key: cord-0820815-8du5lbem authors: Archana, K. M.; Rajagopal, Revathy; Krishnaswamy, Veena Gayathri; Aishwarya, S. title: Application of green synthesised Copper iodide particles on cotton fabric- Protective face mask material against COVID-19 pandemic date: 2021-09-14 journal: Journal of Materials Research and Technology DOI: 10.1016/j.jmrt.2021.09.020 sha: 900ea6cf7bb9e4907f188261628e31093cdb3114 doc_id: 820815 cord_uid: 8du5lbem Microorganisms cause variety of diseases that constitutes a severe threat to mankind. Due to the upsurge of many infectious diseases, there is a high requirement and demand for the development of safety products finished with antimicrobial properties. The study involves the antimicrobial activity of natural cotton coated with copper iodide capped with Hibiscus rosa-sinensis L. flower extract (CuI-FE) which is rich in anthocyanin, cyanidine-3-sophoroside by ultrasonication method. The coated and uncoated cotton fabric was characterised through XRD, SEM, AFM, tensile strength and UV-Visible spectroscopic techniques. XRD confirmed the formation of CuI particles, SEM showed that CuI-FE was prismatic in shape. The average size of CuI-FE particles was found to be 552.45 nm. Anti-bacterial studies showed copper iodide particles to be a potent antimicrobial agent. AFM images confirmed the rupture of bacterial cell walls in the presence of prismatic CuI-FE. In-vitro cytotoxicity investigation of CuI-FE was performed against cancer and spleen cell lines to evaluate the cell viability. Cytotoxicity analysis revealed the IC50 value of 233.93 μg/mL in the presence of CuI-FE. Molecular docking study was also carried out to understand the interaction of CuI-FE with COVID-19 main protease. This paper has given an insight on the usage of CuI-FE coated on the cotton fabric that has proved to have strong inhibition against the nano ranged bacterial, cancerous cell line and a strong interaction with the COVID-19 protease.Such eco-friendly material will provide a safe environment even after the disposable of medical waste from the infectious diseases like influenza and current pandemic like COVID-19. Cotton fabric plays an important role in our everyday life. It has been extensively used for clothing applications since many years. The flexibility and comfort of cotton fabric make it an outstanding and far & widely used textile in the world owing to its exclusive qualities such as restoration, eco-friendly, gentleness, hypoallergenic and hygroscopic nature [1, 2] . Cotton is the most significant textile crop in numerous countries around the globe. Wool, silk, nylon may have been around longer in some countries, but cotton has excelled them all in adaptability and versatility [3] . In the course of Corona Virus Disease-2019 (COVID- 19) , there has been rapid rise in the use of Personal Protective Equipment (PPE) kits by the frontline workers and sanitation bodies to decrease the probability of infections [4] . The efficient management of corona virus infectious waste including PPEs has been recognized as a major area of concern worldwide. PPE kit, include face masks, face shields, goggles, hand gloves, gowns, head and shoe cover which are all made of plastic and they decrease the danger of an individual using them from contracting the infection. However, the extreme usage and consumption of such single-use plastics have become a severe threat to human health and natural ecosystem. In view of immediate and urgent preventive measures taken, it is apparent that the used PPEs waste is expected to multiply in many folds and will create hassle in the present waste management systems posing a serious threat to the environment if not handled properly on time. [5, 6] . Therefore, there is a need of the hour to find an eco-friendly alternative which can effectively replace the single use plastics and control the pollution in the environment. The infection caused by various pathogens should be controlled as the proliferation of microorganisms on fabric could lead to dramatic effects such as deterioration of textile strength, mutilation and odors which can contaminate the fabric as well as endanger the wearer with diseases such as serious skin infections [7] . Generally, the influence of the microorganism on textile material could be understood by two major approaches, that is assimilation and degradation. In fact, their metabolites disclosed the spots on the surface of fabric, the generation of bubbles on colored surface of the fabric, stimulation of breaking of bonds in fibrous materials and adverse effects on mass loss, mechanical strength, variation of chemical properties, etc [8] . Health care concern related with the upsurge of infectious diseases by various microorganisms has augmented the need for the fabrication of an effective multifunctional textile. In this context, J o u r n a l P r e -p r o o f antimicrobial textile has been established to forbid the growth of bacteria on the textile to avoid adverse hygiene effect on wearer. Despite the excellent properties of cotton materials, they also provide a good atmosphere for microbial proliferation, due to their wide surface area and capability to maintain dampness. In order to control such issues, a broad range of chemicals have been used to produce antimicrobial property to cotton materials [9] [10] [11] . Metal-based nanostructures provided novel functionality and capability to develop pristine textile in terms of physical, chemical, and biological properties have been studied as one of the most hopeful routes to make antimicrobial textile. Currently, metallic nanoparticles