View Article Online

View Journal

NJC
New Journal of Chemistry A journal for new directions in chemistry

Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: S. Shams, W.
Ahmad, A. H. Memon, S. Shams, Y. Wei, Q. Yuan and H. Liang, New J. Chem., 2020, DOI:
10.1039/D0NJ04120C.
Volume 42
Number 6
21 March 2018
This is an Accepted Manuscript, which has been through the
Pages 3963-4776
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry A journal for new directions in chemistry Accepted Manuscripts are published online shortly after acceptance,
rsc.li/njc

before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.

You can find more information about Accepted Manuscripts in the

Information for Authors.

Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
ISSN 1144-2546 Terms & Conditions and the Ethical guidelines still apply. In no event
PAPER
Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
shall the Royal Society of Chemistry be held responsible for any errors
prepared via a microwave-assisted hydrothermal route using
H2O2 and KMnO4 as oxidizing agents
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.

rsc.li/njc
 Page 1 of 8 Please
New do not adjust
Journal margins
of Chemistry

1
2
3 View Article Online

4 DOI: 10.1039/D0NJ04120C

5
6
7 ARTICLE
8
9

New Journal of Chemistry Accepted Manuscript

10
11
12
Cu/H3BTC MOF as a potential antibacterial therapeutic agent
13 against Staphylococcus aureus and Escherichia coli
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

14 Received 00th January 20xx,

15 Accepted 00th January 20xx

16 DOI: 10.1039/x0xx00000x
17
18 Saira Shamsa, Waqas Ahmada, Amjad Hussain Memona, Sumaira Shamsb, Yun Weia, Qipeng Yuana*,
19 Hao Lianga*
20 Abstract
21
22 The objective of this paper was to design a more effective antibacterial agent to overcome the problem of fast-growing
23 bacterial resistance. The results showed that Cu/H3BTC metal organic frame work (MOF) exhibited notable antibacterial
24 performance towards Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and could significantly disrupt the cell
25 membrane, resulting in discharge of cell constituents. In addition, morphological changes were elucidated by using
26 scanning electron microscope (SEM). SEM photographs demonstrated noticeable and apparent changes in the morphology
27 of bacterial cells, signifying cell damage which was further confirmed by confocal laser scanning microscopy (CLSM) test.
28 Furthermore, a result of agarose gel electrophoresis (AGE) has shown that Cu/H3BTC MOF could enter to the bacterial cells
29 by impaired cell membranes and inhibits the synthesis of DNA. These findings indicated that Cu/H3BTC MOF is a potent
30 antimicrobial material that can be applied to hinder the growth of pathogenic microbes.
31
32
33
34
35
36 have fascinated increasing attention as potent antimicrobial
37 1. Introduction materials for the control of health related diseases [6][7].
MOFs are a multi-functional specific type of crystalline
38 Bacterial infections are a public health issue throughout the
hybrid constituents comprised of metal ions and organic
39 world and cause a potent and significant economic concern on linkers and are capable for a broad range of applications
40 the health care of a state. Many human diseases including [8][9][10][11][12]. Biological roles of MOFs have since been
41 septicaemia, extraintestinal diseases, neonatal meningitis, studied in fields of cosmetics, bio sensing, drug delivery and
42 diarrheal and urinary tract infections are induced by E. coli enzyme encapsulation [13][14][15][16][17]. One of the most
43 [1][2]. Moreover, various infections are caused by S. aureus significant application of MOFs is its usage as antimicrobial
44 such as pneumonia, infective endocarditis, Septicaemia, ocular material [18]. Several porous materials like mesoporous silica,
45 infections, and central nervous system infections [3][4]. zeolites and activated carbon have also been examined for
46 The growing number of antibacterial drug resistance have storage and release of antimicrobial substances. However,
47 become critical health care concerns [5]. However, control of MOFs remain valuable and are advantageous due to their
48 biocompatibility, ordered structures, large pore size and
bacterial contamination is definitely among the most
bactericidal nature because of their metal ions [1][18]. A
49 important and appropriate approach to mitigate the incidence
probable cause for this might be due to reactivity of organisms
50 of disease. In this context, nanocomposites and nanomaterials with active metals and the compositional degradation of
51 MOFs, along with discharge of metal ions. Moreover, MOFs
52 have potential to depolarize and oxidize the outer membranes
53 a. StateKey Laboratory of Chemical Resource Engineering, Beijing University of of microorganisms that results in inhibition of protein [19].
54 Chemical Technology, Beijing 100029, P. R. China
b. Department
Subsequently, the antibacterial function of MOFs is commonly
of Zoology, Abdul Wali Khan University, Mardan, Pakistan
55 Corresponding Author: *Prof Hao Liang, *Prof Qipeng Yuan.
ascribed to the existence of metallic ions such as Cu, Fe, Ca, Ag
56 State Key Laboratory of Chemical Resource Engineering, Beijing University of and Zn [20][21]. For instance, Ag containing MOFs have been
57 Chemical Technology, Beisanhuan Donglu 15 Hao, Beijing, P. R. China synthesized having antibacterial function towards E. coli, S.
Tel.: +86 10 64431557; Fax: +86 10 64437610.
58 aureus and P. aeruginosa [22][23]. Similarly, Co imidazolate
E-mail address: lianghao@mail.buct.edu.cn, yuanqp@mail.buct.edu.cn
59 MOF has been studied to inhibit the growth of p. putida and E.
60

Please do not adjust margins

 Please
New do not adjust
Journal margins
of Chemistry Page 2 of 8

1 ARTICLE Journal Name

2
3 coli [24][25]. In another study, the antimicrobial activity of silk 2.4. Diameter of inhibition Zone View Article Online

