Synthesis of ZnO nanoparticles using Mussaenda frondose and its Applications in Sensor with Antibacterial Studies

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The synthesized ZnO nanoparticles (ZnO NPs) were examined using UV-Visible spectrophotometry (UV-VIS), X-ray diffractometry (XRD), Field emission scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDX), Fourier-transform infrared spectroscopy (FTIR) and Transmission electron microscopy illustrating its Hexagonal Wurtzite structure. Electrochemical behaviour of the dopamine was studied using modified ZnO NPs/CPE. These studies revealed high sensitivity and selectivity for the dopamine(DA) detection in presence of 0.2M phosphate buffer solution of pH 7.4. Antibacterial studies revealed activity against the test pathogens evaluated namely Klebsiella sp. (Gram negative) and S. aureus (Gram positive) by assessing the zone of inhibition in mms following the agar cup diffusion assay. Mussaenda frondose Zinc Oxide Nanoparticle Cyclic voltammetry Electrochemical sensor Modified carbon paste electrode Antibacterial Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1.Introduction Nanotechnology is an interdisciplinary field of research involving allied disciplines namely physics, chemistry, biology, electronics, medical, food and agriculture. The synthesis of metal oxide nanoparticles especially ZnO has received gigantic attention due to their global medical and industrial applications [ 1 , 2 ]. Zinc oxide is an n-type semiconductor which has broad band gap (~ 3.37eV) and substantial exciton binding energy ~ 60 meV at room temperature. It is especially attractive for biological applications like sensing, biological labelling, gene delivery, cell imaging and so on. The other properties of relevance like high electron mobility, high thermal conductivity and good transparency make them candidates of choice in the field of electrochemistry [ 3 , 4 ]. Number of chemical and physical processes such as co-precipitation, thermal decomposition, sol-gel, wet chemical, direct precipitation, microwave assisted combustion have been used for the synthesis of ZnO nanoparticles [ 5 – 8 ]. Among the various methods, green synthesis is promoted as it is cost effective, bio safe, biocompatible and eco-friendly approach employing micro-organisms, extract of different parts of plant, DNA and proteins. By virtue of their eco-friendly and sustainable production, biological methods are now being used to synthesise ZnO nanoparticles with desired characteristics [ 9 – 11 ]. Different plant extracts namely Cnidoscolus aconitifolius [ 12 ], Ricinus communis [ 13 ], Calotropis gigantea [ 14 ], Plectranthus amboinicus [ 15 ], Vitex negundo [ 16 ] among others have been used to carry out the green synthesis of ZnO NPs. The phytochemicals such as flavones, terpenoids, polysaccharides, ketones, aldehydes, carboxylic acids, and amides found in plants are involved in the bio-reduction and stability of nanoparticles. The functional groups present increases their capacity to reduce metal ions [ 17 ]. Mussaenda frondosa is a significant tropical plant of family Rubiaceae. The plant has several medicinal benefits and is extensively used as astringent, expectorant in jaundice, wound healing, leprosy, ulcers, hyperacidity, antimicrobial, and protective asthma [ 18 ]. Dopamine (DA) is an important neurotransmitter present in mammalian central nervous system which affects brain processes that control movement, emotional response. Changes in concentration levels of DA may give rise to several diseases such as Parkinson’s disease, Schizophrenia and HIV infection senile dementia. Detection of Dopamine has become an interesting field of research [ 19 – 21 ]. Therefore, it is in demand to develop simple and fast methods to determine Dopamine. Since Dopamine is an electrochemically active compound, its electrochemical behaviour can be studied using voltametric methods. The voltametric techniques are mostly used due to their very good selectivity, high sensitivity, rapid response, good accuracy, their simplicity and low cost. Generally, the rate of electron transfer is slow at the bare electrode surface, therefore the fabrication of cheap modified electrode for determination of Dopamine is investigated by researchers. The modification has to be done by mixing the carbon paste with the modifier in a mortar. It is possible to use various inorganic or organic substances as a modifier. Hence, fabrication of modified carbon paste electrode with excellent catalytic applications is of great importance [ 22 – 24 ]. In the present work, we successfully synthesised ZnO NPs using Mussaenda frondose leaf extract. The fabrication of ZnO modified carbon paste electrode and the study of electrochemical behaviour of Dopamine was carried out using cyclic voltammetry. The ZnO modified carbon paste electrode showed excellent electrocatalytic oxidation of dopamine showing its application in the development of sensors. In these times, greater the functionality of a product greater the acceptance in an ever-demanding society. Hence, we aimed at testing the antibacterial activity of these nanoparticles against bacterial pathogens namely Klebsiella sp. and S. aureus . Klebsiella sp. is a Gram-negative bacteria implicated in healthcare-associated infections, such as pneumonia, bloodstream infections, wound or surgical site infections, and meningitis [ 25 ]. Staphylococcus aureus on the other hand is an infectious agent implicated for the worldwide causes of morbidity and mortality. It causes diseases, ranging from moderately severe skin infections to fatal pneumonia and sepsis [ 26 ]. The biocidal activity of the nanoparticles was ascertained using the agar cup diffusion assay. 2.EXPERIMENTAL SEGMENT 2.1. Preparation of extract: Mussaenda frondose plant leaves were freshly collected from forests of North Goa, India. The leaves were washed thoroughly under the tap water for 5 minutes in the laboratory and air-dried for 2 hours. 