In fact, we used in the study of direct adsorption of AuNP with Anti-SARS-CoV-2 spike to form the Au@ Anti-SARS-CoV-2 28 . In order to ensure the binding of AuNP to the Anti-SARS-CoV-2 spike, we used MV-gal1@ SARS-CoV-2 Antigen@Anti-SARS-CoV-2 (Without AuNP as control) in the electrochemical reaction of DPV, (Fig. S5) .

In research of Dianyun showed that gold nanoparticle (AuNP) can act as an electrochemical amplifier to detection human chorionic gonadotropin (hCG) which it is used as a label with the second AB 29 . Not only, adding the Au@Anti-SARS-CoV-2 spike on the MV-gal1@SARS-CoV-2 Antigen show the specific SARS-CoV-2 Antigen (specific detection) with the increase in the electrochemical reaction of AuNP with Fe(CN) 6 ] −3/−4 probe, but only it can prove the connection of MV-gal1 with glycans of SARS-CoV-2 which happened in first step, too. So at this point, it may confirm the connection of gal-1 on the MV with the spike of SARS-CoV-2 based on the NTD-domains. We also verified the effect of bonding of glycans in attachment with receptors based on the electrochemical reactions. We use as the EIS to identify the modified electrode with surface properties. The impedance behaviors of the respective layers are reported in Fig. 2B . Two equivalent circuits viz., Rs(Qdl(RCTW)) and Rs(Qdl(RCTW)(CRL)) were used as model to show the impedance data. Rs(Qdl(RCTW)) will use for further analysis if the Rs(Qdl(RCTW)(CRL)) circuit does not fit well with all surfaces studied. So, Rs shows the solution resistance; Qdl and R CT are capacitance (constant phase element) and charge transfer resistance of the gold electrode respectively; RL is the layer resistance and W is Warburg element. In corroboration with the DPV results, (Fig. 4B(b) ). To identify the interaction of each component on the modified SCPE/GNP sensor, TEM images have been used. Figure 4A panel (a) shows the TEM images of the modified MV-gal1 at higher magnification, ~ 300 nm. The interaction of SARS-CoV-2 virus on MV-gal1 has shown with the increased size of the ~ 650 nm. (Fig. 4A(b) .

To investigate the performance of the MV-gal1/SCPE-GNP sensor, we evaluated the response of the sensor to SARS-CoV-2 Antigen Protein. First, we showed the LOD of sensor for spike protein when it connected to MV-gal1. Our devise sensed to 500 fg/mL of SARS-CoV-2 spike protein in phosphate buffer saline (Fig. 5A) . The j value increases linearly with increasing the concentration of SARS-CoV-2 spike protein ranged from 1 µg/mL to 500 fg/mL. A regression equation of y = 10.973x + 30.456 (R 2 = 0.976) was obtained, where y is the j value in μA cm −2 and x is the logarithmic concentration of SARS-CoV-2 spike protein in µg/mL. The sensor responded the LOD (1 fg/mL) with lower sensing of SARS-CoV-2 spike protein in phosphate buffer saline when the Au@Anti-SARS-CoV-2 spike was added on the SCPE-GNP electrode. A regression equation of y = 12.763x + 30.456 (R 2 = 0.976) was obtained, where y is the j value in μA cm −2 and x is the logarithmic concentration of SARS-CoV-2 spike protein in fg/Ml (Fig. 5C,D) . So, it can indicate high sensitivity and specificity for detection of the SARS-CoV-2 spike antigen. Also, it can be proved that the connection of MV-gal1 with glycan of SARS-CoV-2 spike protein which it can cause the connection of Au@Anti-SARS-CoV-2 spike via another specific part of SARS-CoV-2 spike protein. To diagnosis of COVID-19 is performed using nasopharyngeal swabs suspended in transport medium (VTM). So, we used as our sensor to detection of SARS-CoV-2 Antigen protein in Universal Transport Medium (UTM). However, the presence of various reagents such as salts and non-specific factors can affect the performances of the sensor, but our sensor can sense SRS-CoV-2 spike proteins in 0.01 × VTM with starting from a concentration of 1 µg/mL when it attached to the MV-gal1/SCPE-GNP sensor and 500 fg/mL when adding of the Au@Anti-SARS-CoV-2 spike was occurred (Fig. S2A,B) . So, our device can screen and specific detection of the COVID-19 samples in two steps without any preparation or preprocessing.

Our sensor was used to show the functionality of COVID-19 in clinical samples (Fig. 5) . So, we collected the nasopharyngeal swab specimens from COVID-19 patients (Emad laboratory) and normal subjects and stored them in VTM (Table S1 ). We optimize the nasopharyngeal swab samples relate to normal subjects with DPV analysis to determine the basal signal (Fig. S3) . Then, our sensor responded to patient samples diluted as much as 2:1 × 10 5 (610 copies/mL) with the overall regression equation of y = 11.321x + 29.512 (R 2 = 0.973) (Fig. 5E,F) . At end, sensor diluted as much as 1:4 × 10 5 and sensed (457 copies/mL) when faced with specific antibody (Au@Anti-SARS-CoV-2 spike) in following the regression equation of y = 12.124x + 20.512 (R 2 = 0.989) (Fig. 5G,H) . Because of the various reagents and generates noise signals of VTM includes, we consider the LOD of the COVID-19 sensor to be low enough for practical Table 1 .

Reproducibility and stability of MV-gal1@Au/Anti-SARS-CoV-2 spike to sense the SARS-CoV-2 virus on the SCPE-GNP. The virus concertation of 1 × 10 5 (virus particle/mL −1 ) relate to the MV-gal1 sensing and 1:4 × 10 5 (virus particle/mL −1 ) relate to the Au/Anti-SARS-CoV-2 spike, was showed the standard deviation of 4.9% and 5.3% examined for five measurements, respectively with a showing good reproducibility (Fig. S4a ). Our results also show the perfect response after 35 successively scanning, suggesting the acceptable durability of this method (Fig. S4b) . Calibration results were reported in (Table S1) for MV-gal1@SARS-CoV-2 Antigen@Au/Anti-SARS-CoV-2 spike sensors. Statement on human guidelines. The clinical samples used in this were collected who suspicious patients referred to Emad laboratory were used. Ethical committed of Iran medical university confirmed that all experiments were performed in accordance with relevant guidelines as the declaration of Helsinki and regulations with registration code as: 99-1-6-8-17943. We also confirmed that experimental protocols were approved by Iran University of medical science and with financial committee by including a statement in the methods section to this effect, including any relevant details. We explained about human samples in section "Clinical sample preparation" and Table S2 .

Consent for publication. The graphic figure was prepared by Dr. Elham Ghazizadeh.

Statement confirming. All our results have been achieved realistically and with great effort during the Corona pandemic in IRAN and our research were carried out in compliance with the ARRIVE guidelines.

In the development of the COVID-19 pandemic, it is necessary to design the sensors to enhance screening and specific detection for COVID-19 in a short time 31, 32 . For the first time, we designed a dual-sensor based on MV-gal1/Au@Anti-SARS-CoV-2 spike to detection SARS-CoV-2 Antigen. We used of the MV-gal1 to general screening of any spike virus. We introduce the natural bed-receptor (MV-gal1) with electrochemical reactions in solid sensor with can bind to glycan of any virus. At the end, using the Au@Antibody-SARS-CoV-2 spike cause to specific bind with the SARS-CoV-2 Antigen. So, our dual-platform has the ability of screening for any spike virus (at the first step) and specific detection of SARS-CoV-2 with high sensitivity (second step) in the solid biosensor. However, there are need more in vivo studies with more sample size to validate this sensor for another spike virus and SARS-CoV-2. Table 1 . Comparison the biosensor methods to detection of surface antigen of SARS-COV2. 1 The sensitivity of a clinical test refers to the ability to correctly identify those patient samples (also called the true positive rate) (Lalkhen and McCluskey 30 ). 2 The specificity of a clinical test refers to the ability to correctly identify those non-patient samples (also called true negative rate) ( www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints.

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Authors thank Iran University of Medical Sciences for support this project as project (99-1-6-8-17943) and EMAD laboratory and Pasteur Institute to provide samples of inactive Covid-19 patients.

Everyone contributed equally to this article. A.N.z is corresponding, and E.G. do this research, A.D. helped in analyzing and E.S. prepare lab, S.E. prepared figures and M.K.Y. wrote this research. Figure 3 was created by E.G.

The authors declare no competing interests.

The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-021-02029-0.