4 fibers coated with Cu-BTC MOF has been testified [26]. DOI: 10.1039/D0NJ04120C
The antibacterial performance of Cu/H3BTC MOF in contrast to
Recently, our group has synthesized mimic laccase Cu/H3BTC
5 S. aureus and E. coli were tested by employing a qualitative
MOF, using Cu ions and trimesic acid (1,3,5-H3BTC), which
6 technique of zone of inhibition as previously described with
show significant ability in degradation of azo dye and enzyme
7 some modifications [30][31][32]. A volume of 100 µL of the
catalysis [27]. In this material, the organic part is Cu ions which
8 bacterial suspension (nearly 1 × 106 CFU/mL) was evenly
have powerful antibacterial activity and the carboxylic groups
9 spread over agar Luria Bertani (LB) plate. Using sterilized metal
which are classified as hard bases are present [26]. These features

New Journal of Chemistry Accepted Manuscript

borer, holes (6 mm in diameter) was made on LB agar plates
10 have enthused us to assess the antimicrobial properties of
and various concentration (2, 1, 0.5 and 0.25 mg/mL) of
11 Cu/H3BTC MOF. Hence, in present study the inhibitory impact
Cu/H3BTC MOF (dissolved in DMSO) in the pipette was added
12 of Cu/H3BTC MOF was evaluated using S. aureus and E. coli. The
to the holes. DMSO was taken as a negative control as this
13 antibacterial mechanism of Cu/H3BTC MOF on growth of E. coli
polar aprotic solvent has no antibacterial properties and has
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

14 and S. aureus was evaluated via cell membrane integrity, SEM,

ability to dissolve Cu/H3BTC MOF. These inoculated plates
CLSM and AGE.
15 were incubated for 24 h at 37 °C. Subsequent microbial
16 inhibitory zones around the holes were assessed visually which
17 is a sign of the bacteria sensitivity to Cu/H3BTC MOF. The
2. Materials and methods diameters of the inhibition zone were measured with a scale in
18
19 2.1. Materials millimetre.
20 All the reagents and solvents were of analytical grade and
2.5. Elucidation of minimum inhibitory concentration (MIC)
21 were employed without further purification. HEPES (4- (2-
22 hydroxyethyl) -1- piperazine ethane. sulfonic acid) was MIC was measured as reported by previous method with
23 obtained from Tianjin, Heowns Biochem. L1c. (Shanghai particular modification in order to determine the lowest
China). H3BTC (1, 3, 5-benzenetricarboxylic acid) was concentration of Cu/H3BTC MOF that prevents visual growth of
24
purchased from J and K Chemical Ltd. (Beijing., China) and selected microorganisms [33][34]. 100 µL of individual
25
CuCl2, DMSO (Dimethyl sulfoxide) were obtained from Aladdin bacterial suspension having final standardized concentration
26 of 1 ⅹ 106 CFU/mL prepared in nutrient broth was added to
Inc, (Shanghai, China). Bacterial strains such as E. coli (ATCC
27 25922) and S. aureus (CMCC (B) 26003) were bought from the each well. 60 µL of different concentration of Cu/H3BTC MOF
28 CGMCC (China General Microbiological Culture Collection was transferred to the wells containing bacterial suspension
29 Center, Beijing, China). PBS (phosphate buffer saline (0.1 M, pH and placed at temperature 37 °C for 24 h to assess
30 7.2) was purchased from Beijing Kehua Jingwei Technology antibacterial activity. The final concentrations of Cu/H3BTC
31 Co., Ltd whereas AO (acridine orange) and PI (propidium MOF in the wells were from 18.75 to 600 µg/160 µL. Similarly;
32 iodide) were from Sigma–Aldrich. Milli-Q water has been the wells without Cu/H3BTC MOF were also tested as a
33 utilized in the preparation of all the buffers and solutions. negative control to examine their possible antibacterial
34 activity. After incubation, the antimicrobial activity was
35 2.2. Synthesis of Cu/H3BTC MOF measured by assessing absorbance at a wavelength of 600 nm
and the well holding the minimum concentration of Cu/H3BTC
36 The Cu/H3BTC MOF was synthesized using a facile and
MOF that exhibit no visible growth sign are considered as the
37 effectual method according to our prior reported study [27].
MIC.
38 The Cu/H3BTC MOF was prepared by mixing of H3BTC (200 μL,
39 25 mM), HEPES buffer (700 μL, pH 8.0, 10 mM) and CuCl2 (100
2.6. Time- kill assay
40 μL, 50 mM). Furthermore, incubate the samples at room-
temperature for 1 h followed by centrifugation (10,000 rpm, 5 Time kill assay, a more effective qualitative test was performed
41 to determine the interaction between Cu/H3BTC MOF and
min) to take out the supernatant. The obtained precipitates
42 were mixed with sterilized water (1 mL) for volume balance. bacteria according to previous reported method [35]. Briefly,
43 For the subsequent experimental step, samples were freshly suspensions of individually S. aureus and E. coli in log-phase
44 prepared in triplicates. growth were prepared in LB and diluted to obtain final
45 2.3. Bacterial strains and cultures concentration of 1 ⅹ 105 ∼ 106 CFU/mL. Subsequently,
46 Cu/H3BTC MOF at a concentration of 1ⅹ MIC was added with
The antimicrobial properties were assessed with one gram-
47 positive and one gram-negative bacterium namely S. aureus, E. individually bacterial strains and incubated at 37 °C for 24 h
48 coli. Both strains were preserved at -20 °C. Same quantity of with continuous agitation. After every 2 h of time-interval,
49 individual strain was transferred to test tubes holding 4 mL of aliquot of each bacterial suspension was taken and measured
50 LB and incubated for 24 h at 37 °C. At regular time interval, the OD values of collected supernatant at 600 nm wavelength.
51 bacterial suspension was taken out in order to measure its Bacterial culture containing DMSO without the addition of
52 OD600. Subsequently, bacterial suspension was further diluted Cu/H3BTC MOF was used as a negative control. The
and their concentration was studied through cell counting assessments were conducted in triplicate and the results were
53
plate. Then, a standard curve between bacterial concentration noted as mean ± SD (standard deviation).
54
55 and OD600 was plotted to confirm the concentration of bacteria
2.7. Scanning electron microscopy (SEM)
56 to about 105 ∼ 106 CFU/mL for further studies as reported
57 [28][29]. SEM was preformed to study the physical and morphological
58 variations in bacterial cells after treated with Cu/H3BTC MOF.
59 For SEM, bacterial strains were processed according to
60