such as Ag [12, 13] , ZnO [14] , Cu based compounds [15, 16] and TiO2 [17] utilized in the development of antimicrobial textile owing to its potential antimicrobial activity. The coating and deposition of these nanoparticles onto the substrate involved various procedures such as dip coating, ultrasonication, pad-dry cure method, in-situ chemical reduction, covalent bonding methods etc [12-17, 18] It has been reported the ultrasonication coating is of high practical advantage of being rapid, simple and economically viable single-step reaction. It is also eco-friendly and involves 'green' chemistry principles as the method is free from toxic materials [13, 17] generate CuI of even size and good crystalline nature, but the process require toxic raw materials, chemical reducing agents, complex synthetic steps or high temperature which leads to ecological and biological menace. Therefore, it is essential to investigate a neat, facile, and nonpolluting route to produce CuI at viable conditions. Green synthesis is economical, expeditious, J o u r n a l P r e -p r o o f and efficient which characteristically produces nanostructures with a wide range of shapes. The usage of extracts from plants or fruits as reducing, stabilizing and capping agents have become the majorly concentrated area as they effectively produce copper iodide in a much greener and ecologically friendly ways [42] [43] [44] [45] [46] . The present study involves the coating of Hibiscus rosa-sinensis L. flower extract reduced CuI (CuI-FE) as an antimicrobial finish onto natural cotton by ultrasonication method. CuI-FE coated cotton has been characterized by various physical methodologies and the antibacterial activity of it was tested against major skin infection causing bacteria such as E. coli and S. faecalis by Agar Disc-diffusion method. The effect of CuI-FE on bacterial morphology was analysed by Atomic Force Microscopy. Invitro cytotoxicity was performed to study the impact CuI-FE on cell viability of EAC, DLA and rat spleen cells. Furthermore, studies on molecular docking were performed to understand the binding interaction between CuI-FE and COVID-19 main protease. Hibiscus flowers were collected from Stella Maris College campus. AnalR-Grade CuSO4.5H2O and KI were procured from Spectrum and Fischer Scientific India Pvt. Ltd respectively and were employed as obtained. Muller-Hinton agar and Nutrient broth were secured from Hi-Media, India. The bacterial cultures were purchased from Hubert enviro care systems Pvt. Ltd. Natural cotton fiber was purchased from a local store. The extract of Hibiscus flower was prepared in water by the same procedure previously reported by us [47-49]. CuI was synthesized using Hibiscus flower extract by reacting 1:2 molar solutions of CuSO4. The presence of some phytoconstituents in the extract was studied by standard phytochemical methods. Phytochemical analysis of alkaloids, anthocyanins, flavonoids, polyphenols, saponins and tannins were performed according to the tests described in the literature [50, 51] . Table 1 represents the tests conducted for confirming the presence of phytochemicals. The separation of chemicals was done using Helium as the carrier gas at a constant flow rate of 1mL/min on a fused silica column packed with Elite-5MS (5 percent biphenyl 95 percent dimethylpolysiloxane, 30m × 0.25 mm IDx 250m df).The injector temperature was kept at 260°C throughout the chromatographic run. 1 litre of floral extract was put into the instrument, with the following oven temperature: 60°C for 2 minutes, followed by 300°C at a rate of 10°C min-1 and maintained at 300°C for 6 minutes.The mass detector was set to 230°C for the transfer line& the ion source and 70 eV for the ionisation mode electron impact, with a scan length of 0.2 s and a scan interval of 0.1s.The fragment from 40 to 600 Da were collected. Using the GC-MS NIST(2008) library, the spectra of the separated compounds was compared to the spectrum of standard compounds in a database. J o u r n a l P r e -p r o o f CuI-FE was analysed for short term in vitro cytotoxicity using Ehrlich Ascites Carcinoma (EAC) Amala Cancer Research Centre Society (A Society registered T.C.Act, XII of 1955sl.No. 56 of 1984), it is a registered society which follows all rules of its licensing committee for performing of we confirm that all methods were carried out in accordance with regulations. We also confirm that all experimental protocols were approved by a licensing committee. The tumour cells were washed three times with PBS or normal saline after being removed from the peritoneal cavity of tumor-bearing mice. The cell viability was determined using the trypan blue exclusion method. Phosphate buffered saline was used to make up to 1 mL of viable cell suspension (1x10 6 cells in 0.1 mL) in the tubes holding varied amounts of the substances. As a control, a tube holding simply the cell suspension was employed. At 37°C, the test tubes were incubated for 3 hours. The cell solution was then mixed with 0.1mL of 1% trypan blue for 2-3 minutes before being put into a hemocytometer. The dye colour of trypan blue is taken up by the dead cells, while the dye colour is not taken up by the living cells. Separate counts of labeled and unstained cells were made. For rat spleen cells, the rat was sacrificed using carbon dioxide anesthesia and the spleen tissue was taken out. In RPMI complete medium, it was shattered into single cell suspension and filtered with a mesh fabric. The cells thus collected were washed thrice and suspended in known volume of RPMI complete medium containing antibiotics and counted. Viable cell suspension (1x10 6 cells in 0.1 mL) was added to tubes containing various sample concentrations, and the volume was increased to 1 mL using RPMI medium. The tube containing only the cell suspension was used as the control. The test tubes were incubated for 3 hours at 37 o C. The cell solution was then mixed with 0.1mL of 1% trypan blue for 2-3 minutes before being put into a hemocytometer. The number of stained and unstained cells were individually counted [52] . . * 100 J o u r n a l P r e -p r o o f The cotton fabric was made into 5x5 cm 2 The plates were left for 24h at 37 o C for incubation. The zone of inhibition produced by the indicator bacterial strains was measured. The effect of CuI-FE on the cell membrane was investigated by AFM. 40 µL of bacterial suspension treated with CuI-FE (sample) and the untreated one (control) was applied onto a sterilized glass surface and air dried. The sample spots were then rinsed gently with de-ionized water to get rid of any impurities and air dried again [55]. Cyanidine The prepared Hibiscus flower extract was analysed using GC-MS (Perkin Elmer, Clarus 680, Clarus 600(EI)). The fabricated CuI-FE coated and uncoated cotton was delineated using Bruker The results from the qualitative tests performed for Hibiscus rosa-sinensis L. flower extract represented in Table 2 ., indicated that it contained alkaloids, anthocyanins, flavonoids, polyphenols and terpenoids which are the main phytochemical groups [50]. 3. 5. 7. (Fig 1) . Table 3 The surface morphology and elemental analysis was done by SEM and EDAX. CuI-FE appeared to be triangular prism-like in shape with sharp edges Figure 3 . The mean size of the prismatic particles was found to be 552.45nm. The EDAX analysis (inset Figure 3) of CuI-FE revealed the occurrence of Cu and I elements, signifying that the synthesized product is of high purity [47]. The short-term in vitro cytotoxicity analysis results of CuI-FE at five different concentrations in ethanol are given in Table 4 . Linear trendline was drawn for each graph which was used to calculate the IC50 value against each cell line by regression analysis (Figure 4) The cytotoxicity of CuI-FE against the normal cell line was less, that its propensity in biomedical applications can be promising. As a proof of the concept, it was thought worthwhile to coat it onto natural cotton as an antibacterial finish and study its antibacterial property. The surface morphology of CuI-FE coated cotton was probed with scanning electron microscope (SEM). The SEM images of CuI-FE treated cotton and untreated cotton are shown in Fig 7. The untreated cotton (Figure 7 a) and b)) appear as smooth fibers whereas (Fig 7 c) The UV-Vis spectral analysis in the wavelength range of 400-800nm was used to evaluate the coating of CuI-FE on cotton surface. Figure 8 shows the UV-Vis spectra of CuI-FE treated and untreated cotton. The Surface Plasmon resonance peak corresponding to CuI has been noticed in the UV-Vis spectra which revealed the incorporation of CuI-FE in the cotton . Fig 8 a) shows the reflectance of CuI-FE treated and untreated cotton. It is inferred from the spectra that the uncoated sample exhibited greater reflectance values covering from 40-80%. For CuI-FE treated specimen, the value of reflectance was reduced to 20-50%, owing to the decrease in refractive index and roughness of the cotton surface. From Fig 8 b) it was observed that the intensity of absorbance was lesser for uncoated cotton than that of the coated one. A wide absorption peak appeared around 405nm for CuI-FE coated cotton, confirming the good adhesion of CuI-FE [67]. A force-extension plot is used to depict the tensile properties of a material. The tensile strength studies of the natural cotton before and after coating of CuI-FE are shown in Figure 9 a) and b). The extension is directly proportional to the force applied. For both bare cotton and CuI-FE coated cotton, the fabric deforms initially with extension leading to increase in force. Thereafter, at the breakpoint, the force decreases as the fabric can no longer be elongated [68] . J o u r n a l P r e -p r o o f determined to be 36mm and 30mm for E.coli and S. faecalis respectively which shows the higher sensitivity of E.coli towards CuI-FE coated cotton. The changes observed in bacterial cell after treatment with CuI-FE were analyzed using AFM technique. Figure J o u r n a l P r e -p r o o f activity and anti-cancerous activity, hence it was thought worthwhile to do molecular docking study with the CuI-FE synthesized to act against COVID-19 virus. The first stage of treatment is to eliminate the binding S protein to ACE2 receptor [76] which is E. coli expedited by transmembrane serine protease 2 (TMPRSS2) via protease activity. In the COVID-19 main protease, 15 Cu binding sites were identified. The residues and the score of the same is shown in Table 5 . The score of the normal residues were identified as 0.22, whereas the Cu bound residues scored from 3.02 to 5.64. The highest score of 5.64 was obtained with 264 Met, upon Cu binding. The least score 3.02 was seen in 208 Leu, 264 His and 250 Leu. The lollipop plot shown in Figure 12 a) shows the binding affinity of Cu with COVID-19 main protease and Figure 12 b) shows the bound CuI with COVID-19 main protease. Table 6 . The structure of cyanidine-3-sophoroside capped CuI and the docked complex are represented in Figure 13a) J o u r n a l P r e -p r o o f The synthesized CuI-FE was deposited onto the natural cotton to improve its multifunctional properties. 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