10 gm of leaves were weighed, cut into small pieces and rinsed with distilled water. These 10 gm of Mussaenda frondose leaves were placed in a 250ml beaker containing 100mL distilled water and heated at 60 o C for 2 hours until the colour of the extract changes to light yellow. The Mussaenda frondose leaf extract was then cooled, filtered using whatman filter paper and stored in the refrigerator for further procedure of synthesis of ZnO NPs [ 27 , 28 ]. 2.2. Synthesis of ZnO NPs: 2.0 gm of Zinc Nitrate Hexahydrate (purchased from Merck) was dissolved in 50mL of distilled water and kept on magnetic stirrer with hot plate at 60 o C. 50mL of Mussaenda frondose leaf extract was added dropwise with constant stirring. The light-yellow coloured mixture was allowed to heat overnight until yellow paste was obtained. The paste was then dried and calcined in the muffle furnace at 400 o C for 2hours to obtain white colour ZnO NPs [ 29 , 30 ]. 2.3 Medium, chemicals and bacterial strains: Mueller Hinton agar [300 g/l beef infusion, 17.5 g/l casein hydrolysate, 1.5 g/l starch, 17 g/l agar with pH adjusted to 7.4 at 25°C] was used as a growth medium for the maintenance of bacterial isolates and as a medium to assess the antibacterial effects of chemically synthesized nanoparticles. The bacterial strains employed in this study were clinical isolates obtained from the Microbiology laboratory of Goa Medical College Bambolim, Goa; namely Klebsiella sp. and S. aureus. Stock cultures were maintained at 4°C on slopes of Muller Hinton agar medium. 2.4 Antibacterial activity of ZnO nanoparticles The bactericidal potency of the chemically synthesized nanoparticles was evaluated against Klebsiella sp. (Gram negative) and S. aureus (Gram positive) by assessing the zone of inhibition in mms following the agar cup diffusion assay. The nanoparticles were individually dispersed by ultrasonication [Digital Ultrasonic Cleaner (Watts:170W, 220VAC Frequency 42KHz/80W)] in DMSO 50 and 100 µg/ml). This was followed by the inoculation of 0.1 ml of suspension of bacterial cells (10 5 CFU/ml) in 5 ml of Muller Hinton broth and incubation on an orbital shaker [Scigenics Biotech-Orbitek (LT)] at 150 rpm, 37°C for 24 h. Following incubation, 100 µl was spread plated onto a solid agar medium to obtain a matt. An agar-cup or cylinder was cut aseptically with a sterile cork borer and 10 µl of the respective nanoparticle suspension dispensed in the cup, subjected to diffusion at 4°C followed by incubation at 37°C for 24 h. After 24 h, the growth of bacteria was monitored and finally, the zone of inhibition for bacterial growth was determined in a mm scale. 3.RESULTS AND DISCUSSION 3.1. Characterisation of ZnO NPs 3.1.1. X-ray diffraction X-ray powder diffraction was employed to study the phase identification of crystalline structure of ZnO NPs using Rigaku Miniflex 600 X-Ray Diffractometer. XRD diffraction peaks of ZnO NPs synthesised from Mussaenda frondose plant extract are shown in Fig. 1 . The XRD pattern illustrates the noticeable reflection plane such as 100, 002, 101, 102, 110, 103, 200, 112 and 201 which corresponds to the diffraction angles 31.82 o , 34.49 o , 36.31 o , 47.62 o , 56.65 o , 62.97 o , 66.64 o , 68.04 o and 69.14 o respectively. The observed peaks agree well with JCPDS No. 36-1451. The sharp and narrow diffraction peaks confirm Hexagonal Wurtzite crystalline structure without any impurities [ 31 , 32 ]. The diffraction peak maximum observed at plane (101) and the average crystallite size was calculated by using Scherrer equation. $$\text{D}=\frac{\text{k}{\lambda }}{{\beta }\text{c}\text{o}\text{s}{\theta }}$$ where D = Particle size of the crystal K = Scherrer constant (0.9) \({\lambda }\) = x-ray wavelength (0.154nm) β = is the width of the XRD peak at half height θ = Bragg diffraction angle The average crystallite size of the ZnO NPs was calculated as 14.03 nm. 3.1.2. UV-Visible spectrometry The optical property of synthesized ZnO NPs was studied using UV-Visible spectrophotometry. The characterization was done using Thermo scientific Evolution 201 UV-Visible spectrophotometer scanned at the wavelength ranging from 200-800nm. ZnO NPs exhibit strong ultra violet absorption peak at 369.81nm confirmed the formation of ZnO NPs synthesized using Mussaenda frondose leaf extract as in Fig. 2 . The band gap was calculated using formula 1240/λ eV and was found to be 3.35 eV which can be compared with previously reported data [ 33 , 34 ]. . 3.1.3. FTIR spectroscopy FTIR spectroscopy was recorded on Perkin Elmer FTIR Spectrometric analyzer with KBr pellets. FTIR analysis was carried out to determine various functional groups responsible for the formation of ZnO NPs. FTIR spectrum of ZnO NPs from Mussaenda frondose leaf extract is shown in Fig. 3 . It shows broad absorption bands at 3437cm − 1 is assigned to hydroxyl group -OH stretching mode. The absorption bands at 1632.1cm − 1 and 1502.6cm − 1 is ascribed to the C = C stretching vibrations and C = O stretch in polyphenols. The peak at 1384cm − 1 is attributed to stretching vibration of C-N bond. The peaks at 1106.9cm − 1 and 1044.6cm − 1 are assigned to the stretching vibration of C-O bond. The absorption band at 841.7cm − 1 corresponds for alkane C-H stretching mode. The peaks at 515cm − 1 and 467cm − 1 are assigned to Zn-O stretching mode of vibration which confirms the formation of ZnO NPs [ 35 – 37 ]. 3.1.4. Field Emission Scanning electron microscopy and Energy dispersive X-ray FE-SEM was recorded on Quanta FEG 250 Field Emission Scanning Electron Microscope to detect the morphology and the size of a sample by scanning it using a high energy electron beam. FE-SEM image clearly shows individual ZnO NPs predominantly uniform spherical shaped and well dispersed. The Fig. 4 a and 4 b also illustrates the aggregation of nanoparticles into larger particles with well-defined morphology and some of the crystals are more visible. The diameter of particles is ranging from 23-25nm. The nanoparticles are stabilized because of the bioactive reducing, capping and stabilizing agents in Mussaenda frondose extract [ 38 , 39 ]. Energy dispersive X-ray spectroscopy (EDX) revealed the elemental composition of synthesised ZnO NPs. In the fig. Zinc and Oxygen signals were observed which reveals that the synthesised nanoparticles are in pure state of chemical nature. Three sharp signals of Zn and one clear signal O, C, Na, K were observed. The existence of small signals of K, Na in EDX spectrum confirmed the existence of the bioactive compounds of the Mussaenda frondose leaves on the surface of synthesised ZnO nanoparticles [ 40 , 41 ]. 