2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

 Page 3 of 8 Please
New do not adjust
Journal margins
of Chemistry

1 Journal Name ARTICLE

2
3 previously described procedure [36][28]. S. aureus and E. coli were collected by centrifugation and extracted their genomic
View Article Online

4 (around 105 ∼ 108 CFU/mL) were incubated at 37 °C with DNA using EZNA Bacterial DNA kit (Omega Bio-tek, Inc.)
DOI: 10.1039/D0NJ04120C

5 Cu/H3BTC MOF at concentrations of either 1 × MIC and 2 × MIC ensuing the precepts. The quantity and quality of DNA samples
6 in nutrient broth for 24 h. After incubations, bacterial cells were tested by a Nano Drop 2000 (Thermo Electron North
7 were centrifuged, washed thrice with PBS (0.1 M, pH 7.2) America LLC, FL). Finally, 5 µL of each sample was intermixed
8 followed by glutaraldehyde (2.5%, v/v) fixation for 12 h in PBS. with 1.5 µL of loading dye and run on agarose gel stained with
9 Bacterial cells were again centrifuged (10,000 rpm, 10 min), EB (ethidium bromide).

New Journal of Chemistry Accepted Manuscript

10 dehydrated in ethanol (25, 50, 75, 100%, v/v) and rinsed with
11 t-butanol for 20 min. Finally, bacterial cells were treated with
12 platinum (90 sec) and studied by SEM (JSM-6360, JEOL, Japan). 3. Results and Discussion
13 The unprocessed bacterial cells were taken as control. 3.1. Diameter of Inhibition Zone of Cu/H3BTC MOF
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

14 2.8. Confocal laser scanning microscopy (CLSM) study

To analyze the antibacterial properties of Cu/H3BTC MOF, S.
15 To determine the damaging effect of Cu/H3BTC MOF on the aureus and E. coli were chosen for antibacterial experiments as
16 cell membranes of S. aureus and E. coli, CLSM (LEICA SP8, pathogenic and infectious strains. Figure 1, clearly revealed
17 Germany) analysis was carried out by double fluorescent that Cu/H3BTC MOF had more active inhibitory influence on
18 staining AO and PI dyes as described previously [37][38]. AO gram-positive and gram-negative microbes. The results also
19 fluoresce green whereas PI produces red fluorescence and was demonstrated a relationship between antibacterial activity and
20 employed to mark live and dead cells respectively. PI and AO Cu/H3BTC MOF concentration that activity increased as the
were prepared by mixing of PI (10 mg) and AO (5 mg) in PBS concentration of Cu/H3BTC MOF increased. The diameter of
21
(10 mL, 0.01 M, pH 7.2). In short, E. coli and S. aureus cells zone of inhibition (DIZ) with S. aureus and E. coli was 22 mm
22 were treated with Cu/H3BTC MOF during the logarithmic and 16 mm respectively, showing that S. aureus has greater
23 growth phase at different concentrations (1 × MIC and 2 × sensitivity to Cu/H3BTC MOF as compared to E. coli. Such result
24 MIC) for 1 h. Afterwards, the bacteria were stained with PI and might be explained by the distinctive dissimilarities in the cell
25 AO (100 μL) in the absence of light for 30 min using moderate membrane of gram positive and gram negative bacteria [40].
26 shaking. Subsequently, bacterial cells were splashed with PBS, Gram positive bacteria are devoid of an outer membrane. The
27 centrifuged and studied with the help of CLSM at 488/552 nm. absence of outer membrane provokes foreign particles to be
28 The control assay was conducted without Cu/H3BTC MOF able to diffuse by the cell wall [41][42]. Thus, gram positive
29 treatment. bacteria are less resistant to Cu/H3BTC MOF in comparison
30 with gram negative bacteria. It is known that antimicrobial
31 2.9. Integrity of cell membrane activity of copper’s are complex and diverse so it was
32 Cell membrane integrity is described as the condition or presumed that the observed inhibition activity of Cu/H3BTC
MOF was probably a result of release of Cu2+ ions that act as
33 quality of the membrane in perfect state. The integrity of S.
aureus and E. coli strains cell membrane was studied by the the oxidizer for the organic moieties and causes rupture of the
34 cell wall and cell membrane leading to cell lysis [43][44][45].
35 nucleic acids leakage according to the previously described
procedure [34][39]. To measure the nucleic acids leakage, We also hypothesize that the oxidized form of copper in
36 Cu/H3BTC MOF structure could cause the damage of
bacterial suspensions at logarithmic phase were centrifuged,
37 membrane and could boost the antimicrobial potential of
rinsed with PBS (0.1 M, pH 7.2) and re-dissolved in PBS.
38 copper. Subsequently, MIC experiments were performed for
Cu/H3BTC MOF at three different concentrations (control, 1 ⅹ
39 further validation.
MIC, 1.5 ⅹ MIC and 2 ⅹ MIC) was mixed with bacterial
40 suspensions and incubated with constant stirring at 37 °C. At
41 regular interval of time, the sample was collected and
42 centrifuged (8000 rpm, 5 min) to acquire the supernatant.
43 UV/Vis spectrophotometer (UV-2600, UNICO Instrument Co.
44 Ltd., Shanghai, China) at wavelength of 260 nm was used to
45 elucidate nucleic acid concentration in the supernatant. For
46 correction of absorption, PBS was used containing the same
47 concentration of Cu/H3BTC MOF after 2 min of contact with
48 both strains. Bacterial strains without treatment were
corrected with PBS and used as control. Experiment was
49
carried out in triplicate and the results were specified as
50
mean ± SD (standard deviation).
51
52 2.10. Agarose gel electrophoresis for DNA disintegration
53
DNA disintegration and destruction were analyzed by agarose
54
gel electrophoresis for assessing cell death by detecting DNA
55
fragments. Strains of S. aureus and E. coli in logarithmic growth
56
phase were treated with Cu/H3BTC MOF at different
57 Figure 1 Diameters and (inset) images of zones of inhibition for Cu/H3BTC MOF towards
concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 × MIC) at 37 °C
58 S. aureus (B) and E. coli (D). Control groups (A, C).
for 24 h. After 24 h, the bacterial cells of S. aureus and E. coli
59
60

