Journal Pre-proof

Synthesis of Zinc oxide nanoparticles and its conjugation with antibiotic:

antibacterial and morphological characterization

Pankaj Kumar Tyagi (Conceptualization) (Methodology) (Funding

acquisition), Deepak Gola (Investigation) (Writing - original draft)
(Writing - review and editing), Shruti Tyagi (Conceptualization)
(Methodology) (Funding acquisition), Ankit Kumar Mishra
(Investigation) (Writing - original draft) (Writing - review and editing),
Arvind Kumar (Investigation) (Writing - original draft) (Writing -
review and editing), Nitin Chauhan (Investigation) (Writing - original
draft) (Writing - review and editing), Anami Ahuja (Validation)
(Formal analysis), Sandeep Sirohi (Validation) (Formal analysis)

PII: S2215-1532(20)30373-1
DOI: https://doi.org/10.1016/j.enmm.2020.100391
Reference: ENMM 100391

To appear in: Environmental Nanotechnology, Monitoring & Management

Received Date: 17 June 2020

Revised Date: 11 September 2020
Accepted Date: 20 October 2020

Please cite this article as: Tyagi PK, Gola D, Tyagi S, Mishra AK, Kumar A, Chauhan N, Ahuja
A, Sirohi S, Synthesis of Zinc oxide nanoparticles and its conjugation with antibiotic:
antibacterial and morphological characterization, Environmental Nanotechnology, Monitoring
and amp; Management (2020), doi: https://doi.org/10.1016/j.enmm.2020.100391
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© 2020 Published by Elsevier.

Synthesis of Zinc oxide nanoparticles and its conjugation with antibiotic: antibacterial and

morphological characterization.

Pankaj Kumar Tyagi1* pktgenetics@gmail.com, Deepak Gola1, Shruti Tyagi1, Ankit Kumar

Mishra1, Arvind Kumar1, Nitin Chauhan2, Anami Ahuja3 and Sandeep Sirohi4
1*
Noida Institute of Engineering and Technology, Greater Noida, Uttar Pradesh, India
2
Department of Microbiology, Shaheed Rajguru College of Applied Sciences for Women, University of Delhi,

Delhi, India
3
Dr. A.P.J. Abdul Kalam Technical University, Lucknow, Uttar Pradesh.

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2
Meerut Institute of Engineering and Technology, Meerut, Uttar Pradesh, India

*
Corresponding Author: PK Tyagi

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Highlights

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 ZnO-NP were synthesized by chemical method in alkaline condition.

 ZnO-NP were characterized using UV-vis spectroscopy, SEM, TEM, FTIR

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 ZnO-NP, ciprofloxacin and ZnO-NP conjugated- ciprofloxacin were tested against
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pathogenic bacteria

 ZnO-NP conjugated- ciprofloxacin produces synergetic antibacterial activity

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Abstract

In the present study zinc oxide nanoparticles were synthesized through chemical method in
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alkaline condition. The initial formation of monodispersed zinc oxide nanoparticles was
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confirmed by a sharp peak at 390 nm via UV-Vis spectroscopy. Further, microscopic techniques

such as SEM and TEM established the formation of multi shaped zinc oxide nanoparticles with

an average size of 20-24 nm. The FTIR, analysis confirmed the presence of multiple functional

groups on zinc oxide nanoparticles. In addition to this, zinc oxide nanoparticles were conjugated

with ciprofloxacin (antibiotic) through chemical method. Later, antibacterial potential of zinc
oxide nanoparticles, ciprofloxacin and zinc oxide nanoparticles - ciprofloxacin conjugate were

compared against multiple bacterial pathogens (E. coli, Klebsiella spp., B. subtilis and

Streptococcus spp.). It was observed that, conjugation of zinc oxide nanoparticles with

ciprofloxacin produces synergetic effect in term of antibacterial activity. Results confirmed that,

there was 2.9 fold increase in antibacterial activity of nanoparticle-ciprofloxacin conjugates

against E.coli and 2.8 fold increase for Streptococcus spp. as compared to ciprofloxacin alone.

The present formulation clearly indicates the potential of metallic nanoparticles in the area of

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healthcare.