3.1.5. Transmission electron microscopy and Selected area diffraction analysis TEM was employed using JEOL JEM-2100 Plus Electron Microscope to study the morphology of ZnO NPs synthesised using Mussaenda frondose leaf extract. The Fig. 5 a and 5 b shows the TEM images of ZnO NPs. TEM images revealed the agglomerated ZnO NPs with particle size as 21.54nm. SAED pattern includes the planes 100, 002, 101, 102, 110, 103, 200 and 112 which proves the crystallinity and the hexagonal shape of ZnO NPs synthesised from Mussaenda frondose leaf extract [ 42 , 43 ]. 3.2. Zeta Potential Analysis Figure 6 . shows the zeta potential value of ZnO NPs as -20mV. The zeta potential was recorded on Anton Paar Litesizer to analyse the stability and surface charge of ZnO NPs synthesised by using Mussaenda frondose leaf extract. The zeta potential value − 20mV indicates the negative surface charge on synthesised nanoparticles which can be considered as strongly anionic. The binding affinity of Mussaenda frondose extract compounds with the synthesised ZnO NPs gives stability to the ZnO NPs and therefore reduces the potential responsible for aggregation of the particles. Generally, the nanoparticles having zeta potential values more negative than − 30mV and also more positive than + 30mV are considered as stable. [ 44 – 46 ] 3.3 Electrochemical behaviour of Dopamine 3.3.1. The response of DA at the bare CPE, and ZnO NPs / CPE Cyclic voltammetric experiments were performed with a Model 660c (CH Instruments) Potentialstat/Galvanostat. A conventional three electrode cell is employed and the bare or ZnO modified carbon paste electrode (ZnO NPs/CPE) with 3.0 mm diameter as a working electrode, a saturated calomel electrode as a reference electrode and a platinum electrode as a counter electrode. Figure 7 shows the electrochemical responses of 1 x 10 − 5 M DA in 0.2 M phosphate buffer solution (PBS) of pH 7.4 at the Bare carbon paste electrode (BCPE) and the ZnO NPs/CPE with scan rate 0.1 Vs- 1 . At the BCPE, the difference between the anodic peak potential (Epa) 0.136 V and the cathodic peak potential (Epc) 0.085V is reversible wave with ΔE p 0.051 V. However, Dopamine peak currents significantly increased at the ZnO NPs/CPE, with the anodic peak potential as 0.124 V and the corresponding cathodic peak potential as 0.087 V and corresponding ΔE p 0.037V. Compared with BCPE the remarkable enhancement in the peak currents with reduction of over potential showed catalytic effects of the ZnO NPs. The mechanism may be ZnO NPs combined with the hydrogen bond of the hydroxyl of DA, which activated hydroxyl, weakened the bond energy of O–H and improved the electron transfer rate. At the same time, high surface area of the ZnO NPs improved the electrode contact area of DA. 3.3.2. Effect of scan rate on the peak currents The effect of scan rate for 1 x 10 − 5 M DA in 0.2M PBS at pH 7.4 was studied by cyclic voltammetry at ZnO NPs/CPE showed an increase in the redox peak current with increasing scan rate (0.05 to 0.45 Vs _1 ). It has good linearity between the scan rate and redox peak current which indicates the electrode reaction was adsorption controlled which was supported by previously reported literatures [ 47 – 49 ]. 3.4. Antibacterial activity of ZnO NPs : The antimicrobial activity of ZnO NPs is shown in Fig. 8 . The growth rate of Klebsiella sp., Staphylococcus aureus decreased with increasing the concentration of ZnO NPs and the highest inhibition of growth was attained at 100 µg/ml. Metal oxides readily undergo redox reactions catalysed by a specific wavelength of light radiation. The occupied conduction band (CB) and a vacant valence band (VB) enables metal oxides to undergo redox reactions following exposure [ 50 ]. The electron and hole pairs generated on the CB and VB react with O 2 and H 2 O adsorbed on the surface of the metal oxides. The chain redox reactions, produces ROS such as hydroxyl radical, hydrogen peroxide and superoxide. These free radicals accompanied by electrostatic interaction, are responsible for the lysis of the bacterial pathogens. The ROS induces oxidative stress and damages the cellular components like DNA and protein [ 51 ]. The antimicrobial activity of ZnO NPs has been tested against both Gram-positive and Gram-negative bacteria [ 52 ]. The cell membrane of Gram positive has a less negative charge that allows penetration of negatively charged ROS. The outer membrane of Gram-negative restricts the penetration of negatively charged ROS. This is illustrative of the better activity of Gram-positive bacteria then Gram- negative bacteria. The antibacterial activities of ZnO NPs have been reported to be dependent on its morphology, particle size, concentration, surface area, etc. [ 53 ]. Conclusion In this study, facile green approach has been explored using Mussaenda frondose plant for the synthesis of ZnO NPs. The result indicated that the method is very simple, cost effective, less toxic and eco-friendly. The XRD pattern showed well defined peaks which confirmed that ZnO NPs have hexagonal wurtzite structure with average crystallite size 14.03 nm. FESEM and TEM analysis revealed that the morphology of the ZnO NPs was found to be spherical. The UV-visible spectra confirmed the maximum absorption at 369.81nm indicating the formation of ZnO NPs. The zeta potential value of ZnO NPs was recorded as -20mV. The biosynthesised ZnO NPs/CPE exhibits highly electrocatalytic activity making it more effective in studying the electrochemical behaviour of Dopamine in 0.2 M Phosphate buffer solution of pH 7.4 by using cyclic voltametric