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Please do not adjust margins

 Please
New do not adjust
Journal margins
of Chemistry Page 4 of 8

1 ARTICLE Journal Name

2
3 View Article Online

4 3.2. Minimum inhibitory concentration (MIC) 3.4. Scanning electron microscope observation
DOI: 10.1039/D0NJ04120C

5 The MICs of the Cu/H3BTC MOF with their concentration SEM micrographs in Figure 3 represented the structural
6 ranges studied against the target microorganisms are shown in alterations following treatment of bacterial cells with
7 Table 1. Results indicated that antibacterial activity of Cu/H3BTC MOF. These images demonstrated the obvious
8 Cu/H3BTC MOF was more pronounced against S. aureus than destructive and negative impact towards the treated S. aureus
9 for E. coli as compared to the control groups. This feature and E. coli as compared to untreated or control cells. Results

New Journal of Chemistry Accepted Manuscript

10 could be associated to the distinct features of the cell wall showed that control sample of S. aureus had a rigid and
11 composition which results in increased resistance to antibiotics complete cell wall with round appearance (Figure. 3A).
12 and higher MIC of gram-negative bacteria [46]. Moreover, the Following treatment with Cu/H3BTC MOF at 1 × MIC,
13 noticeable sunken cells and vesicular structures were detected
time-kill test of Cu/H3BTC MOF against two bacterial species
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

in the SEM microphotograph of S. aureus (Figure 3B).

14 has studied.
Furthermore, the cells processed with Cu/H3BTC MOF at 2 ×
15
MIC showed severe damaging effect and had more membrane
16 depression (Figure 3C). In addition, untreated E. coli cells had
Table 1 MIC of Cu/H3BTC MOF against tested bacteria.
17 normal morphology with regular rod shape and smooth
18 surface (Figure 3D). However, the bacterial cells processed
Strains Concentration (µg)
19 with Cu/H3BTC MOF at 1 × MIC as shown in Figure 3E were
20 Control 18.75 37.5 75 150 300 600 shriveled, partially deformed with several visible cracks on the
21 S. +++ ++ + - - - - surface. Furthermore, when exposed to Cu/H3BTC MOF at 2 ×
22 aureus MIC, the E. coli cells were demolished with impaired
23 E. coli +++ +++ ++ + - - - membrane and lost their regularly arranged surface layer
24 (Figure 3F). The SEM results suggested that Cu/H3BTC MOF
- No growth. damaged the bacterial cells membrane permeability in a
25
+ Significant growth inhibition. concentration dependent manner. Subsequently, these
26
+ + Partial growth inhibition. changes might also cause disruption of internal structure of
27 cells [47]. Our SEM results were also in accordance with
+ + + Strong growth.
28 previous literature of testing other antibacterial agents on S.
29 aureus and E. coli [48][49][50].
3.3. Time-kill assay
30
31 The bactericidal effects towards S. aureus and E. coli were
32 verified by a time-kill assay following the MIC values. It is
33 shown in Figure 2 that as compared to control group,
Cu/H3BTC MOF significantly affected the production of S.
34
aureus and E. coli. Furthermore, results also revealed that
35
after appropriate stage, the two species of bacteria entered
36 the rapid growth logarithmic stage and after 12 h stepped into
37 the static growth stage. Although, the bacterial growth was
38 inhibit greatly by incubation with Cu/H3BTC MOF at
39 concentration of 1 × MIC for the entire 24 h. The results of
40 time-kill assay confirmed the impact of Cu/H3BTC MOF at
41 relatively low concentration towards S. aureus and E. coli.
42
43
44
45
Figure 3 SEM micrographs of S. aureus (A, B, C) and E. coli (D, E, F). Control groups (A,
46 D), Cu/H3BTC MOF treatments at 1 × MIC (B, E) and 2 × MIC (C, F).
47
48 3.5. Confocal laser scanning microscopy
49 Aforementioned SEM results demonstrated the death and
50 morphological variations in S. aureus and E. coli cells.
51 Moreover, CLSM was performed to further determine the
52 overall viability of the bacterial cells. Based on the principle of
53 staining, AO can stain both live and dead bacteria which results
54 in the production of green fluorescence whereas bacteria
Figure 2 Inhibitory effect of Cu/H3BTC MOF on the growth curve of (A) S. aureus (B) E. having impaired and injured cell membranes produce yellow
55
coli.
56 fluorescence. Similarly, dead bacteria emit red fluorescence
57 upon staining with PI [51]. CLSM photos in Figure 4A and 4D
described that untreated cells produced only sharp green light,
58
signifying that cells were alive and intact. On the other hand,
59
60

4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

 Page 5 of 8 Please
New do not adjust
Journal margins
of Chemistry

1 Journal Name ARTICLE

2
3 the portion of cells marked with yellow and red fluorescence View Article Online

4 has risen with Cu/H3BTC MOF at 1 × MIC (Figure. 4B and 4E). 3.7. Agarose gel electrophoresis for DNA fragmentation
DOI: 10.1039/D0NJ04120C
This might be due to the increased permeability of the cell
5 Influence of Cu/H3BTC MOF on DNA of S. aureus and E. coli
membrane after treated with 1 × MIC Cu/H3BTC MOF [52].
6 However, after treatment with Cu/H3BTC MOF at 2 × MIC, was studied by agarose gel electrophoresis technique. The
7 number of bacterial cells marked by red fluorescent material electrophoretogram of the genomic DNA of Cu/H3BTC MOF
8 has elevated, while green fluorescence approximately treated bacterial strains as shown in Figure 6 demonstrated
9 vanished (Figure 4C and 4F). These results revealed that major the bright and clear DNA band of the untreated groups.