Keywords: Zinc oxide nanoparticles; Antibiotic; Conjugate; Antibacterial activity

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1. Introduction

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The emergence of new infectious diseases along with an increase in antibiotic resistance among

bacterial pathogen poses a great health risk to human health (Gill et al., 2015; Zeinali Aghdam
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et al., 2019). Developing a drug that can work simultaneously on multiple pathogenic bacteria

remains a challenge in the sector of health care (Garg et al., 2020; Saravanan et al., 2018; Tyagi
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et al., 2019). In past few years, nanotechnology has acquired a great amount of attention due to

its potential application in a wide area of science such as environment, healthcare, electronics,
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bio-sensors etc (Bansal et al., 2019; Jain et al., 2020; Tyagi et al., 2020). Different chemical and

physical approaches have been investigated for the synthesis of metallic (Ag, Au, Cd, Zn, etc.)
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nanoparticles (Balashanmugam et al., 2016; Barbosa et al., 2014; Thiyagarajan et al., 2018;
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Tyagi et al., 2020). Now a day, development of metal oxide nanoparticles has drawn attention as

an antimicrobial agent because of their safety, stability and application against a wide range of

pathogenic bacteria [20] (Roopan et al., 2019). In addition to this, development of zinc oxide

nanoparticles via various methods gained great attention due to their unique properties and

widespread application (Dimapilis et al., 2018; Lingaraju et al., 2016; Nazoori and Kariminik,
2018; Siddiqi et al., 2018). The main benefit of using zinc oxide nanoparticles is that they can

deter the growth of multiple bacterial species even at very small concentration or dose.

Recently, numerous works have been reported on antibacterial activity of zinc oxide

nanoparticles. For example, high and significant antibacterial activity of zinc oxide

nanoparticles was investigated against multiple pathogenic bacteria such as K. aerogenes, P.

aeruginosa, E. coli, and S. aureus (Happy Agarwal et al., 2018; Kadiyala et al., 2018; Slavin et

al., 2017). Exposure of bacterial agents to metallic nanoparticles led to the formation of reactive

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oxygen species (ROS) and hence inhibit the growth of the bacteria (Baker et al., 2017;

Gangishetty et al., 2012) . Researchers have also investigated the antibacterial efficacy of

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nanoparticles in conjugation with antibiotic. Conjugation of antibiotic with nanoparticles may or

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may not produce a synergetic effect (Banoee et al., 2010). Understanding the antimicrobial

potential of zinc oxide nanoparticle in conjugation with ciprofloxacin is very limited and needs
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more scientific attention (Kadam et al., 2019; Patra et al., 2014).

Therefore, in the present study, we describe a chemical method for the synthesis of zinc
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oxide nanoparticles. Further, antibiotic ciprofloxacin was conjugated with amine functionalized

nanoparticles by using a saline coupling agent aminopropyltriethoxysilane under refluxing

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condition using EDC/NHS (3-ethyldimethylaminopropyl carbodiimide/N-hydroxysuccinimide).

The antibacterial potential of zinc oxide nanoparticles, antibiotic and conjugate were compared
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against multiple pathogenic bacteria species. In addition to this, microscopic techniques such as
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transmission Electron Microscopy (TEM), Fourier Transform Infra-Red (FTIR), Scanning

Electron Microscopy (SEM) and X-Ray Diffraction (XRD) patterns were employed to

understand the morphological feature of zinc oxide nanoparticles.

2. Material and methods

2.1 Chemicals and reagent preparation:

All the chemicals required for the synthesis of zinc oxide nanoparticles such as zinc nitrate

(ZnNO3), sodium hydroxides (NaOH), starch, dimethyl sulfoxide (DMSO), 3-

ethyldimethylaminopropyl carbodiimide (EDC), N-hydroxysuccinimide (NHS) were obtained

from Merck. Double distilled water was used to prepare the stock solutions and reagents.

2.2 Synthesis of zinc oxide nanoparticles

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Zinc oxide nanoparticles were chemically synthesized by top down method under strong

alkaline conditions maintained by sodium hydroxide (0.2 M) solution. In the first step, 1000 mL

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soluble starch solution (1%) was prepared (45-45 °C) and 14.47 gm salt of Zinc nitrate was

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added to the above solution. The mixture was stirred continuously for 5 min to form a

homogenous solution. To the above solution, 0.2 M sodium hydroxide was added dropwise with
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continuous stirring for 2h to complete the reaction of zinc oxide nanoparticles synthesis. The

above reaction solution was centrifuged at 10,000 rpm for 10 min and pellets were obtained.
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The pellets were washed 3-4 times with deionized distilled water to remove any starch or

chemical bounds to the zinc oxide nanoparticles. After washing with double distilled water, the
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pellet is washed again using ethanol. The pellet obtained after ethanol washing was dried at

