2.1. 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 1mM [Fe(CN)6] -3/-4 in phosphate buffer saline in the potential window -0.4 V to +0.4 V at a scan rate 50Mv s 1, and impedance measurement was done between 100 kHz to 1Hz 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 532nm.
2.2. Experimental Autoimmune Encephalomyelitis (EAE) induction
We purchased the female C57BL/6 mice at 6–8 weeks old from Pasteur Institute, Iran. Animals were kept under pathogen-free conditions at the animal house of IPIU (Institute Physiology of IRAN University). They were treated according to the National Institute of Health Guide for Care and Use of Laboratory Animals. We have done the EAE induction based on the previous protocol (21).
2. 3. Isolation and characterization of Microparticle Vesicles (MSC)
Marrow-derived mesenchymal stem cells (MSC) were harvested from the Tibia and Femurs of healthy C57BL/6 mice by flushing method. Cells were taken to plate in T75 flasks using low-glucose Dulbecco’s Modified Eagle’s Medium (LG-DMEM; Invitrogen, Carlsbad, CA), after centrifugation at 1500 rpm for 5 min in Hank’s Balanced Salt Solution buffer (HBSS; Invitrogen, Carlsbad, CA). Then we plated the cells containing 15% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and antibiotics. Non-adherent cells were removed after 6 h incubation at 37 ◦C and humidified 5% CO2. When cultured showed >70 % confluence, adherent cells were gathered using 10 min with incubation at 37 ◦C with 0.05% trypsin (Invitrogen, Carlsbad, CA) solution containing 0.02% ethylene diamine tetraacetic acid (EDTA; Sigma–Aldrich, St. Louis, MO) and washed twice with phosphate buffer saline. Harvested cells were transferred into the T25 flasks for sub-culturing (22). We used the 3rd passage of MSCs (adherent cells) for flow cytometry analysis. Surface expression of stem cell markers characterized using anti-mouse monoclonal antibodies against CD90-PE, and CD73-FITC (all purchased from eBioscience, San Diego, CA). Flow cytometric analyses were performed using a PAS flow cytometer (Partec GmbH, Germany). Cell Quest software was used for data analysis.
2.4. Isolation and characterization MV
Isolation of MVs were done as previously published protocol (23). After collecting the supernatant of MSC culture, they were centrifuged at 300×g for 10 min, 1000×g for 20 min, and 10,000×g for 30 min. Then, the final centrifuged supernatant was ultra-centrifuged at 100,000 g for 2 h in the ultracentrifuge (Beckman coulter optima TMXL-100K ultracentrifuge. The pelleted MV was washed in saline and again centrifuged at 100,000 g for 2 h. The suspension pellet was quantified by Bradford assay (Sigma–Aldrich, St. Louis, MO).
2.5. Flow Cytometry analysis for MV/gal-1
The MVs (40 µg) were incubated with 4 µm diameter aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA), for 4h at 37 ºC with gentle mixing. We use as 100 mM glycine to fill reactive sites on the beads’ surface to prevent the coupling reaction was stopped. To form pellet MV-coated beads, the mixture was centrifuged at 3000× g for 20 min (24). Then, the suspension of the pellet in phosphate buffer saline was occurred and then washed three times. MV-coated beads were stained using specific antibodies to CD9 FITC, CD63 Biotin followed by streptavidin PE and Anti-GAL1 FITC (MyBiotech Co).
2.6. Virus Culture.
The infection of corona virus was done in a biosafety level 3 laboratory at Pasteur institute. We use as an african green monkey kidney Vero E6 cells with a clinical isolate of SARS-CoV-2 (https://wwwnc.cdc.gov/travel/notices/covid-4/coronavirus-iran). We collected the culture medium containing mature infectious virus (virus medium), and titration were done by plaque assay. Live virus was inactivated by heating at 100 °C for 15 min and was stored at −80 °C for further use.
2.7. Clinical Sample Preparation.
The clinical samples used in this were colltecd who Suspicious patients referred to Emad laboratory were used. They provided written informed consent as registration number: EHW 2020-04-07). Nasopharyngeal swabs from COVID-19 patients and healthy subjects were stored in VTM (Nedashimi, IRAN). Viral copy number was determined by real-time RT-PCR. Clinical samples were inactivated by heating at 100 °C for 10 min and were stored at −80 °C for further use.
2.8. Preparation of MV-gal1/ SARS-CoV-2 Antigen on the SCPE-GNP
Immobilization of MV-gal1was 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) 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 5µl 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.
2.9. Bioconjugation of gold nanoparticle to Anti-SARS-CoV-2 spike
A mixture of 100 µL of Anti-SARS-CoV-2 spike (50 µg/mL in 5 mM KH2PO4, pH 7.5) (MyBiotech Co) and 700 µL of 0.1% Au nanoparticle solution was prepared an kept for 10 min at room temperature. We add 50 µL of 1% PEG in 5 mM KH2PO4 solution (pH= 7.5) and 100 µL of 10% BSA in 50 mM KH2PO4 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 (8000 g 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% NaN3 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.
2.10. Sandwiched Au /Anti-SARS-CoV-2 spike on the MV-gal1/ SARS-CoV-2 Antigen Protein SCPE-GNP
Au /Anti-SARS-CoV-2 spike stock solution was diluted to 100fold and 6 µL of this diluted solution was dropped onto the MV-gal1/ SARS-CoV-2 Antigen Protein. After incubation for 30 min at room temperature, the surface was left for 1h and washed with blank phosphate buffer saline. So, the electrochemical tests were done, again.