New Journal of Chemistry Accepted Manuscript

10 portion of S. aureus and E. coli cell membrane was seriously Whereas, Cu/H3BTC MOF treated bacterial DNA bands became
11 destroyed following treatment with Cu/H3BTC MOF at 2 × MIC, dimmer or even extinct, indicating that morphology of E. coli
12 which was in accordance with the SEM assay results. and S. aureus DNA were damaged by the Cu/H3BTC MOF. In
13 addition, this disruptive effect increased with increasing
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

14 concentration of Cu/H3BTC MOF, e.g. the higher the

15 concentration, the greater indistinct the DNA bands. Previous
16 studies also verified similar results of some bacteriostatic
17 agents that might responsible to induce changes in
18 morphology and structure of DNA [53],[54]. According to these
19 results, it could be shown that Cu/H3BTC MOF could also affect
20 and hinder the biological function of DNA along with targeting
21 the cell membrane. On the basis of these results, the
22 significant antibacterial mechanism of Cu/H3BTC MOF was
23 briefed in the following ways: The Cu/H3BTC MOF interrupted
24 the stability of cell membrane which resulted in collapse of
25 active transport [55][56]. Furthermore, this compound entered
26 the impaired membrane of bacteria, causing degradation of
27 DNA.
28
Figure 4 CLSM of S. aureus (A, B, C) and E. coli (D, E, F). Control groups (A, D), Cu/H3BTC
29
MOF treatments at 1 ×MIC (B, E) and 2 ×MIC (C, F).
30
31
32 3.6. Integrity of cell membrane
33
The leakage of nucleic acids was evaluated to reveal the
34
integrity of cell membrane. As shown in Figure 5, released
35
contents of the bacterial cells were measured from 1 h to 7 h.
36
The results exhibited that the absorbance values for release of
37
nucleic acids into bacterial
38
suspension enhanced considerably as a consequence of
39
greater Cu/H3BTC MOF concentration (1 × MIC, 1.5 × MIC, 2 ×
40
MIC). In both strains of S. aureus and E. coli, high leakage of Figure 6 DNA agarose gel electrophoresis of (A) S. aureus (B) E. coli treated with various
41
contents was detected after 1 h of treatment and the concentrations of Cu/H3BTC MOF.
42
absorption continued to increase throughout treatment of 7 h
43
period as compared to control groups. The results clarified
44 4. Conclusion
that Cu/H3BTC MOF triggered severe ravage to the cell
45
membrane, resulting in the intracellular components leakage In conclusion, the present work demonstrated that it is
46
and ultimately cell death. These results were in consistent with possible to use Cu/H3BTC MOF as an antibacterial agent due to
47
the results of SEM assay. potent and significant antibacterial activity. The antibacterial
48
mechanism of Cu/H3BTC MOF was suggested to be that
49
Cu/H3BTC MOF disrupted the integrity of bacterial cell
50
membrane, destroyed DNA and eventually following to the
51
bacterial cell death. Another benefit of Cu/H3BTC MOF is their
52
preparation with quite low-priced, simple and easily available
53
ligand, leading to a more reasonable future application as
54
antimicrobial materials. Such emancipation of bactericidal
55
species uncovers novel guidelines and ideas for biological
56
applications of MOFs.
57
58
59
60
Figure 5 Release of intracellular nucleic acids from (A) S. aureus and (B) E. coli treated
with Cu/H
This 3BTCis
journal MOF. Values
© The shown
Royal are means
Society ± S.D.
of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5

Please do not adjust margins

 Please
New do not adjust
Journal margins
of Chemistry Page 6 of 8

1 ARTICLE Journal Name

2
3 [12] S.K. Bhardwaj, A.L. Sharma, N. Bhardwaj, M. Kukkar, A.A.S.
View Article Online

4 Conflicts of interest Gill, K.H. Kim, A. Deep, TCNQ-dopedDOI: Cu-metal organic

10.1039/D0NJ04120C

5 Authors have declared for not existence of conflict of concern. framework as a novel conductometric immunosensing
6 platform for the quantification of prostate cancer antigen,
7 Acknowledgements Sensors and Actuators, B: Chemical. 240 (2017) 10–17.
8 doi:10.1016/j.snb.2016.08.138.
The authors acknowledged financial assistance from the CSC [13] D. Cunha, M. Ben Yahia, S. Hall, S.R. Miller, H. Chevreau, E.
9
(2016GXYS68), National Natural Science Foundation of China

New Journal of Chemistry Accepted Manuscript

10 Elkaïm, G. Maurin, P. Horcajada, C. Serre, Rationale of drug
(21878014), the Beijing Natural Sciences Foundation encapsulation and release from biocompatible porous
11
(2162030), and Beijing Municipal Education Commission Joint metal-organic frameworks, Chemistry of Materials. 25
12
Funding Project (KZ201710020014), the Double First-rate (2013) 2767–2776. doi:10.1021/cm400798p.
13
Program (ylkxj03) and the 111 Project (B13005).
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

14 [14] P. Horcajada, T. Chalati, C. Serre, B. Gillet, C. Sebrie, T.