80oC in hot air oven for 2h. The samples were stored at 4oC for further experiments and
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analysis.
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2.3 Characterization of zinc oxide nanoparticles

The zinc oxide nanoparticles were dissolved in double distilled water and primarily

characterized by scanning the nanoparticle solution between 300-700 nm via UV-Vis

spectrophotometer (JASCO-V-530). For FTIR analysis, powder samples of zinc oxide

nanoparticles were taken and spectra were obtained in the range of 4000-500cm-1 (PerkinElmer

One spectrum). For morphological characterization (size, shape and structure) of zinc oxide

nanoparticles, scanning electron microscopy (SEM), Transmission electron microscopy (TEM)

and XRD micrographs were obtained via ZEISS EVO 50 and JEOL JEM-2100F, respectively.

2.4 Amine functionalization of zinc oxide nanoparticles and formation of zinc oxide

nanoparticles-ciprofloxacin conjugates

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Amine functionalization of zinc oxide nanoparticles was obtained through a chemical process,

using EDC/NHS with some modifications in previous reported protocol (Kadam et al., 2019). In

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the first step antibiotic was activated by suspending 10 mg of ciprofloxacin antibiotic in 20 mL

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DMSO: water (1:1) solution with sonication. This was followed by addition of 5 mg EDC with

continuous stirring. To this solution 5 mg of NHS was added and pH of the solution was
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adjusted to the 6. The solution was thereafter kept on stirring for 3 h in dark (Solution 1). In the

second step, 20 mg of amine functionalized zinc oxide nanoparticles were suspended in 5 mL of

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DMSO: water (1:1) solution and was added to solution 1. The pH of the resultant solution was

adjusted to 8 and was kept on stirring for 12 h. To obtain zinc oxide nanoparticles-ciprofloxacin
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conjugates, the solution was centrifuged at 5000 rpm for 10 min. After, centrifugation

supernatant was discarded and pellet was obtained. Pellets were washed 3 times with DMSO
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followed by washing with ethanol. The pellets were dried at 600C for 2 h to obtain zinc oxide

nanoparticles-ciprofloxacin conjugates. The conjugates were stored at 40C for further

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experiments.

2.5 Antibacterial potential

The antibacterial potential of synthesized zinc oxide nanoparticles was tested against pathogenic

bacteria [Gram negative (E.coli and Klebsiella spp.) and Gram positive (B. subtilis and

Streptococcus spp.]. The antibacterial potential of Zinc oxide nanoparticle (5 µg/ml)) was

compared with the standard antibiotic ciprofloxacin (5 µg/ml)). In addition to this, to study the

synergistic effect of nanoparticles and antibiotic combination, the antibacterial potential of zinc

oxide nanoparticle and ciprofloxacin conjugates was examined against above bacteria using disc

diffusion method. Sterile nutrient agar plates were prepared and these plates were inoculated

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with overnight grown bacterial culture (50 μL). A sterilized disc of zinc oxide nanoparticle and

conjugate was prepared via dipping the disc into zinc oxide nanoparticles solution and conjugate

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solution (Zinc nanoparticles+ antibiotic). The standard antibiotic disc was used as positive

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control in the experiment. Afterward, all the plates were incubated at 37°C for 36 h. After

incubation, the petri plates were examined for the zone of inhibition, which appear as a clear
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area around the disc. The diameter of zone of inhibition was calculated in centimetre via scale

for each set in duplicate and the mean value was recorded.
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2.6 Bacterial cell membrane damage assay

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Application of zinc oxide nanoparticles, antibiotic and conjugate cause bacterial cell and hence

releases the cytoplasmic content. The release of content such as DNA can be measured via
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absorbance at 260 nm. For the same, bacterial suspension of E. coli and B. subtilis were
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prepared in several flasks. After that, zinc oxide nanoparticles, ciprofloxacin and conjugate of

concentration 20μg/mL were added to separate flask. Sample volume of 2 mL was withdrawn

from each flask at a regular interval of 1h and filtered via 0.2 μm syringe filter to removal

bacterial cell and debris. The optical density of filtered sample was measured at 260 nm for

DNA content.
3. Results and discussion

3.1 UV visible spectroscopy of zinc oxide nanoparticles

The synthesized zinc oxide nanoparticles were analysed by scanning UV visible spectra (300-

700 nm) of the solution containing zinc oxide nanoparticles. Figure 1 illustrates the UV visible

spectra for synthesized nanoparticles at different time interval. A sharp plasmon peak was

observed after 2 h at 390 nm indicating the presence of zinc oxide nanoparticles in the solution.