technique. The modified ZnO NPs/CPE showed excellent sensitivity which is expected to hold applications in the field of electroanalytical chemistry and biosensors. Antibacterial studies revealed that the as synthesized ZnO NPs were active against both the test pathogens tested upon namely Klebsiella sp. and S. aureus. Declarations There is no conflict of Interest Ethical Approval : Not Applicable Funding : No Funding Author Contribution Mamata C. Naik : Extraction of ZnO nanoparticles, experiments, Formal analysis, Writing - original draft B.E.Kumara Swamy : Conceptualization, Supervision, Writing - review and editing.Jyothi Kini : Writing - review and editingSheryanne Velho-Pereira : Antibacterial Studies Acknowledgement Only on subscription publication and we don’t want to publish in golden open access because of no grants Data Availability : It’s on request and permission form the Journal References A.A. Annu, Shakeel, Ahmed, Handbook of Ecomaterials 2018 (2018) 1–45. https://doi.org/10.1007/978-3-319-48281-1_115-1 M. Manokari, M.S. Shekhawat, Br. Int. J. Res. Stud. Microbiol. Biotechnol. 1 , 20–24 (2015) T. Karnan, S.A.S. Selvakumar, J. Mol. Struct. 1125 , 358–365 (2016). http://dx.doi.org/10.1016/j.molstruc.2016.07.029 Fu, Li, Z. Fu, Ceram. Int. 41 , 2492–2496 (2015). http://dx.doi.org/10.1016/j.ceramint.2014.10.069 S. Gebre, Hagos, M.G. Sendeku, SN Appl. 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Uttara Kannada,","correspondingAuthor":false,"prefix":"","firstName":"Jyothi","middleName":"","lastName":"Kini","suffix":""},{"id":309205010,"identity":"b9f72dd2-acd4-4fc9-a40d-472fcd7859f1","order_by":2,"name":"Sheryanne Velho-Pereira","email":"","orcid":"","institution":"Goa University","correspondingAuthor":false,"prefix":"","firstName":"Sheryanne","middleName":"","lastName":"Velho-Pereira","suffix":""},{"id":309205011,"identity":"c7da8261-cf5d-42f4-ae04-432626142291","order_by":3,"name":"B.E.Kumara Swamy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYBACCSQ244OECiDFzNxAtBZmgwdnQBQj8VrYJB+2gW3Dr0VyRnbi54qausT+aWcMJBLn1UbztwO1/KjYhlOLtETuZskzxw4nzridY2CQuO147ozDjA2MPWdu49QiJ5G7QbKB7UBuA1BLQuK2Y7kNQC3MjG14tWz+2fCvLnc+UMuBxDnHcucT0gJ02DbJxjbm3A23cwwbEhtqcjcQ0iLZ83abZWPf4fqNt9OKGRKOHcjdCNRyEJ9fJI7nbr7Z8K3OWO528vafP2rqcuedP3zwwY8K3FqQAIcBkDgMZh4gRj0QsD8AEnVEKh4Fo2AUjIKRBAArxWP3tZqCKQAAAABJRU5ErkJggg==","orcid":"","institution":"Kuvempu University","correspondingAuthor":true,"prefix":"","firstName":"B.E.Kumara","middleName":"","lastName":"Swamy","suffix":""}],"badges":[],"createdAt":"2024-05-23 10:18:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4466127/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4466127/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57953216,"identity":"e50f71ae-dcae-484b-b145-cc422229453b","added_by":"auto","created_at":"2024-06-07 23:01:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65844,"visible":true,"origin":"","legend":"\u003cp\u003eXRD pattern of ZnO NPs synthesised from \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/1db7c234549d39a99d2cbb5c.png"},{"id":57953649,"identity":"e6fc0ce1-61fa-4fd0-88ee-aad2467060c7","added_by":"auto","created_at":"2024-06-07 23:09:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44278,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Visible spectra of ZnO nanoparticles\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/b496ceee2b968cdc274e2339.png"},{"id":57953218,"identity":"8031ccc7-8658-4177-be58-e6bc3ce7a2e0","added_by":"auto","created_at":"2024-06-07 23:01:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":88417,"visible":true,"origin":"","legend":"\u003cp\u003eIR spectra of ZnO nanoparticles\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/b6c3f6e3cc3593516417a9d2.png"},{"id":57953650,"identity":"1e8ecaef-8f66-49d9-81f2-ed22568292d0","added_by":"auto","created_at":"2024-06-07 23:09:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":723459,"visible":true,"origin":"","legend":"\u003cp\u003e4a and 4b: FE-SEM images of ZnO NPs\u003c/p\u003e\n\u003cp\u003e4c: EDX of ZnO NPs\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/732af8e06af287d88d063e48.png"},{"id":57953651,"identity":"49e0f07b-3ace-472f-81f5-ada1a170b886","added_by":"auto","created_at":"2024-06-07 23:09:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":727069,"visible":true,"origin":"","legend":"\u003cp\u003e5a,5b: TEM images of ZnO NPs\u003c/p\u003e\n\u003cp\u003e5c: SAED image of ZnO NPs\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/44bdfe695c0aaeea069bfe63.png"},{"id":57953219,"identity":"b506aa11-8efc-45e0-9d0b-d4e9a73e6882","added_by":"auto","created_at":"2024-06-07 23:01:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65057,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential of ZnO NPs synthesised from leaf extract of \u003cem\u003eMussaenda frondose\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/4d9d0eb564a430ab7be225bf.png"},{"id":57953222,"identity":"ffa85f8d-6717-4276-a5c3-b38e9d4c6e16","added_by":"auto","created_at":"2024-06-07 23:01:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":61996,"visible":true,"origin":"","legend":"\u003cp\u003eThe cyclic voltammograms of bare and modified carbon paste electrode\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/9e108dfa3cab3c0400fc2f22.png"},{"id":57953223,"identity":"8e53eb97-c3b0-4be8-92ed-ae35e495cad5","added_by":"auto","created_at":"2024-06-07 23:01:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":776353,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial activity of ZnO NPs synthesized against test cultures\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/9014fcb248f8f21dfbfb543c.png"},{"id":58044401,"identity":"de80c24e-0e6a-4b64-831b-f0a9043958bc","added_by":"auto","created_at":"2024-06-10 11:15:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3139184,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4466127/v1/34f5c6db-89f0-48f6-9a0c-0009dcf86ae8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis of ZnO nanoparticles using Mussaenda frondose and its Applications in Sensor