References Baati, J.F. Eubank, D. Heurtaux, P. Clayette, C. Kreuz, J.S.
15
16 [1] N. Bhardwaj, S.K. Pandey, J. Mehta, S.K. Bhardwaj, K.H. Chang, Y.K. Hwang, V. Marsaud, P.N. Bories, L. Cynober, S.
17 Kim, A. Deep, Bioactive nano-metal-organic frameworks as Gil, G. Férey, P. Couvreur, R. Gref, Porous metal-organic-
18 antimicrobials against Gram-positive and Gram-negative framework nanoscale carriers as a potential platform for
19 bacteria, Toxicology Research. 7 (2018) 931–941. drug deliveryand imaging, Nature Materials. 9 (2010) 172–
20 doi:10.1039/c8tx00087e. 178. doi:10.1038/nmat2608.
21 [2] M.A. Croxen, R.J. Law, R. Scholz, K.M. Keeney, M. [15] P. Horcajada, R. Gref, T. Baati, P.K. Allan, G. Maurin, P.
22 Wlodarska, B.B. Finlay, Recent advances in understanding Couvreur, G. Férey, R.E. Morris, C. Serre, Metal-organic
23 enteric pathogenic Escherichia coli, Clinical Microbiology frameworks in biomedicine, Chemical Reviews. 112 (2012)
24 Reviews. 26 (2013) 822–880. doi:10.1128/CMR.00022-13. 1232–1268. doi:10.1021/cr200256v.
25 [3] J. S., M. C., T. J., L. G., M. H., F. F., N. X., E. J., V. F., [16] S. Keskin, S. Kizilel, Biomedical Applications of Metal
26 Relationships between Staphylococcus aureus genetic Organic Frameworks, Industrial and Engineering Chemistry
27 background, virulence factors, agr groups (alleles), and Research. 50 (2011) 1799–1812. doi:10.1021/ie101312k.
28 human disease, Infection and Immunity. 70 (2002) 631– [17] V. Lykourinou, Y. Chen, X. Sen Wang, L. Meng, T. Hoang,
29 641. doi:10.1128/IAI.70.2.631. L.J. Ming, R.L. Musselman, S. Ma, Immobilization of MP-11
30 [4] Z.R. Ghalehnoo, Diseases caused by Staphylococcus aureus into a mesoporous metal-organic framework, MP-
31 Diseases caused by Staphylococcus aureus, (2019). 11@mesoMOF: A new platform for enzymatic catalysis,
32 [5] G. Wyszogrodzka, B. Marszałek, B. Gil, P. Dorozyński, Journal of the American Chemical Society. 133 (2011)
33 Metal-organic frameworks: Mechanisms of antibacterial 10382–10385. doi:10.1021/ja2038003.
34 action and potential applications, Drug Discovery Today. 21 [18] J. Quirós, K. Boltes, S. Aguado, R.G. de Villoria, J.J. Vilatela,
35 (2016) 1009–1018. doi:10.1016/j.drudis.2016.04.009. R. Rosal, Antimicrobial metal-organic frameworks
36 [6] S.M. Dizaj, F. Lotfipour, M. Barzegar-Jalali, M.H. Zarrintan, incorporated into electrospun fibers, Chemical Engineering
37 K. Adibkia, Antimicrobial activity of the metals and metal Journal. 262 (2015) 189–197.
38 oxide nanoparticles, Materials Science and Engineering C. doi:10.1016/j.cej.2014.09.104.
39 44 (2014) 278–284. doi:10.1016/j.msec.2014.08.031. [19] R. Karimi Alavijeh, S. Beheshti, K. Akhbari, A. Morsali,
40 [7] A.J. Huh, Y.J. Kwon, “Nanoantibiotics”: A new paradigm for Investigation of reasons for metal–organic framework’s
41 treating infectious diseases using nanomaterials in the antibacterial activities, Polyhedron. 156 (2018) 257–278.
42 antibiotics resistant era, Journal of Controlled Release. 156 doi:10.1016/j.poly.2018.09.028.
43 (2011) 128–145. doi:10.1016/j.jconrel.2011.07.002. [20] C.E. Santo, D. Quaranta, G. Grass, Antimicrobial metallic
44 [8] R.J. Kuppler, D.J. Timmons, Q.R. Fang, J.R. Li, T.A. Makal, copper surfaces kill Staphylococcus haemolyticus via
45 M.D. Young, D. Yuan, D. Zhao, W. Zhuang, H.C. Zhou, membrane damage, MicrobiologyOpen. 1 (2012) 46–52.
46 Potential applications of metal-organic frameworks, doi:10.1002/mbo3.2.
47 Coordination Chemistry Reviews. 253 (2009) 3042–3066. [21] A.L. Casey, D. Adams, T.J. Karpanen, P.A. Lambert, B.D.
48 doi:10.1016/j.ccr.2009.05.019. Cookson, P. Nightingale, L. Miruszenko, R. Shillam, P.
49 [9] S.L. James, Metal-organic frameworks, Chemical Society Christian, T.S.J. Elliott, Role of copper in reducing hospital
50 Reviews. 32 (2003) 276–288. doi:10.1039/b200393g. environment contamination, Journal of Hospital Infection.
51 [10] J. Mehta, N. Bhardwaj, S.K. Bhardwaj, K.H. Kim, A. Deep, 74 (2010) 72–77. doi:10.1016/j.jhin.2009.08.018.
52 Recent advances in enzyme immobilization techniques: [22] M. Berchel, T. Le Gall, C. Denis, S. Le Hir, F. Quentel, C.
53 Metal-organic frameworks as novel substrates, Elléouet, T. Montier, J.M. Rueff, J.Y. Salaün, J.P. Haelters,
54 Coordination Chemistry Reviews. 322 (2016) 30–40. G.B. Hix, P. Lehn, P.A. Jaffrès, A silver-based metal-organic
55 doi:10.1016/j.ccr.2016.05.007. framework material as a “reservoir” of bactericidal metal
56 [11] R. Kaur, K.H. Kim, A. Deep, A convenient electrolytic ions, New Journal of Chemistry. 35 (2011) 1000–1003.
57 assembly of graphene-MOF composite thin film and its doi:10.1039/c1nj20202b.
58 photoanodic application, Applied Surface Science. 396 [23] Y. Liu, X. Xu, Q. Xia, G. Yuan, Q. He, Y. Cui, Multiple
59 (2017) 1303–1309. doi:10.1016/j.apsusc.2016.11.150. topological isomerism of three-connected networks in
60

6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

 Page 7 of 8 Please
New do not adjust
Journal margins
of Chemistry

1 Journal Name ARTICLE

2
3 silver-based metal-organoboron frameworks, Chemical anti-diabetic potential, International Journal of Biological
View Article Online

4 Communications. 46 (2010) 2608–2610. Macromolecules. 62 (2013) 321–329. DOI: 10.1039/D0NJ04120C

5 doi:10.1039/b923365b. doi:10.1016/j.ijbiomac.2013.09.032.
6 [24] S. Aguado, J. Quirós, J. Canivet, D. Farrusseng, K. Boltes, R. [36] V.K. Bajpai, A. Sharma, K.H. Baek, Antibacterial mode of
7 Rosal, Antimicrobial activity of cobalt imidazolate metal- action of Cudrania tricuspidata fruit essential oil, affecting
8 organic frameworks, Chemosphere. 113 (2014) 188–192. membrane permeability and surface characteristics of
9 doi:10.1016/j.chemosphere.2014.05.029. food-borne pathogens, Food Control. 32 (2013) 582–590.