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The peak gets further sharpened after 3 h, demonstrating the synthesis process accelerated

between 2h to 3 h. Presence of the single peak clearly indicates the presence of mono-dispersed

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nanoparticles (Tyagi et al., 2019).

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3.2 FTIR characterization of zinc oxide nanoparticles and conjugates
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Further, zinc oxide nanoparticle and zinc oxide nanoparticle-ciprofloxacin conjugates were

characterized via FTIR spectroscopy. Figure S1 illustrate the FTIR spectra for zinc oxide
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nanoparticles and it was observed that multiple peaks were obtained between 4000-400 cm-1.

The sharp peaks at 3169 cm-1 and 3119 cm-1 could be due to bonded O-H (alcohol) and N-H
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(amines) stretching. The peaks between 2429 cm-1 to 1919 cm-1 indicates primarily presence of

C ≡ C stretching of alkynes. The peaks between 1764 cm-1 to 1509 cm-1 signifies the stretching
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due to C = O functional group and presence of aldehydes functional group. The peak at 1384

cm-1 results from the nitro compounds functional groups such as NO2, whereas the peak
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between 1019 cm-1 to 934 cm-1 designates the presence C-N stretching. The peaks of below 834

cm-1 and above 614 cm-1 indicate the presence of alkanes and C-H bending (aromatic

compounds), respectively.
 Further, the FTIR spectrum of nanoparticle-ciprofloxacin conjugates shows multiple

peaks between 3444 cm-1 to 738 cm-1 indicated the stretching and vibration of various

functional groups (Figure S2). The FTIR peaks between 3444 cm-1 to 3201 cm-1 indicated the

presence of bonded O-H functional group (alcohol). The peaks between 2973 cm-1 to 1767 cm-1

designated the peaks due to C-H stretching (aromatic compounds). Prominent peaks between

1638 cm-1 to 1591 cm-1 clearly indicated the presence of NH2 group (amino group), which was

not present in the zinc oxide nanoparticles. Further peaks between 1383 cm-1 to 738 cm-1

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signifies the presence of CH3-(alkyl) and C-O-C (esters) bounded groups on the zinc oxide

nanoparticles + ciprofloxacin conjugates. Presence of similar functional groups on zinc oxide

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nanoparticles and conjugates was observed by multiple authors (Kadam et al., 2004;

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Shanmuganathan et al., 2018).

3.3 Optical characterization of zinc oxide nanoparticles

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The XRD micrograph of the zinc oxide nanoparticles clearly indicates the presence of well-

defined peaks located at Bragg angles (2θ = 20–80 degrees). The zinc oxide nanoparticles
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showed peak at value of 31.75º (100), 34.440 º (002), 36.252 º (101), 47.543 º (102), 56.555 º

(110), 62.870 º (103), 67.917 º (112) and 76.95 º (202), indicating pure and crystalline nature of
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zinc oxide nanoparticles as shown in Figure 2. The average size of zinc oxide nanoparticle

crystal was found to be 24.84 nm using Debye–Scherrer equation and above crystalline value lie
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in the range of 20-26nm.

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Further, TEM micrograph of selected area diffraction pattern of zinc oxide nanoparticles

confirmed the similar size and crystalline nature of nanoparticles. Synthesized nanoparticles

were sticking to one another and agglomeration was observed in TEM micrographs (Figure 3).

Figure 4 shows SEM micrograph of zinc oxide nanoparticles. Multiple shapes of nanoparticles
were observed in SEM micrograph i.e spherical, hexagonal, oval etc. with an average diameter

of 20-26 nm.