with Antibacterial Studies","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eNanotechnology is an interdisciplinary field of research involving allied disciplines namely physics, chemistry, biology, electronics, medical, food and agriculture. The synthesis of metal oxide nanoparticles especially ZnO has received gigantic attention due to their global medical and industrial applications [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Zinc oxide is an n-type semiconductor which has broad band gap (~\u0026thinsp;3.37eV) and substantial exciton binding energy\u0026thinsp;~\u0026thinsp;60 meV at room temperature. It is especially attractive for biological applications like sensing, biological labelling, gene delivery, cell imaging and so on. The other properties of relevance like high electron mobility, high thermal conductivity and good transparency make them candidates of choice in the field of electrochemistry [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNumber of chemical and physical processes such as co-precipitation, thermal decomposition, sol-gel, wet chemical, direct precipitation, microwave assisted combustion have been used for the synthesis of ZnO nanoparticles [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among the various methods, green synthesis is promoted as it is cost effective, bio safe, biocompatible and eco-friendly approach employing micro-organisms, extract of different parts of plant, DNA and proteins. By virtue of their eco-friendly and sustainable production, biological methods are now being used to synthesise ZnO nanoparticles with desired characteristics [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent plant extracts namely \u003cem\u003eCnidoscolus aconitifolius\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eRicinus communis\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], \u003cem\u003eCalotropis gigantea\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], \u003cem\u003ePlectranthus amboinicus\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003eVitex negundo\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] among others have been used to carry out the green synthesis of ZnO NPs. The phytochemicals such as flavones, terpenoids, polysaccharides, ketones, aldehydes, carboxylic acids, and amides found in plants are involved in the bio-reduction and stability of nanoparticles. The functional groups present increases their capacity to reduce metal ions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eMussaenda frondosa\u003c/em\u003e is a significant tropical plant of family Rubiaceae. The plant has several medicinal benefits and is extensively used as astringent, expectorant in jaundice, wound healing, leprosy, ulcers, hyperacidity, antimicrobial, and protective asthma [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDopamine (DA) is an important neurotransmitter present in mammalian central nervous system which affects brain processes that control movement, emotional response. Changes in concentration levels of DA may give rise to several diseases such as Parkinson\u0026rsquo;s disease, Schizophrenia and HIV infection senile dementia. Detection of Dopamine has become an interesting field of research [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, it is in demand to develop simple and fast methods to determine Dopamine. Since Dopamine is an electrochemically active compound, its electrochemical behaviour can be studied using voltametric methods. The voltametric techniques are mostly used due to their very good selectivity, high sensitivity, rapid response, good accuracy, their simplicity and low cost. Generally, the rate of electron transfer is slow at the bare electrode surface, therefore the fabrication of cheap modified electrode for determination of Dopamine is investigated by researchers. The modification has to be done by mixing the carbon paste with the modifier in a mortar. It is possible to use various inorganic or organic substances as a modifier. Hence, fabrication of modified carbon paste electrode with excellent catalytic applications is of great importance [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present work, we successfully synthesised ZnO NPs using \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract. The fabrication of ZnO modified carbon paste electrode and the study of electrochemical behaviour of Dopamine was carried out using cyclic voltammetry. The ZnO modified carbon paste electrode showed excellent electrocatalytic oxidation of dopamine showing its application in the development of sensors. In these times, greater the functionality of a product greater the acceptance in an ever-demanding society. Hence, we aimed at testing the antibacterial activity of these nanoparticles against bacterial pathogens namely \u003cem\u003eKlebsiella\u003c/em\u003e sp. and \u003cem\u003eS. aureus\u003c/em\u003e. \u003cem\u003eKlebsiella\u003c/em\u003e sp. is a Gram-negative bacteria implicated in healthcare-associated infections, such as pneumonia, bloodstream infections, wound or surgical site infections, and meningitis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e on the other hand is an infectious agent implicated for the worldwide causes of morbidity and mortality. It causes diseases, ranging from moderately severe skin infections to fatal pneumonia and sepsis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The biocidal activity of the nanoparticles was ascertained using the agar cup diffusion assay.\u003c/p\u003e"},{"header":"2.EXPERIMENTAL SEGMENT","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of extract:\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMussaenda frondose\u003c/em\u003e plant leaves were freshly collected from forests of North Goa, India. The leaves were washed thoroughly under the tap water for 5 minutes in the laboratory and air-dried for 2 hours. 10 gm of leaves were weighed, cut into small pieces and rinsed with distilled water. These 10 gm of \u003cem\u003eMussaenda frondose\u003c/em\u003e leaves were placed in a 250ml beaker containing 100mL distilled water and heated at 60\u003csup\u003eo\u003c/sup\u003eC for 2 hours until the colour of the extract changes to light yellow. The \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract was then cooled, filtered using whatman filter paper and stored in the refrigerator for further procedure of synthesis of ZnO NPs [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Synthesis of ZnO NPs:\u003c/h2\u003e \u003cp\u003e2.0 gm of Zinc Nitrate Hexahydrate (purchased from Merck) was dissolved in 50mL of distilled water and kept on magnetic stirrer with hot plate at 60\u003csup\u003eo\u003c/sup\u003eC. 50mL of \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract was added dropwise with constant stirring. The light-yellow coloured mixture was allowed to heat overnight until yellow paste was obtained. The paste was then dried and calcined in the muffle furnace at 400\u003csup\u003eo\u003c/sup\u003eC for 2hours to obtain white colour ZnO NPs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Medium, chemicals and bacterial strains:\u003c/h2\u003e \u003cp\u003eMueller Hinton agar [300 g/l beef infusion, 17.5 g/l casein hydrolysate, 1.5 g/l starch, 17 g/l agar with pH adjusted to 7.4 at 25\u0026deg;C] was used as a growth medium for the maintenance of bacterial isolates and as a medium to assess the antibacterial effects of chemically synthesized nanoparticles. The bacterial strains employed in this study were clinical isolates obtained from the Microbiology laboratory of Goa Medical College Bambolim, Goa; namely \u003cem\u003eKlebsiella\u003c/em\u003e sp. and \u003cem\u003eS. aureus.\u003c/em\u003e Stock cultures were maintained at 4\u0026deg;C on slopes of Muller Hinton agar medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Antibacterial activity of ZnO nanoparticles\u003c/h2\u003e \u003cp\u003eThe bactericidal potency of the chemically synthesized nanoparticles was evaluated against \u003cem\u003eKlebsiella\u003c/em\u003e sp. (Gram negative) and \u003cem\u003eS. aureus\u003c/em\u003e (Gram positive) by assessing the zone of inhibition in mms following the agar cup diffusion assay.\u003c/p\u003e \u003cp\u003eThe nanoparticles were individually dispersed by ultrasonication [Digital Ultrasonic Cleaner (Watts:170W, 220VAC Frequency 42KHz/80W)] in DMSO 50 and 100 \u0026micro;g/ml). This was followed by the inoculation of 0.1 ml of suspension of bacterial cells (10\u003csup\u003e5\u003c/sup\u003e CFU/ml) in 5 ml of Muller Hinton broth and incubation on an orbital shaker [Scigenics Biotech-Orbitek (LT)] at 150 rpm, 37\u0026deg;C for 24 h. Following incubation, 100 \u0026micro;l was spread plated onto a solid agar medium to obtain a matt. An agar-cup or cylinder was cut aseptically with a sterile cork borer and 10 \u0026micro;l of the respective nanoparticle suspension dispensed in the cup, subjected to diffusion at 4\u0026deg;C followed by incubation at 37\u0026deg;C for 24 h. After 24 h, the growth of bacteria was monitored and finally, the zone of inhibition for bacterial growth was determined in a mm scale.\u003c/p\u003e \u003c/div\u003e"},{"header":"3.RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterisation of ZnO NPs\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1. X-ray diffraction\u003c/h2\u003e \u003cp\u003eX-ray powder diffraction was employed to study the phase identification of crystalline structure of ZnO NPs using Rigaku Miniflex 600 X-Ray Diffractometer. XRD diffraction peaks of ZnO NPs synthesised from \u003cem\u003eMussaenda frondose\u003c/em\u003e plant extract are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The XRD pattern illustrates the noticeable reflection plane such as 100, 002, 101, 102, 110, 103, 200, 112 and 201 which corresponds to the diffraction angles 31.82\u003csup\u003eo\u003c/sup\u003e, 34.49\u003csup\u003eo\u003c/sup\u003e, 36.31\u003csup\u003eo\u003c/sup\u003e, 47.62\u003csup\u003eo\u003c/sup\u003e, 56.65\u003csup\u003eo\u003c/sup\u003e, 62.97\u003csup\u003eo\u003c/sup\u003e, 66.64\u003csup\u003eo\u003c/sup\u003e, 68.04\u003csup\u003eo\u003c/sup\u003e and 69.14\u003csup\u003eo\u003c/sup\u003e respectively. The observed peaks agree well with JCPDS No. 36-1451. The sharp and narrow diffraction peaks confirm Hexagonal Wurtzite crystalline structure without any impurities [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe diffraction peak maximum observed at plane (101) and the average crystallite size was calculated by using Scherrer equation.\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{D}=\\frac{\\text{k}{\\lambda }}{{\\beta }\\text{c}\\text{o}\\text{s}{\\theta }}$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003cp\u003ewhere D = Particle size of the crystal\u003c/p\u003e \u003cp\u003eK = Scherrer constant (0.9)\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({\\lambda }\\)\u003c/span\u003e \u003c/span\u003e = x-ray wavelength (0.154nm)\u003c/p\u003e \u003cp\u003eβ = is the width of the XRD peak at half height\u003c/p\u003e \u003cp\u003eθ = Bragg diffraction angle\u003c/p\u003e \u003cp\u003eThe average crystallite size of the ZnO NPs was calculated as 14.03 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2. UV-Visible spectrometry\u003c/h2\u003e \u003cp\u003eThe optical property of synthesized ZnO NPs was studied using UV-Visible spectrophotometry. The characterization was done using Thermo scientific Evolution 201 UV-Visible spectrophotometer scanned at the wavelength ranging from 200-800nm. ZnO NPs exhibit strong ultra violet absorption peak at 369.81nm confirmed the formation of ZnO NPs synthesized using \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract as in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The band gap was calculated using formula 1240/λ eV and was found to be 3.35 eV which can be compared with previously reported data [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e.\u003cb\u003e3.1.3. FTIR spectroscopy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFTIR spectroscopy was recorded on Perkin Elmer FTIR Spectrometric analyzer with KBr pellets. FTIR analysis was carried out to determine various functional groups responsible for the formation of ZnO NPs. FTIR spectrum of ZnO NPs from \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It shows broad absorption bands at 3437cm\u003csup\u003e− 1\u003c/sup\u003e is assigned to hydroxyl group -OH stretching mode. The absorption bands at 1632.1cm\u003csup\u003e− 1\u003c/sup\u003e and 1502.6cm\u003csup\u003e− 1\u003c/sup\u003e is ascribed to the C = C stretching vibrations and C = O stretch in polyphenols. The peak at 1384cm\u003csup\u003e− 1\u003c/sup\u003e is attributed to stretching vibration of C-N bond. The peaks at 1106.9cm\u003csup\u003e− 1\u003c/sup\u003e and 1044.6cm\u003csup\u003e− 1\u003c/sup\u003e are assigned to the stretching vibration of C-O bond. The absorption band at 841.7cm\u003csup\u003e− 1\u003c/sup\u003e corresponds for alkane C-H stretching mode. The peaks at 515cm\u003csup\u003e− 1\u003c/sup\u003e and 467cm\u003csup\u003e− 1\u003c/sup\u003eare assigned to Zn-O stretching mode of vibration which confirms the formation of ZnO NPs [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4. Field Emission Scanning electron microscopy and Energy dispersive X-ray\u003c/h2\u003e \u003cp\u003eFE-SEM was recorded on Quanta FEG 250 Field Emission Scanning Electron Microscope to detect the morphology and the size of a sample by scanning it using a high energy electron beam. FE-SEM image clearly shows individual ZnO NPs predominantly uniform spherical shaped and well dispersed. The Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb also illustrates the aggregation of nanoparticles into larger particles with well-defined morphology and some of the crystals are more visible. The diameter of particles is ranging from 23-25nm. The nanoparticles are stabilized because of the bioactive reducing, capping and stabilizing agents in \u003cem\u003eMussaenda frondose\u003c/em\u003e extract [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEnergy dispersive X-ray spectroscopy (EDX) revealed the elemental composition of synthesised ZnO NPs. In the fig. Zinc and Oxygen signals were observed which reveals that the synthesised nanoparticles are in pure state of chemical nature. Three sharp signals of Zn and one clear signal O, C, Na, K were observed. The existence of small signals of K, Na in EDX spectrum confirmed the existence of the bioactive compounds of the \u003cem\u003eMussaenda frondose\u003c/em\u003e leaves on the surface of synthesised ZnO nanoparticles [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1.5. Transmission electron microscopy and Selected area diffraction analysis\u003c/h2\u003e \u003cp\u003eTEM was employed using JEOL JEM-2100 Plus Electron Microscope to study the morphology of ZnO NPs synthesised using \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract. The Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eb shows the TEM images of ZnO NPs. TEM images revealed the agglomerated ZnO NPs with particle size as 21.54nm. SAED pattern includes the planes 100, 002, 101, 102, 110, 103, 200 and 112 which proves the crystallinity and the hexagonal shape of ZnO NPs synthesised from \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Zeta Potential Analysis\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e. shows the zeta potential value of ZnO NPs as -20mV. The zeta potential was recorded on Anton Paar Litesizer to analyse the stability and surface charge of ZnO NPs synthesised by using \u003cem\u003eMussaenda frondose\u003c/em\u003e leaf extract. The zeta potential value − 20mV indicates the negative surface charge on synthesised nanoparticles which can be considered as strongly anionic. The binding affinity of \u003cem\u003eMussaenda frondose\u003c/em\u003e extract compounds with the synthesised ZnO NPs gives stability to the ZnO NPs and therefore reduces the potential responsible for aggregation of the particles. Generally, the nanoparticles having zeta potential values more negative than − 30mV and also more positive than + 30mV are considered as stable. [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e–\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Electrochemical behaviour of Dopamine\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. The response of DA at the bare CPE, and ZnO NPs / CPE\u003c/h2\u003e \u003cp\u003eCyclic voltammetric experiments were performed with a Model 660c (CH Instruments) Potentialstat/Galvanostat. A conventional three electrode cell is employed and the bare or ZnO modified carbon paste electrode (ZnO NPs/CPE) with 3.0 mm diameter as a working electrode, a saturated calomel electrode as a reference electrode and a platinum electrode as a counter electrode. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the electrochemical responses of 1 x 10\u003csup\u003e− 5\u003c/sup\u003e M DA in 0.2 M phosphate buffer solution (PBS) of pH 7.4 at the Bare carbon paste electrode (BCPE) and the ZnO NPs/CPE with scan rate 0.1 Vs-\u003csup\u003e1\u003c/sup\u003e. At the BCPE, the difference between the anodic peak potential (Epa) 0.136 V and the cathodic peak potential (Epc) 0.085V is reversible wave with ΔE\u003csub\u003ep\u003c/sub\u003e 0.051 V. However, Dopamine peak currents significantly increased at the ZnO NPs/CPE, with the anodic peak potential as 0.124 V and the corresponding cathodic peak potential as 0.087 V and corresponding ΔE\u003csub\u003ep\u003c/sub\u003e 0.037V. Compared with BCPE the remarkable enhancement in the peak currents with reduction of over potential showed catalytic effects of the ZnO NPs. The mechanism may be ZnO NPs combined with the hydrogen bond of the hydroxyl of DA, which activated hydroxyl, weakened the bond energy of O–H and improved the electron transfer rate. At the same time, high surface area of the ZnO NPs improved the electrode contact area of DA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Effect of scan rate on the peak currents\u003c/h2\u003e \u003cp\u003eThe effect of scan rate for 1 x 10\u003csup\u003e− 5\u003c/sup\u003e M DA in 0.2M PBS at pH 7.4 was studied by cyclic voltammetry at ZnO NPs/CPE showed an increase in the redox peak current with increasing scan rate (0.05 to 0.45 Vs\u003csup\u003e_1\u003c/sup\u003e). It has good linearity between the scan rate and redox peak current which indicates the electrode reaction was adsorption controlled which was supported by previously reported literatures [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e–\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Antibacterial activity of ZnO NPs :\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of ZnO NPs is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The growth rate of \u003cem\u003eKlebsiella\u003c/em\u003e sp., \u003cem\u003eStaphylococcus aureus\u003c/em\u003e decreased with increasing the concentration of ZnO NPs and the highest inhibition of growth was attained at 100 µg/ml.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMetal oxides readily undergo redox reactions catalysed by a specific wavelength of light radiation. The occupied conduction band (CB) and a vacant valence band (VB) enables metal oxides to undergo redox reactions following exposure [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe electron and hole pairs generated on the CB and VB react with O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO adsorbed on the surface of the metal oxides. The chain redox reactions, produces ROS such as hydroxyl radical, hydrogen peroxide and superoxide. These free radicals accompanied by electrostatic interaction, are responsible for the lysis of the bacterial pathogens. The ROS induces oxidative stress and damages the cellular components like DNA and protein [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe antimicrobial activity of ZnO NPs has been tested against both Gram-positive and Gram-negative bacteria [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The cell membrane of Gram positive has a less negative charge that allows penetration of negatively charged ROS. The outer membrane of Gram-negative restricts the penetration of negatively charged ROS. This is illustrative of the better activity of Gram-positive bacteria then Gram- negative bacteria. The antibacterial activities of ZnO NPs have been reported to be dependent on its morphology, particle size, concentration, surface area, etc. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, facile green approach has been explored using \u003cem\u003eMussaenda frondose\u003c/em\u003e plant for the synthesis of ZnO NPs. The result indicated that the method is very simple, cost effective, less toxic and eco-friendly. The XRD pattern showed well defined peaks which confirmed that ZnO NPs have hexagonal wurtzite structure with average crystallite size 14.03 nm. FESEM and TEM analysis revealed that the morphology of the ZnO NPs was found to be spherical. The UV-visible spectra confirmed the maximum absorption at 369.81nm indicating the formation of ZnO NPs. The zeta potential value of ZnO NPs was recorded as -20mV. The biosynthesised ZnO NPs/CPE exhibits highly electrocatalytic activity making it more effective in studying the electrochemical behaviour of Dopamine in 0.2 M Phosphate buffer solution of pH 7.4 by using cyclic voltametric technique. The modified ZnO NPs/CPE showed excellent sensitivity which is expected to hold applications in the field of electroanalytical chemistry and biosensors. Antibacterial studies revealed that the as synthesized ZnO NPs were active against both the test pathogens tested upon namely \u003cem\u003eKlebsiella\u003c/em\u003e sp. and \u003cem\u003eS. aureus.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThere is no conflict of Interest\u003c/p\u003e\n\u003cp\u003e Ethical Approval :\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003eFunding :\u003c/p\u003e\n\u003cp\u003eNo Funding\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003eMamata C. Naik : Extraction of ZnO nanoparticles, experiments, Formal analysis, Writing - original draft B.E.Kumara Swamy : Conceptualization, Supervision, Writing - review and editing.Jyothi Kini : Writing - review and editingSheryanne Velho-Pereira : Antibacterial Studies\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eOnly on subscription publication and we don\u0026rsquo;t want to publish in golden open access because of no grants\u003c/p\u003e\n\u003cp\u003eData Availability :\u003c/p\u003e\n\u003cp\u003eIt\u0026rsquo;s on request and permission form the Journal\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eA.A. 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The synthesized ZnO nanoparticles (ZnO NPs) were examined using UV-Visible spectrophotometry (UV-VIS), X-ray diffractometry (XRD), Field emission scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDX), Fourier-transform infrared spectroscopy (FTIR) and Transmission electron microscopy illustrating its Hexagonal Wurtzite structure. Electrochemical behaviour of the dopamine was studied using modified ZnO NPs/CPE. These studies revealed high sensitivity and selectivity for the dopamine(DA) detection in presence of 0.2M phosphate buffer solution of pH 7.4. Antibacterial studies revealed activity against the test pathogens evaluated namely \u003cem\u003eKlebsiella\u003c/em\u003e sp. (Gram negative) and \u003cem\u003eS. aureus\u003c/em\u003e (Gram positive) by assessing the zone of inhibition in mms following the agar cup diffusion assay.\u003c/p\u003e","manuscriptTitle":"Synthesis of ZnO nanoparticles using Mussaenda frondose and its Applications in Sensor with Antibacterial Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 23:00:55","doi":"10.21203/rs.3.rs-4466127/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8980791c-a7d4-478f-ba8e-7bb61bfd35bc","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-10T11:07:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 23:00:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4466127","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4466127","identity":"rs-4466127","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}