New Journal of Chemistry Accepted Manuscript

10 [25] A. Alonso, X. Muñoz-Berbel, N. Vigués, J. MacAnás, M. doi:10.1016/j.foodcont.2013.01.032.
11 Muñoz, J. Mas, D.N. Muraviev, Characterization of fibrous [37] K. Li, G. Guan, J. Zhu, H. Wu, Q. Sun, Antibacterial activity
12 polymer silver/cobalt nanocomposite with enhanced and mechanism of a laccase-catalyzed chitosan–gallic acid
13 bactericide activity, Langmuir. 28 (2012) 783–790. derivative against Escherichia coli and Staphylococcus
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

14 doi:10.1021/la203239d. aureus, Food Control. 96 (2019) 234–243.

15 [26] A.R. Abbasi, K. Akhbari, A. Morsali, Dense coating of doi:10.1016/j.foodcont.2018.09.021.
16 surface mounted CuBTC Metal-Organic Framework [38] L. Mei, Z. Lu, W. Zhang, Z. Wu, X. Zhang, Y. Wang, Y. Luo, C.
17 nanostructures on silk fibers, prepared by layer-by-layer Li, Y. Jia, Bioconjugated nanoparticles for attachment and
18 method under ultrasound irradiation with antibacterial penetration into pathogenic bacteria, Biomaterials. 34
19 activity, Ultrasonics Sonochemistry. 19 (2012) 846–852. (2013) 10328–10337.
20 doi:10.1016/j.ultsonch.2011.11.016. doi:10.1016/j.biomaterials.2013.09.045.
21 [27] B.T.C. Mof, Facile synthesis of laccase mimic Cu / H 3 BTC [39] Y. Lin, X. Tang, L. Xu, S. Wang, Antibacterial properties and
22 MOF for e ffi cient dye degradation and detection of possible action mechanism of chelating peptides-zinc
23 phenolic pollutants †, (2019) 40845–40854. nanocomposite against Escherichia coli, Food Control. 106
24 doi:10.1039/c9ra07473b. (2019) 106675. doi:10.1016/j.foodcont.2019.06.001.
25 [28] Y. Zhang, X. Liu, Y. Wang, P. Jiang, S.Y. Quek, Antibacterial [40] Ó.L. Ramos, A.C. Santos, M. V. Leão, J.O. Pereira, S.I. Silva,
26 activity and mechanism of cinnamon essential oil against J.C. Fernandes, M.I. Franco, M.E. Pintado, F.X. Malcata,
27 Escherichia coli and Staphylococcus aureus, Food Control. Antimicrobial activity of edible coatings prepared from
28 59 (2016) 282–289. doi:10.1016/j.foodcont.2015.05.032. whey protein isolate and formulated with various
29 [29] W. Ahmad, A.U. Khan, S. Shams, L. Qin, A.U. Rahman, Jo ur, antimicrobial agents, International Dairy Journal. 25 (2012)
30 Journal of Photochemistry & Photobiology, B: Biology. 132–141. doi:10.1016/j.idairyj.2012.02.008.
31 (2020) 111821. doi:10.1016/j.jphotobiol.2020.111821. [41] N.B.A. Prasetya, Ngadiwiyana, Ismiyarto, P.R. Sarjono,
32 [30] D. Lee, S. Eom, Y. Kim, H.S. Kim, M. Yim, S. Lee, D. Kim, J. Synthesis and study of antibacterial activity of polyeugenol,
33 Je, Antibacterial and synergic effects of gallic acid- grafted IOP Conference Series: Materials Science and Engineering.
34 chitosan, Can. J. Microbiol. 60 (2014) 629–638. 509 (2019). doi:10.1088/1757-899X/509/1/012101.
35 [31] S. Shams, A.U. Khan, Q. Yuan, W. Ahmad, Y. Wei, Z.U.H. [42] J.C. Lopez-Romero, H. González-Ríos, A. Borges, M. Simões,
36 Khan, S. Shams, A. Ahmad, A.U. Rahman, S. Ullah, Facile Antibacterial Effects and Mode of Action of Selected
37 and eco-benign synthesis of Au@Fe2O3 nanocomposite: Essential Oils Components against Escherichia coli and
38 Efficient photocatalytic, antibacterial and antioxidant Staphylococcus aureus, Evidence-Based Complementary
39 agent, Journal of Photochemistry and Photobiology B: and Alternative Medicine. 2015 (2015).
40 Biology. 199 (2019) 111632. doi:10.1155/2015/795435.
41 doi:10.1016/j.jphotobiol.2019.111632. [43] A.R. Abbasi, K. Akhbari, A. Morsali, Ultrasonics
42 [32] W. Ahmad, S. Shams, A. Ahmad, Y. Wei, Q. Yuan, A.U. Sonochemistry Dense coating of surface mounted CuBTC
43 Khan, M.S. Khan, A. Ur Rahman, M. Iqbal, Synthesis of Metal – Organic Framework nanostructures on silk fibers ,
44 selenium–silver nanostructures with enhanced prepared by layer-by-layer method under ultrasound
45 antibacterial, photocatalytic and antioxidant activities, irradiation with antibacterial activity, Ultrasonics -
46 Applied Nanoscience (Switzerland). (2019). Sonochemistry. 19 (2012) 846–852.
47 doi:10.1007/s13204-019-01213-z. doi:10.1016/j.ultsonch.2011.11.016.
48 [33] R. Moghimi, A. Aliahmadi, D.J. McClements, H. Rafati, [44] G. Wyszogrodzka, B. Marszałek, B. Gil, P. Dorozyński,
49 Investigations of the effectiveness of nanoemulsions from Metal-organic frameworks: Mechanisms of antibacterial
50 sage oil as antibacterial agents on some food borne action and potential applications, Drug Discovery Today. 21
51 pathogens, LWT - Food Science and Technology. 71 (2016) (2016) 1009–1018. doi:10.1016/j.drudis.2016.04.009.
52 69–76. doi:10.1016/j.lwt.2016.03.018. [45] K. Singbumrung, K. Motina, P. Pisitsak, P. Chitichotpanya, S.
53 [34] C. Xu, J. Li, L. Yang, F. Shi, L. Yang, M. Ye, Antibacterial Wongkasemjit, T. Inprasit, Preparation of Cu-BTC/PVA
54 activity and a membrane damage mechanism of Lachnum Fibers with Antibacterial Applications, Fibers and Polymers.
55 YM30 melanin against Vibrio parahaemolyticus and 19 (2018) 1373–1378. doi:10.1007/s12221-018-8072-8.
56 Staphylococcus aureus, Food Control. 73 (2017) 1445– [46] L. Ponnusamy, N. Xu, G. Stav, D.M. Wesson, C. Schal, C.S.
57 1451. doi:10.1016/j.foodcont.2016.10.048. Apperson, Diversity of bacterial communities in container
58 [35] J. Liu, J. feng Lu, J. Kan, C. hai Jin, Synthesis of chitosan- habitats of mosquitoes, Microbial Ecology. 56 (2008) 593–
59 gallic acid conjugate: Structure characterization and in vitro 603. doi:10.1007/s00248-008-9379-6.
60