3.4 Antibacterial potential of zinc oxide nanoparticles, ciprofloxacin and zinc oxide

nanoparticles- ciprofloxacin conjugate

The antibacterial potential of zinc oxide nanoparticles was confirmed against multiple

pathogenic bacteria species (B. subtilis, Streptococcus spp., Klebsiella spp. and E. coli) in term

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of the zone of inhibition or clear zone via disk diffusion method. Zinc oxide nanoparticles

showed maximum antibacterial activity against Klebsiella spp. with 3.13 cm (diameter) of zone

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of inhibition, whereas least antibacterial potential of nanoparticles was observed for E. coli

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having zone of inhibition 0.4 cm (diameter). It was observed that in comparison to antibiotic

ciprofloxacin, the antibacterial potential of zinc oxide nanoparticles was less against all the
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bacterial species, except Streptococcus spp. Ciprofloxacin also showed maximum antibacterial

potential against Klebsiella spp. followed by B. subtilis, Streptococcus spp and E. coli as
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illustrated in Figure 5.

The above results clearly indicated that the antibiotic ciprofloxacin are more efficient to
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kill pathogenic bacteria as compared to zinc oxide nanoparticles. However, synergistic effects

were observed in antibacterial activity when zinc oxide nanoparticles were used in conjugation
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with antibiotic against all the bacterial species. Nanoparticle-ciprofloxacin conjugates showed
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maximum antibacterial activity against Klebsiella spp, with 5.03 cm (diameter) zone of

inhibition. The minimum zones of inhibition for nanoparticle-ciprofloxacin conjugates were

obtained against Streptococcus spp i.e 2.8 cm (diameter). In summary, there were 2.9 fold

increases in antibacterial activity of nanoparticle-ciprofloxacin conjugates against E.coli and 2.8

fold increase for Streptococcus spp. as compared to ciprofloxacin alone as shown in Table 1.
Maximum 4.3 fold increment in antimicrobial activity against Klebsiella spp. was observed for

conjugate as compared to nanoparticles alone, while the minimum activity was observed for

Streptococcus spp. Whereas as compared to ciprofloxacin alone, conjugate exhibit 2.9 fold

increment in antibacterial activity for E. coli, followed by 2.7 fold increment against

Streptococcus spp. The conjugation between the nanoparticles and antibiotic is probably based

on the adsorption phenomenon due to intermolecular forces. This intermolecular force cause

high local density of antibiotic molecules attached to the surface of nanoparticle and produced

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polyvalent effect. Due to this nanoparticles also act as a delivery system for the antibiotic

internalization inside bacterial cell and hence causing synergetic effect (Monti et al., 2019; Saha

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et al., 2007). Moreover, the conjugated molecules provides more stability in comparison to the

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individual molecules/mixture of molecules (Saha et al., 2007).

Few previous studies i.e., Auger et al., (2018) combined mixed nano-Zn-MgO with
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ciprofloxacin and observed an increased in antibiotic activity towards B. subtilis and E. coli.

Similarly, Banoee et al. (2010) showed that ZnO nanoparticles enhanced antibacterial activity of
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ciprofloxacin against S. aureus and E. coli. Similar, synergetic effect in antibacterial activity of

metallic nanoparticles in conjugation with different antibiotics was observed against B. cereus,
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S. aureus, K. pneumoniae, P. aeruginosa, S. typhi and P. fluorescens (Agrawal and Kulkarni,

2015; Harshiny et al., 2015). Investigator stated that conjugate (nanoparticle+ ceftriaxone)
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displayed greatest antimicrobial potential against multiple pathogenic bacteria compared to

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antibiotic alone (Patra et al., 2015). The possible synergistic effect might be due rupturing of

the cell wall via silver nanoparticles and increase in reactive oxygen species due to ceftriaxone

by ultimately causing cell death. In the present study, the small sized zinc oxide nanoparticles

are speculated to act as the primary agents in damaging the bacterial cell membrane and

generating reactive oxygen species, responsible for the antibacterial activity. Further, when
conjugates were used, cell membrane damaged by nanoparticles might have created an easy

entry passage for antibiotic thereby, inhibiting bacterial growth with higher potential (Happy

Agarwal et al., 2018; Nazoori and Kariminik, 2018). Cell membrane damage caused by both

nanoparticles and conjugates was confirmed by 260 nm release assay (Kadam et al., 2019;

Tiwari et al., 2018). Damage in cell membrane cause release of cytoplasmic material such as

DNA, RNA and other material (Chatterjee et al., 2014; Tiwari et al., 2018). Hence, absorbance

at 260 nm would increase, if leakage of cytoplasmic material occurred due to damaged cell

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membrane. Figure 6 (A and B) illustrates the absorbance peak for E. coli and B. subtilis at 260

nm was maximum for conjugates as compared to nanoparticles and antibiotic alone, hence

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confirming high antibacterial activity of conjugates. It was observed that, absorbance at 260 nm

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was more for E. coli as compared to B. subtilis. The presence of thick peptidoglycan layer and

anionic glycopolymers (teichoic acids) on gram positive bacteria, might be the reason for the
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above observation as these constituent of cell membrane can provide resistance to the toxic

environment (Tyagi et al., 2019).

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4. Conclusion
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Zinc oxide nanoparticles in powder form were synthesized through chemical method.

Synthesized nano-powder of zinc oxide nanoparticles were characterize through UV-Vis

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spectroscopy, Fourier Transform Infra-Red (FTIR), Transmission Electron Microscopy (TEM),

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Scanning Electron Microscopy (SEM) and X-Ray Diffraction (XRD) patterns. The antibacterial

efficacy of zinc oxide nanoparticles, ciprofloxacin and zinc oxide nanoparticles-ciprofloxacin

conjugate were checked against gram negative and positive pathogenic bacteria. The sharp

plasmon peak of zinc oxide nanoparticles was observed at 390 nm. FTIR results confirm

successful formation of conjugates of zinc oxide nanoparticle-ciprofloxacin. Newly formed zinc

oxide nanoparticles-ciprofloxacin conjugates were tested against pathogenic bacteria for

antibacterial activity and a zone of inhibition of (5.2 cm) in Klebsiella spp. followed by 3.2 cm

in E. coli, 2.9 in B. subtilis and 2.8 in Streptococcus spp. was observed. Finding of this work

clearly indicates that the conjugate of zinc oxide nanoparticles-ciprofloxacin was successfully

able to produce a strong response against pathogenic bacteria species.

Author Statement

Pankaj Kumar Tyagi and Shruti Tyagi: Conceptualization, Methodology and Funding

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acquisition

Deepak Gola, Ankit Kumar Mishra, Arvind Kumar and Nitin Chauhan: Investigation, Writing -

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Original Draft and Writing - Review & Editing

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Anami Ahuja and Sandeep Sirohi: Validation and Formal analysis

Author’s Contribution
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All authors contribute equally.

Conflict of interest statement

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The authors declare that they have no conflict of interest.

Acknowledgement
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Authors gratefully acknowledge CST, Uttar Pradesh [Grant no: CST/8276 (Young Scientist
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Scheme) and CST/1586 (Adhoc Research Grant)] for funds. We are thankful to the AIIMS

(New Delhi, India) facilities for their technical support in SEM and TEM analysis.
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Figures 1: Figure 1: UV-Vis absorption spectra for chemically synthesized zinc oxide

nanoparticles.

Figure 2: XRD micrograph of zinc oxide nanoparticles.

Figure 3: TEM micrograph of zinc oxide nanoparticles.

Figure 4: SEM micrograph of zinc oxide nanoparticles.

Figure 5: Zone of inhibition cause by zinc oxide nanoparticles, ciprofloxacin and conjugate.

Figure 6: Mean absorbance against time to study release DNA from zinc oxide nanoparticles,

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ciprofloxacin and conjugate (zinc oxide nanoparticles-ciprofloxacin) treated E. coli (A) and B.

subtilis (B) at 260 nm at 0, 1, 2, 3 and 4 h time intervals.

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Figure 1: UV-Vis absorption spectra for chemically synthesized zinc oxide nanoparticles.
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Figure 2: XRD micrograph of zinc oxide nanoparticles.

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Figure 3: TEM micrograph of zinc oxide nanoparticles.
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Figure 4: SEM micrograph of zinc oxide nanoparticles.
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Figure 5: Zone of inhibition cause by zinc oxide nanoparticles, ciprofloxacin and conjugate
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Figure 6. Mean absorbance against time to study release DNA from zinc oxide nanoparticles,
ciprofloxacin and conjugate (zinc oxide nanoparticles-ciprofloxacin) treated E. coli (A) and B.
subtilis (B) at 260 nm at 0, 1, 2, 3 and 4 h time intervals.
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Table 1. Fold increase in antibacterial activity of conjugate as compared to zinc oxide

nanoparticles and ciprofloxacin

Fold increase in activity of conjugate

Bacterial species As compared to nanoparticles As compared to Ciprofloxacin

B. Subtilis 2.4 1.8

Streptococcus spp. 2.3 2.7

E. coli 2.7 2.9

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Klebsiella spp. 4.3 1.0

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