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Please do not adjust margins

 Please
New do not adjust
Journal margins
of Chemistry Page 8 of 8

1 ARTICLE Journal Name

2
3 [47] V.K. Bajpai, S.M. Al-reza, U. Kyu, J. Hwi, S. Chul, Chemical View Article Online

4 composition , antibacterial and antioxidant activities of leaf DOI: 10.1039/D0NJ04120C

5 essential oil and extracts of Metasequioa glyptostroboides

6 Miki ex Hu, Food and Chemical Toxicology. 47 (2009) 1876–
7 1883. doi:10.1016/j.fct.2009.04.043.
8 [48] M. Chen, Z. Zhao, H. Meng, S. Yu, LWT - Food Science and
9 Technology The antibiotic activity and mechanisms of

New Journal of Chemistry Accepted Manuscript

10 sugar beet ( Beta vulgaris ) molasses polyphenols against
11 selected food-borne pathogens, LWT - Food Science and
12 Technology. 82 (2017) 354–360.
13 doi:10.1016/j.lwt.2017.04.063.
Published on 23 September 2020. Downloaded by University of New England on 9/25/2020 10:47:45 PM.

14 [49] H. Zhou, J. Ren, Z. Li, Antibacterial activity and mechanism

15 of pinoresinol from Cinnamomum Camphora leaves against
16 food-related bacteria, Food Control. 79 (2017) 192–199.
17 doi:10.1016/j.foodcont.2017.03.041.
18 [50] S. Paul, R.C. Dubey, D.K. Maheswari, S. Chul,
19 Trachyspermum ammi ( L .) fruit essential oil in fl uencing
20 on membrane permeability and surface characteristics in
21 inhibiting food-borne pathogens, Food Control. 22 (2011)
22 725–731. doi:10.1016/j.foodcont.2010.11.003.
23 [51] A.S.H. Hameed, C. Karthikeyan, A.P. Ahamed, N. Thajuddin,
24 N.S. Alharbi, S.A. Alharbi, G. Ravi, In vitro antibacterial
25 activity of ZnO and Nd doped ZnO nanoparticles against
26 ESBL producing Escherichia coli and Klebsiella pneumoniae,
27 Scientific Reports. 6 (2016). doi:10.1038/srep24312.
28 [52] G. Liu, G. Ren, L. Zhao, L. Cheng, C. Wang, B. Sun,
29 Antibacterial activity and mechanism of bifidocin A against
30 Listeria monocytogenes, Food Control. 73 (2017) 854–861.
31 doi:10.1016/j.foodcont.2016.09.036.
32 [53] M. Tao, G. Zhang, J. Pan, C. Xiong, Deciphering the groove
33 binding modes of tau-fluvalinate and flumethrin with calf
34 thymus DNA, Spectrochimica Acta - Part A: Molecular and
35 Biomolecular Spectroscopy. 155 (2016) 28–37.
36 doi:10.1016/j.saa.2015.11.006.
37 [54] L.H. Wang, Z.H. Zhang, X.A. Zeng, D.M. Gong, M.S. Wang,
38 Combination of microbiological, spectroscopic and
39 molecular docking techniques to study the antibacterial
40 mechanism of thymol against Staphylococcus aureus:
41 membrane damage and genomic DNA binding, Analytical
42 and Bioanalytical Chemistry. 409 (2017) 1615–1625.
43 doi:10.1007/s00216-016-0102-z.
44 [55] H.Y. Cui, H. Zhou, L. Lin, C.T. Zhao, X.J. Zhang, Z.H. Xiao, C.Z.
45 Li, Antibacterial activity and mechanism of cinnamon
46 essential oil and its application in milk, Journal of Animal
47 and Plant Sciences. 26 (2016) 532–541.
48 [56] F. Nazzaro, F. Fratianni, L. De Martino, R. Coppola, V. De
49 Feo, Effect of essential oils on pathogenic bacteria,
50 Pharmaceuticals. 6 (2013) 1451–1474.
51 doi:10.3390/ph6121451.
52
53
54
55
56
57
58
59
60

8 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins